Split (1)

train · ~9.11M rows (showing the first 93.5k)

identifier stringlengths 11 32	pdf_url stringlengths 17 4.62k ⌀	lang stringclasses 120 values	error stringclasses 1 value	title stringlengths 2 500 ⌀	source_name stringlengths 1 435 ⌀	publication_year float64 1.9k 2.02k	license stringclasses 3 values	word_count int64 0 1.64M	text stringlengths 1 9.75M
https://openalex.org/W2888588420	https://www.econstor.eu/bitstream/10419/206106/1/23311975.2018.1512833.pdf	English	null	The role of behavioural competences in predicting entrepreneurial funding resource orchestration	Cogent business & management	2,018	cc-by	15,122	Article The role of behavioural competences in predicting entrepreneurial funding resource orchestration Cogent Business & Management Provided in Cooperation with: Taylor & Francis Group Provided in Cooperation with: Taylor & Francis Group Suggested Citation: Parkes, Geoff; Hart, Mark; Rudd, John; Liu, Rebecca (2018) : The role of behavioural competences in predicting entrepreneurial funding resource orchestration, Cogent Business & Management, ISSN 2331-1975, Taylor & Francis, Abingdon, Vol. 5, https://doi.org/10.1080/23311975.2018.1512833 Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Documents in EconStor may be saved and copied for your personal and scholarly purposes. You are not to copy documents for public or commercial purposes, to exhibit the documents publicly, to make them publicly available on the internet, or to distribute or otherwise use the documents in public. Sie dürfen die Dokumente nicht für öffentliche oder kommerzielle Zwecke vervielfältigen, öffentlich ausstellen, öffentlich zugänglich machen, vertreiben oder anderweitig nutzen. If the documents have been made available under an Open Content Licence (especially Creative Commons Licences), you may exercise further usage rights as specified in the indicated licence. Sofern die Verfasser die Dokumente unter Open-Content-Lizenzen (insbesondere CC-Lizenzen) zur Verfügung gestellt haben sollten, gelten abweichend von diesen Nutzungsbedingungen die in der dort genannten Lizenz gewährten Nutzungsrechte. https://creativecommons.org/licenses/by/4.0/ https://creativecommons.o Parkes, Geoff; Hart, Mark; Rudd, John; Liu, Rebecca Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 MANAGEMENT \| RESEARCH ARTICLE Geoff Parkes1, Mark Hart1, John Rudd1 and Rebecca Liu1 Received: 16 August 2017 Accepted: 13 August 2018 First Published: 24 August 2018 Corresponding author: Geoff Parkes, Marketing & Strategy, Aston University,Birmingham, UK E-mail: parkesgs@aston.ac.uk Reviewing editor: Gordon Liu, The Open University, UK Additional information is available at the end of the article Geoff Parkes1*, Mark Hart1, John Rudd1 and Rebecca Liu1 Abstract: This study examines how a psychometric testing tool can be used to explain, predict and measure behavioural competences and how entrepreneurs fund the firm. Reference is made to studies of personality traits. More recent studies have called for research into behaviour and competences and specifically in the finance context of orchestration of resources. The authors take a pragmatic realism perspective using a mixed method study to explore the “reality” of the entrepreneur. Cluster analysis is used to identify the relationship between behavioural competences and funding outcomes. Applying Big 5 Theory of Personality and the Great 8 Competences indicates how behaviour impacts outcomes as entrepreneurs seek to access finance. The identifi- cation of three distinct groups in this longitudinal study means belonging to one of these groups predicts likely behaviour when searching for finance. A strong behavioural characteristic which emerged, validated through interviews and psychometric testing, was an orientation towards engagement and working with © 2018 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. 1. Introduction Behavioural competence is used as a lens through which the differences between entrepreneurs are identified and analysed, examining the impact this has on funding outcomes. A longitudinal metho- dology is adopted to examine how entrepreneurial behaviour influences funding outcomes of the firm. Despite numerous Government initiatives, net lending to businesses has continued to fall, and schemes to encourage lending have made little difference (BOE, 2014). Indeed, “the funding gap has increased exponentially since onset of the financial crisis” (Jones & Jayawarna, 2012). Supply side problems have been exacerbated by banks rebuilding their capital base, becoming more risk averse and being unable to resume adequate lending to creditworthy businesses. Venture capital has also declined, as fund managers seek to carefully manage current investments. Demand side factors have also had an impact on the flow of funds to small firms. The British Bankers Association commented “Every time that people read that banks aren’t lending, they don’t apply”, (Management Today, April 2013). This is consistent with the trend towards discouragement and the “rise of the non-seeker of advice” (SME Finance Monitor, 2013). At the same time, there has been growth in more asset-based lending, and tax incentive schemes have made investments more efficient. This has resulted in the growth of business angel finance. There is also evidence that Peer-to-Peer funding models, now prevalent throughout the world, are beginning to develop in the UK through both debt and equity models. In order to minimise the effect of situational conditions, the study focuses on a single sector— Creative Industries thus controlling for proximal explanations (Magnusson & Endler, 1977). This sector is growing at twice the rate of the rest of the economy and there is a greater reliance on equity finance. However, there is also a lack of data, a greater perception of risk, a lack of collateral and a concern amongst investors that entrepreneurs in this sector lack the commercial acumen to generate value within the venture (Deakins, North, Baldock, & Whittam, 2008). Both demand and supply side factors have therefore resulted in fewer firms chasing diminished funding sources. Despite this environment, 28% of firms in the SME Finance Monitor (2013) expected to try and raise finance and a further 17% were “would be,” and therefore wanted to raise finance if barriers could be removed. Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 other organisations. In a funding context, this manifested itself in using networks, seeking advice and sharing equity. These co-operative, collaborative characteristics are different to the classic image of the entrepreneur as a risk-taker or extrovert. other organisations. In a funding context, this manifested itself in using networks, seeking advice and sharing equity. These co-operative, collaborative characteristics are different to the classic image of the entrepreneur as a risk-taker or extrovert. The study identifies entrepreneurs who are both successful and unsuccessful in finance applications and compares behavioural competency profiles, thus over- coming the limitations of many studies that are biased towards successful enterprises. Subjects: Testing, Measurement and Assessment; Economic Psychology; Corporate Finance; Banking; Entrepreneurship; Small Business Management; Entrepreneurial Finance Keywords: access to finance; analytic induction; behaviour; clusters; competencies; entrepreneur; longitudinal; mixed methodology; personality; pragmatic realism PUBLIC INTEREST STATEMENT It is difficult for financial institutions to determine the future viability of a business based purely on collateral and track record, resulting in potentially viable young businesses being denied access to growth capital. The 2004 Graham Review (HMT, 2004) concluded that even with new credit scor- ing techniques, the problems associated with asymmetric information remain. My main areas of research interest are interna- tional marketing strategies and small firms. I have a background in practice and am inter- ested in the areas of behaviour and factors influencing outcomes in entrepreneurial ven- tures. This study is in line with further work cur- rently undertaken in language competency and the impact on competitive advantage in small firms. This research provides a methodology which enables the identification of competences which overcome some of the difficulties caused by information asymmetry between stakeholders. Financial Institutions, Business Angels and Accelerator Programmes can use Competency data to differentiate between entrepreneurs and aid decision-making in the allocation of funding and other support. Dr Geoff Parkes is the Associate Dean International, Aston Business School and the Associate Pro Vice Chancellor International at Aston University. He is a Warwick MBA and holds a Doctorate of Business Administration from Aston University. He is currently seconded to Aston Hong Kong where he leads the University's international collaborative activities in China and East Asia. He supervises DBA and PhD candidates and his teaching experience includes a variety of entrepreneurship pro- grammes including the Goldman Sachs 10,000 Small Business programme. He is a visiting lec- turer at VWA-Studienakademie, Stuttgart and Hong Kong Polytechnic University. He is a Senior Fellow of the UK Higher Education Academy. © 2018 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. © 2018 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. Page 1 of 29 Page 1 of 29 2. Literature review The study examines demand side market failure and considers the very different funding land- scape faced by entrepreneurs following the banking crisis of 2008. The literature under review therefore considers funding factors, personality and entrepreneurial behaviour. It considers what individual entrepreneurs can “do” and why in order to access finance. Many of the early studies in entrepreneurship were focused on what makes an entrepreneur (Baum & Locke, 2004; Baum & Silverman, 2004; Brockhaus, 1980; McClelland, 1961; Rauch & Frese, 2007; Sandberg & Hofer, 1987), and lacked definitive conclusions (Gartner, 1989). However, more recently, the debate has re-emerged in using personality traits as a means of predicting activity. The Five Factor Model, in particular, has been used to predict behaviour in the general work setting and Ciavarella (2004) has identified this as a useful tool in the entrepreneurial context More recently, there has been a call to look in more detail at behaviour and “what do entrepre- neurs actually do” (Mueller, Volery, & von Siemens, 2012, p. 1; Bird, Schjoedt, & Baum, 2012). A number of studies have looked at entrepreneurial behaviour and it has been concluded that competences provides a potentially useful lens through which to frame these and other questions (Bridge, O’Neill, & Cromie, 1998; Burgoyne, 1993, 1989; Gherardi, 2003; Gruglis, 1997; Holton & Naquin, 2000; Sadler–Smith, Hampson, Chaston, & Badger, 2003). The study of managerial behaviour has been described as “a missing ﬁeld of research within the small business literature” and authors have highlighted the need for reliable measures (O’Gorman, Bourke, & Murray, 2005, p. 2; Bird et al., 2012). Wright and Stigliani (2013) noted the need to examine in more detail how individual entrepreneurs arrive at the appropriate bundles of resources and capabilities to generate growth (Wright & Stigliani, 2013) and called for more “fine grained work” on how entrepreneurs influence outcomes (Wright & Stigliani, 2013, p.4). Other authors have called for an alternative paradigm (Bygrave & Timmons, 1992), involving more field studies and longitudinal research, and embracing the use of multi-dimensional approaches linked to the real working situation of the owner-manager (e.g. Caird, 1993; Gibb & Davies, 1990). McCarthy, Krueger, and Schoenecker (1990), for example, called for more qualitative longitudinal studies to answer questions of how entrepreneurs leverage social networks in order to access funding sources (Brockhaus, 1980; Moran, 1998). Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 funding the small firm. The research makes a contribution through the confirmation that psycho- metric testing can be used to explain and predict how individuals finance the firm; what Wright and Stigliani (2013) describes as resource orchestration. funding the small firm. The research makes a contribution through the confirmation that psycho- metric testing can be used to explain and predict how individuals finance the firm; what Wright and Stigliani (2013) describes as resource orchestration. 1. Introduction There is also evidence that Small Medium Sized Enterprises (SMEs) are increasingly prepared to consider alternative funding sources to the tradi- tional types of equity and loans. This research makes a contribution through the use of psychometric testing and measuring behavioural competences in entrepreneurs. It is a longitudinal study; three qualitative interviews are conducted over a 3-year period, identifying funding outcomes and behaviour associated with Page 2 of 29 3. Theoretical background In studies on how entrepreneurs develop capital structure for small firms, Pecking Order Theory and Funding Escalator models were used to identify how firms fund themselves as they grow (Myers, 1984). Changes in financial markets have resulted in academics re-examining these tools concluding that a definitive rationale for capital structure in small firms remains elusive. In addition, most research has failed to make use of the potential of inductive analysis to uncover what constitutes entrepreneurs’ behaviour in a holistic manner (Bird et al., 2012). The development of the Big Five model of personality traits (Goldberg, 1990) has provided a commonly accepted taxonomy for classifying personality. The absence of an equivalent taxonomy for classifying performance constructs has been repeatedly identiﬁed as a barrier hindering a better understanding of the relationship between personality and performance. Understanding causation, as in how and why things are related, is necessary for effective intervention in organisations and specifying causal pathways and models is a particular strength of psychology. Kurz and Bartram used the concept of behavioural competency to attempt to integrate diverse theories, concepts and measures into an overall model of individual performance. Behavioural competency is defined as sets of behaviours that are instrumental in the delivery of desired results or outcomes. Woodruffe agrees with the definition that behavioural competency is the set of behaviour patterns that the incumbent needs to bring to a position in order to perform its tasks and functions. These definitions represent a development from the trait-based approach of Boyatzis in his seminal book “The Competent Manager”, where job competency is defined as an underlying characteristic of a person which results in an effective and/or superior performance of a job. So, a competency is not the behaviour or performance itself, but the repertoire of capabilities, activities, processes and responses available that enable a range of work demands to be met more effectively by some people than others. The main factor that distinguishes a competency from other weighted composites of psychological constructs is the fact that a competency is defined in relation to its significance for performance at work (Kurz and Bartram 2002). There were therefore a number of attempts to define the competency concept further and to provide more “finely grained” constructs of competency. Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 their conceptualisation, and particularly, in their operationalisation of behaviour. Mueller et al. (2012) also noted, with the exception of the Panel Study of Entrepreneurial Dynamics, many studies build on self-reports, rely on vague behavioural constructs or capture only one behaviour at a time. 2. Literature review Fraser (2014) recognised a number of policy initiatives which could potentially allow entrepreneurs, who previously had been discour- aged borrowers, to consider bank borrowing. These included more awareness of policy initiatives and better support for SMEs. A number of studies have looked at entrepreneurial behaviour and it has been concluded that competences provide a potentially useful lens through which to frame these and other questions (Bridge et al., 1998; Burgoyne, 1993, 1989; Gherardi, 2003; Gruglis, 1997; Holton & Naquin, 2000; Sadler–Smith et al., 2003). Through better observations of behaviour, small business researchers can therefore make a distinctive contribution to the understanding of how small firms are managed and structured (Bird et al., 2012; Gartner, Bird, & Starr, 1992; Mueller et al., 2012; O’Gorman et al., 2005). This will provide a better understanding as to why some individuals are better than others at exploiting resources, in addition to providing more detail on measures and also more detail on entrepreneurial characteristics. Specifically in the context of finding finance, this study examines behaviour and how does individual resource orchestration arrive at the appropriate bundles of resources and capabilities to generate growth (Wright & Stigliani, 2013)? In identifying these opportunities for future research, Bird et al. (2012) noted the shortcomings in research into entrepreneurial behaviour, and called for future researchers to be more precise in Page 3 of 29 Page 3 of 29 4. Objectives This mixed methods study adopts an interpretist agenda and seeks to make a contribution to knowledge in the study of access to finance and entrepreneurship through the application of academic models developed in the psychology domain. Overall, the research seeks to answer the question: Can psychometric testing be used to explain, predict and measure behavioural competences and the funding resource orchestration of the entrepreneur? The objectives of the study are as follows: 1. Present a behavioural competency profile for a sample group of entrepreneurs and identify the differences between individuals. 2. Explore the use of psychometric testing in explaining and predicting how individual entrepre- neurs seek finance for the firm. 2. Explore the use of psychometric testing in explaining and predicting how individual entrepre- neurs seek finance for the firm. 5. Develop a methodology for entrepreneurs, policymakers and financial institutions to identify competencies in finding finance, and overcome problems of information asymmetry. 5. Develop a methodology for entrepreneurs, policymakers and financial institutions to identify competencies in finding finance, and overcome problems of information asymmetry. The study identifies entrepreneurs who are both successful and unsuccessful in finance applica- tions and those that did not apply, comparing behavioural competency profiles, thus overcoming any bias towards successful enterprises (Rauch, 2007). Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 The top tier is the Big Eight, and importantly, also provides a mechanism for mapping measures of disposition or attainment onto competencies, and a number of studies, including longitudinal studies, have provided further confirmation of the eight factor structure (Kurz and Bartram 2002). The Great Eight competencies (Bartram, 2005) represent a set of factors that underpin job performance. These eight competencies include: leading and deciding; supporting and cooperat- ing; interacting and presenting; analysing and interpreting; creating and conceptualising; organis- ing and executing; adapting and coping; as well as enterprising and performing (see Bartram and SHL Group 2005; Kurz and Bartram 2002). The entrepreneurial behaviour literature therefore calls for more research which is able to both identify behaviour as discrete units and also introduce some element of measurement (Bird et al., 2012). The Great Eight was presented as an attempt to introduce a measurement tool and also identify competency as a lens through which to study behaviour. The Great Eight methodology (Bartram, 2005), grounded in Big Five model of personality (Goldberg (1990), therefore forms the theoretical background to this study. 3. Theoretical background Tett and Burnett, for example, developed a taxonomy of 53 competencies clustered around nine general areas—task orientation, depend- ability, open-mindedness, emotional control, communication, developing self and others, occupa- tional acumen and concerns. Borman and Brush proposed a structure of 1987 behaviours mapping onto 18 dimensions, which in turn map onto four very general dimensions—leadership and supervision; interpersonal relations and communication; technical behaviours and the mechanics of management; and useful beha- viours and skills. Bartram (2005) extended this further adopting a three-tiered structure; bottom tier consisted of 110 components, mapped onto a set of 20 competency dimensions (the middle tier) and this is then loaded onto eight broad competency factors. Page 4 of 29 5. Methodology This study was conducted using a longitudinal, fieldwork process incorporating analytical induction methodology. It adopts a pragmatic realist approach (Watson, 2013). As a sector, Creative Industries is dynamic and therefore more sensitive to unfavourable environments and one which over the course of this 3-year longitudinal study, follows Pettigrew’s recommendation to “go for extreme situations, critical incidents and social drama” and “provides a transparent look at growth, evolution, transformation, and conceivably decay of an organisation over time” (Pettigrew, 1990, p.277–280). Size of business is another factor that might also moderate the eﬀects of the individual. Creative Industries, with a larger proportion of smaller, growing firms, also allows for more expression of individual characteristics (Van Geldern et al. 2000). Page 5 of 29 Page 5 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Table 1. Panel profile of entrepreneurs Turnover (£000s) Employees (No.) Year incorporated <£100 100–500 500–1000 +1,000 <10 10+ 1995– 2000 2000– 2005 2005– 2011 27 20 6 7 46 14 3 14 43 A convenience sample of 60 entrepreneurs was recruited. The participants were screened in order to ensure each individual was the main financial decision maker in the company. In almost all cases, this person was also the owner, managing director, or senior partner (BDRC 2013). The panel was selected through trade networking activity, exhibitions and science park events. In order to participate, the entrepreneur had to evidence: ●A desire to grow coupled with an increase in employment by 20% in at least 1 year in the last 5 years. ●A desire to grow coupled with an increase in employment by 20% in at least 1 year in the last 5 years. ●The raising of funds or the intention to raise funds in the future. ●Active trading (indicated in year of incorporation). ●A minimum of one employee in addition to the entrepreneur. A profile of the sample is summarised as follows: A profile of the sample is summarised as follows: Within the definition of Creative Industries, 16 firms are software developers, 20 are mobile gaming companies, five develop social networking tools, three are commercial designers and five are promotional design agencies. Ten entrepreneurs are females and 50 are male. Age groups are 25–55. None of the firms are more than 20 years’ old and the maximum level of turnover is £1.8 million. Firms are based throughout the UK but predominantly in the Midlands. The panel profile is detailed in Table 1. Competency data on each Entrepreneur was collected using “Trait”, a personality inventory assessment which measures 13 dimensions of personality and nine behavioural competences on a scale 0–10. The nine Trait competencies together with research propositions are detailed in Table 2. Each entrepreneur is given a prefix T 1–60. Table 2. Research propositions Trait competency and proposition References P1 Working with Others: Being able to Work with Others provides opportunities to access finance Social Network Theory; Granovetter (1973) P2 Communicating, Meeting and Presenting: Being a good communicator can facilitate access to finance Baum and Locke (2004); Collins and Porras (1994); Rauch and Frese (2007) P3 Innovating and Creating: Innovating and creative skills open up more opportunities for access to finance Rauch, van Doorn, and Hulsink (2014); Schumpeter (1934) P4 Problem-Solving: An entrepreneur who can problem solve is better able to access finance. Sarasvathy (2001); Dew and Wiltbank (2008); Rauch and Frese (2007); Sarasvathy (2004) P5 Planning and Organising: Planning and organising are key to successful access to finance for the firm (Black (1998); Shapero and Sokol (1982); Ciavarella (2004); Theory of Planned Behaviour Ajzen (1991) P6 Driving for Results: An entrepreneur who is driven can access more finance opportunities Delmar and Wiklund (2008); Locke and Latham (1990) P7 Working with Customers: Working with customers increases opportunities to access finance Social Network Theory, Resource Development Theory Granovetter (1973) P8 Leading Others: Competency in leadership increases access to finance Collins and Porras (1994); Rauch and Frese (2007) P9 Coping with Pressure: Entrepreneurs who are better able to cope with pressure increase access to finance Sarasvathy (2004); Dew and Wiltbank (2008); Rauch and Frese (2007) Page 6 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Trait is grounded in the Big Five Model of personality (Goldberg, 1990) and Bartram’s Great Eight Competency Model (Bartram, 2005). Semi-structured Interviews were recorded taking between 30 min and 1 h with each entrepreneur annually over 3 years. The questionnaires were designed in order to evidence entrepreneurial behaviour, defined as “the concrete enact- ment by individuals (or teams) of tasks or activities” within a funding context (Bird et al., 2012: p.890). Using an analytic induction methodology (Znaniecki, 1934), the research question is examined using propositions with the goal of most accurately representing the reality of the situation. Qualitative, semi-structured interviews explored the funding activities of the entrepreneur; what is the process through which they try and raise funds and what evidence is there of using behavioural competences in order to achieve their funding objectives. Questions are detailed in Appendix 1. Interviews were recorded, transcribed and coded directly using NVivo 10 qualitative research software, seeking to avoid the weaknesses highlighted by Bazeley (2007, p.132) that “too often qualitative researchers rely simply on the presentation of key themes supported by quotes. . .” The “density” of each code (Carter, Shaw, Lam, & Wilson, 2007) is calculated by using the number and percentage of text characters that respondents spend talking about specific codes. Researcher bias was checked through a coding test with another researcher. 6. Results 6.1. Results of behavioural competences using psychometric testing The mean data for the Behavioural Competency Score (BCS), 0–10 for all 60 of the entrepreneurs (T1–T60), is presented as follows: The results from this research indicate a tendency for higher competences in collaborative behaviours, along with business planning and problem-solving. Working with Others (6.07) and Working with Customers (5.2) are the highest scores. Leading Others (3.92) and Driving for Results (4.22) have the lowest BCSs. These results are in line with more recent studies (Zhao & Lumpkin, 2010) that the clichéd view of the swashbuckling entrepreneur emphasising leadership (Brockhaus, 1980) and locus of control (Begley & Boyd, 1987), for example, are at odds with reality. Cluster analysis, using Ward’s method, was then performed to identify groups (clusters) within the 60 cases of entrepreneurs, i.e. those entrepreneurs who share similar characteristics across the nine Behavioural Competences. For ease of clarity of subsequent analysis, each group is given a name and the mean scores for each group are presented as follows: Capables has the highest competence scores in all groups; again Working with Others is the strongest (7.14), followed by Communicating, Meeting and Presenting (6.68), Working with Customers (6.61) and Driving for Results (6.14). Although the remaining competencies have lower scores, they are still higher than the other two groups. On balance, this group is the closest to the traditional view of entrepreneurs. Collaborators has a focus on co-operation with high competency in Working with Others (6.67) and Working with Customers (5.4), followed by lower scores for Innovating and Creating (5.53) and Problem-Solving (5.2). The Low Competences group above displays low scores across all competences; Planning and Organising (4.29) is the strongest competency in this group. The group is the most introverted; less interested in others with few social skills and methodical in approach. Figure 1 illustrates the mean behavioural competence scores and Figure 2 illustrates the data, and the distinctive differences between the three clusters: Page 7 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 6.2. Funding outcomes years 1–3 6.2.1. Funding outcomes by cluster Table 3 details the number of entrepreneurs in each cluster, and among them, the number of entrepreneurs making successful, unsuccessful and non-applications (Didn’t Apply), in each of the 3 years of data collection. Interviews were carried out between September 2011 and August 2014 and as much as possible at 12-month intervals. Figure 2. Competences by cluster. 6. Results Four cases dropped out of the programme after Year 1; 56 cases were analysed in Years 2 and 3. 0 1 2 3 4 5 6 7 8 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Sixty Cases: Behavioural Competences Low Competences Capables Collaborators Figure 2. Competences by cluster. Figure 1. Mean BCSs: 60 entre- preneurs (scale 0–10). Figure 1. Mean BCSs: 60 entre- preneurs (scale 0–10). Figure 1. Mean BCSs: 60 entre- preneurs (scale 0–10). 6.2. Funding outcomes years 1–3 6.2.1. Funding outcomes by cluster Table 3 details the number of entrepreneurs in each cluster, and among them, the number of entrepreneurs making successful, unsuccessful and non-applications (Didn’t Apply), in each of the 3 years of data collection. Interviews were carried out between September 2011 and August 2014 and as much as possible at 12-month intervals. Four cases dropped out of the programme after Year 1; 56 cases were analysed in Years 2 and 3. 0 1 2 3 4 5 6 7 8 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Sixty Cases: Behavioural Competences Low Competences Capables Collaborators P 8 f 29 Sixty Cases: Behavioural Competences 0 1 2 3 4 5 6 7 8 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Sixty Cases: Behavioural Competences 8 Communicating. Meeting and Presenting Planning and Organising Working with Customers Problem Solving Driving for Results Coping with Pressure Working with Others Leading Others Innovating and Creating Low Competences Collaborators Capables Capables 6.2. Funding outcomes years 1–3 6.2.1. Funding outcomes by cluster Table 3 details the number of entrepreneurs in each cluster, and among them, the number of entrepreneurs making successful, unsuccessful and non-applications (Didn’t Apply), in each of the 3 years of data collection. Interviews were carried out between September 2011 and August 2014 and as much as possible at 12-month intervals. Four cases dropped out of the programme after Year 1; 56 cases were analysed in Years 2 and 3. Page 8 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Table 3. Applications versus Clusters Year 1 Finance Applications Capables % Collaborators % Low Competencers % Total 28 15 17 Applied and Successful 11 39% 4 27% 2 12% Applied and Unsuccessful 1 4% 1 7% 4 24% Didn’t Apply 16 57% 10 66% 11 64% Year 2 Finance Applications Capables % Collaborators % Low Competencers % Total 26 15 15 Applied and Successful 13 50% 7 47% 4 27% Applied and Unsuccessful 1 4% 2 13% 6 40% Didn’t Apply 12 46% 6 40% 5 33% Year 3 Finance Applications Capables % Collaborators % Low Competencers % Total 26 15 15 Applied and Successful 13 50% 4 27% 3 20% Applied and Unsuccessful 1 4% 1 7% 3 20% Didn’t Apply 12 46% 10 66% 9 60% Page 9 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Table 4. Funding type by cluster Funding type by cluster Capables Collaborators Low Competencers Self-Funded 31% 49% 34% Equity Funded 31% 27% 34% Secured Funding 38% 24% 32% Figure 3. Successful versus Unsuccessful versus Didn’t Apply. Twenty-eight entrepreneurs in the Capables cluster took part in the study in Year 1. This reduced to 26 who agreed to continue their participation in the study in Years 2 and 3. The Capables cluster was consistently more successful in funding applications over the periods; 11 entrepreneurs (39%) in this cluster made successful applications in Year 1, 13 (50%) in Year 2 and 13 (50%) in Year 3. This group also had the fewest unsuccessful applications; only three over the 3-year period. The number of Capables choosing not to apply for finance was also fairly stable over the period. Fifteen Collaborators participated in the study throughout the 3- year period. Collaborators had mixed results. Figure 3. Successful versus Unsuccessful versus Didn’t Apply. 6.2. Funding outcomes years 1–3 The highest proportion of this cluster making successful applications was in Year 2 at seven (47% of Collaborators); this group had four unsuccessful applications over the 3-year period and also had the highest proportion of non-applications (67%, 40% and 66%, respectively). Seventeen Low Competence entrepreneurs embarked on the study and this reduced to 15 for Years 2 and 3. The Low Competence group had the lowest level of success; 13 unsuccessful applications over the period with a success rate below 27%. Non-applications were also high at 64%, 33% and 60%, respectively. Using the BCS scores, the study also analysed Behavioural Competency by funding outcome and these are illustrated in Figure 3. Unsuccessful applications had lower levels of competencies compared with entrepreneurs, who either chose not to apply, or made successful applications. Successful cases were stronger in Communicating, Meeting, Presenting, Leading Others, Coping with Pressure and Driving for Results. Didn’t Apply cases were stronger in Planning and Organising, Problem-Solving, Working with Others, Innovating and Creating and Working with Customers. Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 confirmed the significance of the relationship between cluster membership and application outcome χ2 (1, n = 172) = 21.488, p < .000). This shows that cluster membership is an indicator of funding outcomes confirmed the significance of the relationship between cluster membership and application outcome χ2 (1, n = 172) = 21.488, p < .000). This shows that cluster membership is an indicator of funding outcomes 6.3.1. Funding type versus cluster group 6.3.1. Funding type versus cluster group Self-Funded are those entrepreneurs who have used only internal resources to fund the firm, either through working capital, director’s loans or qualifying for grants. Equity-funded is those who have shared equity with third-party investors. Secured Funding is loan finance, where entrepreneurs have arranged borrowing using secured forms of finance Capables had the same proportion (31%) of self-funded entrepreneurs and equity-funded, and overall, had a greater proportion of secured funding. Collaborators had a greater proportion of Self Funders (49%). Low Competences were equally spread across all funding types. Funding type by cluster remained very stable for each entrepreneur. Only two entrepreneurs changed funding type over the period of the study. By examining the BCSs across these different groups, Figure 4 presents the differences in competencies across funding types. Equity-funded entrepreneurs have higher BCSs in Coping with Pressure and Communicating Meeting and Presenting, possible indicators of the process of both presenting and subse- quently working with third-party investors. Entrepreneurs who are self-funded or use secured finance have higher BCSs in Planning and Organising and Problem-Solving, possibly due to competences required to both satisfy secured lenders or for problem-solving in a totally self- funded business. Figure 4. Competences versus funding type. 6.3. Funding outcome by cluster: significance test Collecting three tranches of data produced a sufficient sample to make further statistical analysis appropriate. Analysing all applications over the 3-year period, a Chi-Square test was performed and 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Didn’t Apply Unsuccessful Successful Page 10 of 29 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Didn’t Apply Unsuccessful Successful 7 00 Communicating. Meeting and Presenting Planning and Organising Working with Customers Driving for Results Didn’t Apply Coping with Pressure Working with Others Innovating and Creating Leading Others Page 10 of 29 6.4. Funding type by cluster: significance test Funding outcomes by funding type Funding outcome by funding type Self-Funded Equity-Funded Secured Funding Applied and Successful 10% 72% 30% Applied and Unsuccessful 13% 9% 13% Didn’t Apply 78% 19% 57% 6.4.1. Funding outcome by funding type Seventy-eight per cent of Entrepreneurs, who were self-funded at the outset of the research, remained self-funded at the end. Of particular note in Table 5 is the success of equity- funded entrepreneurs to make further successful funding applications, either for new inves- tors or existing investors “following-on”. Secured funders had mixed results with 30% having applied with successful applications and 57% choosing not to apply over the 3-year period. This is detailed in Table 4. 6.5. Funding outcome by funding type: significance test Analysing applications over the 3-year period, a chi-square test was performed and confirmed the significance of the relationship between funding type and funding outcome (χ2 (1, n = 172) = 51.466, p < .000). 6.5.1. Using advisors This is summarised in Figure 5. To provide increased insight into the degree to which entrepreneurs Work with Others, each was asked in every phase of the study to confirm if advisors had been used to assist decision-making, in relation to funding. Table 6 analyses this by cluster: In the study, 77% of Capables reported using advisors in each year of the study. Collaborators also made use of advisors at 60%. Conversely, only 25% of the Low Competency cluster had 0 1 2 3 4 5 6 7 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Advisor Non Advisor Figure 5. Advisors versus non- advisors. Table 5. Funding outcomes by funding type Funding outcome by funding type Self-Funded Equity-Funded Secured Funding Applied and Successful 10% 72% 30% Applied and Unsuccessful 13% 9% 13% Didn’t Apply 78% 19% 57% Table 6. Advisors versus clusters Clusters versus Use of Capables Collaborators Low Competencers 0 1 2 3 4 5 6 7 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Advisor Non Advisor Figure 5. Advisors versus non- advisors. 7 Communicating. Meeting and Presenting Figure 5. Advisors versus non- advisors. Figure 5. Advisors versus non- advisors. 6.4. Funding type by cluster: significance test Working with Customers Planning and Organising Problem Solving Driving for Results Non Advisor Working with Others Coping with Pressure Innovating and Creating Leading Others Table 5. Funding outcomes by funding type Funding outcome by funding type Self-Funded Equity-Funded Secured Funding Applied and Successful 10% 72% 30% Applied and Unsuccessful 13% 9% 13% Didn’t Apply 78% 19% 57% 6.4.1. Funding outcome by funding type g y f g yp Seventy-eight per cent of Entrepreneurs, who were self-funded at the outset of the research, remained self-funded at the end. Of particular note in Table 5 is the success of equity- funded entrepreneurs to make further successful funding applications, either for new inves- tors or existing investors “following-on”. Secured funders had mixed results with 30% having applied with successful applications and 57% choosing not to apply over the 3-year period. This is detailed in Table 4. 6.5. Funding outcome by funding type: significance test Analysing applications over the 3-year period, a chi-square test was performed and confirmed the significance of the relationship between funding type and funding outcome (χ2 (1, n = 172) = 51.466, p < .000). 6.4. Funding type by cluster: significance test Analysing applications over the 3-year period, a chi-square test was performed and confirmed that the relationship between cluster membership and funding type was not significant (χ2 (1, n = 172) = 4.495, p < .343). Entrepreneurs were also asked if, in principle, they would be willing to share equity in the company, i.e. was equity sharing simply not an option in principle. A chi-square test was performed and confirmed that there was no significance between cluster membership and willingness to share equity. 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Self Funded Equity Secured 4. Competences versus type. Page 11 of 29 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Self Funded Equity Secured s 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Self Funded Equity Secured 7 00 Communicating. Meeting and Presenting Planning and Organising Working with Customers Driving for Results Working with Others Coping with Pressure Innovating and Creating Leading Others Page 11 of 29 Page 11 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 6.4.1. Funding outcome by funding type Seventy-eight per cent of Entrepreneurs, who were self-funded at the outset of the research, remained self-funded at the end. Of particular note in Table 5 is the success of equity- funded entrepreneurs to make further successful funding applications, either for new inves- tors or existing investors “following-on”. Secured funders had mixed results with 30% having applied with successful applications and 57% choosing not to apply over the 3-year period. This is detailed in Table 4. 0 1 2 3 4 5 6 7 Communicating. Meeting and Presenting Planning and Organising Problem Solving Working with Others Leading Others Innovating and Creating Coping with Pressure Driving for Results Working with Customers Advisor Non Advisor Figure 5. Advisors versus non- advisors. Table 5. 6.5.1. Using advisors This is summarised in Figure 5. To provide increased insight into the degree to which entrepreneurs Work with Others, each was asked in every phase of the study to confirm if advisors had been used to assist decision-making, in relation to funding. Table 6 analyses this by cluster: In the study, 77% of Capables reported using advisors in each year of the study. Collaborators also made use of advisors at 60%. Conversely, only 25% of the Low Competency cluster had Table 6. Advisors versus clusters Clusters versus Use of Advsiors Capables Collaborators Low Competencers Use Advisors Yes 77% 60% 25% Use Advisors No 23% 40% 75% Page 12 of 29 Page 12 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Table 7. Indicates relationship between the use of advisors and applications outcomes Use of Advisors Yes No Applied and Successful 47% 19% Applied and Unsuccessful 2% 24% Didn’t Apply 52% 57% appointed advisors during the period. Table 7 indicates the relationship between use of advisors and application outcomes. appointed advisors during the period. Table 7 indicates the relationship between use of advisors and application outcomes. appointed advisors during the period. Table 7 indicates the relationship between use of advisors and application outcomes. In the study, 47% of cases with advisors reported successful applications, in contrast with 19% non-advised entrepreneurs. When analysed with the Behavioural Competency scores, this group also outperforms non-advisors across all competences: 6.6. Advisors by cluster: significance test Analysing the use of advisors over the 3-year period, a chi-square test was performed and confirmed the relationship between cluster membership and use of advisors was significant (χ2 (1, n = 172) = 32.974, p < .000). A chi-square test was performed and confirmed the relationship between using advisors and application outcome was significant (χ2 (1, n = 172) = 27.462, p < .000). This would indicate the use of advisors results in more successful funding applications. 6.7. Semi-structured interviews, data collection and analysis “Your (plan) P&L to a degree goes (Bad Planning) out the window . . . all very unpredictable. So it’s guesswork”. Innovating and Creating also remained a strong theme and example of which was T6 (a Table 8. Year 1 coded interview density Capables Collaborators Low Competencers All Cases Working with Others 23% 31% 12% 23% Planning and Organising 24% 13% 10% 19% Communicating, Meeting, Presenting 12% 11% 6% 11% Innovating and Creating 11% 13% 8% 11% Driving for Results 6% 9% 4% 7% Working with Customers 5% 4% 5% 5% Problem-Solving 2% 2% 0% 2% Coping with Pressure 3% 0% 0% 2% Leading Others 2% 1% 0% 1% Behavioural Difficulty 11% 15% 55% 19% Total Code 100% 100% 100% 100% Table 9. Year 2 coded interview density Capables Collaborators Low Competencers Total nodes Working with Others 24% 13% 3% 17% Communicating, Meeting, Presenting 12% 20% 8% 14% Innovating and Creating 11% 18% 4% 12% Problem-Solving 13% 10% 6% 11% Driving for Results 8% 5% 0% 6% Planning and Organising 5% 5% 1% 4% Working with Customers 4% 6% 2% 4% Coping with Pressure 1% 0% 0% 0% Leading Others 0% 1% 0% 0% Behavioural Difficulty 23% 22% 75% 31% Total code 100% 100% 100% 100% 18.1512833 Table 8. Year 1 coded interview density Capables Collaborators Low Competencers All Cases Working with Others 23% 31% 12% 23% Planning and Organising 24% 13% 10% 19% Communicating, Meeting, Presenting 12% 11% 6% 11% Innovating and Creating 11% 13% 8% 11% Driving for Results 6% 9% 4% 7% Working with Customers 5% 4% 5% 5% Problem-Solving 2% 2% 0% 2% Coping with Pressure 3% 0% 0% 2% Leading Others 2% 1% 0% 1% Behavioural Difficulty 11% 15% 55% 19% Total Code 100% 100% 100% 100% Table 9. Year 2 coded interview density Capables Collaborators Low Competencers Total nodes Working with Others 24% 13% 3% 17% Communicating, Meeting, Presenting 12% 20% 8% 14% Innovating and Creating 11% 18% 4% 12% Problem-Solving 13% 10% 6% 11% Driving for Results 8% 5% 0% 6% Planning and Organising 5% 5% 1% 4% Working with Customers 4% 6% 2% 4% Coping with Pressure 1% 0% 0% 0% Leading Others 0% 1% 0% 0% Behavioural Difficulty 23% 22% 75% 31% Total code 100% 100% 100% 100% Low Competency entrepreneurs had a significantly higher density (55%) of codes indicating difficulties associated with sourcing finance. T13, for example, . . . 6.7. Semi-structured interviews, data collection and analysis The nine behavioural competences were used as a guide to frame the semi-structured interviews and explore what the entrepreneur actually “did” in order to fund the firm. In addition, a tenth code was developed—Behavioural Difficulties—in order to explore how each cluster reacted to problems in the funding process. Making up each of the Behavioural Competence Codes are a number of coded themes which emerged during the course of the interviews; descriptions for these are in Appendix 2. 6.7.1. Year 1 coded interviews 6.7.1. Year 1 coded interviews Phase 1 interviews were carried out between May and October 2012. Phase 1 interviews were carried out between May and October 2012. Working with Others accounts for the largest number of words coded in the interviews (All Cases —23%) and is the strongest code for Collaborators (31%). This is detailed in Table 8. An example of this was the emerging theme Serial Networking; T29 (a Collaborator), for example, develops digital games for use in the music industry. The business was established in 2010 and T29 has used private equity and angel finance to fund the business. He talks about how he used his network (coded to Serial Networking) to source funding: “I am an LBS alumni. . . one of my ex-classmates runs an offshore angel group. cooperating with her on the Isle of Man to pitch in front of high net worth individuals”. Planning and Organising, Communicating and Presenting and Innovating and Creating were also strong interview themes. Coded within Planning and Organising, for example, was Capacity Planning. T9 runs a professional architecture practice in the West Midlands. The business has been established 10 years and has now grown to three branches—Midlands, North East, South West—with plans to open more. T9 has used a Government-backed bank loan to start and develop the business and plans to use private equity to expand in the future. He describes how he has developed a method of managing capacity in order to estimate the investment required for the business: “I have detailed capacity planning (Capacity Planning) translated into a spreadsheet which gives us a dynamic target to hit each month”. Page 13 of 29 Page 13 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Low Competency entrepreneurs had a significantly higher density (55%) of codes indicating difficulties associated with sourcing finance. T13, for example, . . . 6.7. Semi-structured interviews, data collection and analysis “Your (plan) P&L to a degree goes (Bad Planning) out the window . . . all very unpredictable. So it’s guesswork”. Innovating and Creating also remained a strong theme and example of which was T6 (a Collaborator) who used a Peer-to-Peer lender to find an alternative to bank funding: “We have used Funding Circle . . . used them because the bank weren’t helpful . . . found through mailer . . . Page 14 of 29 Page 14 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Table 10. Year 3 coded interview density Capables Collaborators Low Competencers All nodes Working with Others 21% 24% 12% 21% Innovating and Creating 15% 23% 12% 17% Driving for Results 12% 15% 6% 13% Communicating, Meeting, Presenting 9% 14% 7% 10% Planning and Organising 11% 0% 0% 8% Problem-Solving 4% 8% 2% 5% Working with Customers 1% 3% 0% 1% Coping with Pressure 2% 0% 0% 1% Leading Others 0% 0% 0% 0% Behavioural Difficulty 25% 13% 61% 25% Total code 100% 100% 100% 100% provided annual P&L plan, predictions, forecasts, unsecured, over three years . . . when the banks rained stuff in got a positive response. Didn’t meet anyone—all over the phone”. provided annual P&L plan, predictions, forecasts, unsecured, over three years . . . when the banks rained stuff in got a positive response. Didn’t meet anyone—all over the phone”. provided annual P&L plan, predictions, forecasts, unsecured, over three years . . . when the banks rained stuff in got a positive response. Didn’t meet anyone—all over the phone”. A more detailed examination of the data revealed Planning and Organising was more prevalent for those entrepreneurs either not applying, or making successful applications. Planning and Organising was a stronger theme for the self-funded and secured group. Innovating and Creating and Communicating, Meeting and Presenting were stronger for equity-funded entrepre- neurs. Working with Customers was a strong theme for Secured entrepreneurs. A strong theme for equity-funded entrepreneurs Communicating, Meeting and Presenting; T19, for example, “(Communicating the Vision) . . . financial data and soft skills that become apparent being con- sistent doing what you said you were going to do . . . and ensuring you communicate and let them know what is happening. So nervous these days . . . any sign people are not communicating things can go pear-shaped very quickly . . .”. Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Compared with Year 1 interviews, Collaborators, in particular, were keener to give examples of problem-solving competences. T29, for example, made changes through a new model which included a revenue share with a partner: “Now more focus on smaller amounts . . . get teamed up with global marketing partner . . . revenue share with them. (Developing a New Business Model) . . . when we are bigger will go back to Broono Mars . . .”. Communicating, Meeting and Presenting was also again discussed in successful cases. T45 (a Capable) approached a number of new private investors: “Did pitches . . . clearly . . . looked at . . . angels who want to invest and make a social impact . . . (Approaching Investors) . . . we did a pitch there and ended up getting £90K from that group . . . got introduced to them in order to give a reference for someone else and they ended up being interested in the business . . .”. Again Low Competences accounted for the largest number of behavioural difficulties, account- ing for 75% of themes coded. Increasing themes over Year 1, however, was Planning and Organising and Problem-Solving, particularly amongst Non-Applying entrepreneurs. Communicating, Meeting and Presenting was an even stronger theme for Equity-Funded entrepreneurs than in Year 1. Problem-Solving was also increasingly important for Self-Funded and Equity-Funded entrepreneurs. Planning and Organising remained a significant theme for Self-Funded entrepreneurs. 6.7.3. Year 3 coded interview analysis This is detailed in Table 10. Again, Working with Others was again the strongest theme, with Capables and Collaborators in particular. Compared with Year 2, Innovating and Creating was also a stronger theme, particularly amongst those that were successful in funding applications. T08 (a Capable) successfully applied for a grant in the year and described the support he received from the SME Educational Programme: “Yeah, it was unbelievable, the training, the coaching, the people that came to the presentation, the follow-up stuff . . . I can just pick up the phone and speak to people, they’re there to basically find someone or find a way. A closer examination of the data also indicated Driving for Results, along with Communicating, Meeting and Presenting and Planning and Organising were also strong themes amongst successful entrepreneurs. Innovating and Creating was a strong theme particularly amongst self-funded entrepreneurs. 6.7. Semi-structured interviews, data collection and analysis In Year 1, therefore, Working with Others and Planning and Organising were the strongest themes emerging in the interviews, particularly with Capables and Collaborators. Low Competence entrepreneurs reported the largest number of behavioural difficulties. Planning and Organising was a strong theme for Non-Applying entrepreneurs, and also for those using self- funded or secured-funded finance. 6.7.2. Year 2 coded interviews This is detailed in Table 9. Working with Others, Communicating Meeting and Presenting, Innovating and Creating and Problem-Solving continued to be strong themes in the interviews and together make up, 54% of coded themes. Working with Others is particularly strong for Capables and those entrepreneurs making suc- cessful applications; T45, for example, expanded into the United States and talks enthusiastically about the use of advisors: “This year we brought in advisors from the West Coast . . . Head of Mobile at Winga . . . she is a new investor one we have brought in (this year) . . .”. Page 15 of 29 Proposition 2: Communicating, Meeting and Presenting: Being a good communicator can facil- itate more access to finance. Positive indicator. Increasingly SMEs are beginning to think more laterally of other potential methods of financing growth. Asset-based lending has increased and Social Lending, Crowdfunding and Peer-to-Peer lending have also increased in relevance through the course of this study. From the literature, therefore, innovative behaviour appears an important competence for an entrepreneur. Innovativeness is a strong competency for both Capables and Collaborators. Again, the study adds to knowledge by recognising some entrepreneurs have higher levels of competencies in Innovation and Creativity. It is clear that competences in these behaviours allow some entrepre- neurs to be more innovative in their consideration of funding, both in terms of the nature of funding and finding the best funding option for the firm, whether they used these options or not. Proposition 3 is therefore modified and as follows: 7. Discussion Data gathered through the Trait test enabled the study to group the entrepreneurs into compe- tency clusters. Further statistical analysis confirmed cluster membership and funding outcome as significant and funding type and funding outcome as also significant. The 3-year qualitative study then allowed for deeper analysis of exactly what entrepreneurs “did” in order to raise finance. For each of the eight propositions where there is a positive indication that the proposition is proven, this has been indicated although to confirm thoroughly from a philosophical perspective will require further studies. In some cases, no positive indication was found in propositions; further work will be required both in formation of propositions and also more data collection. Working with Others has been a strong theme through all three phases of the interviews, particularly with Capables and Collaborators. Key themes emerging from the 3-year study included networking, using advisors and investigating joint ventures. It is also the strongest competence in this group of entrepreneurs and is the strongest competence amongst successfully applying entrepreneurs. However, higher scores also indicate an increased use of self-finance as opposed to equity or secured-funding, indicating collaborative skills may also be used, in some cases, to resource the firm, without the need for external finance. The use of Advisors was also researched Page 16 of 29 Page 16 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 specifically in the study and those entrepreneurs using Advisors were more likely to have success- ful funding outcomes, again statistically significant. specifically in the study and those entrepreneurs using Advisors were more likely to have success- ful funding outcomes, again statistically significant. The methodology of analytic induction allows for modification of propositions as the themes emerge from the data. Working with Others is the highest competency level across all clusters. Capables scores highest across this competency, making most use of Advisors. This leads to a revised Proposition 1: Revised Proposition 1: Working with Others: Being able to Work with Others Provides Opportunities to Access Finance and also Self-Finance. Positive indicator. Communicating, Meeting and Presenting is associated with having social confidence in meeting and speaking, whilst also communicating clearly and persuasively. Business angels have become an important source of equity finance to SMEs and business angel activity and communication skills are key in presenting investment propositions. Themes emerging in the study included presenting to potential international investors and there was evidence of social boldness, the confidence to interact with strangers (Zhang, Souitaris, Soh, & Wong, 2008) and entrepreneurs attempting to send signals to prospective investors (Spence, 1973) at pitching events, for example. In this study, Communicating, Meeting and Presenting was a strong competency amongst Capables and amongst successful applications Proposition 2: Communicating, Meeting and Presenting: Being a good communicator can facil- itate more access to finance. Positive indicator. Revised Proposition 5: Planning and Organising: Entrepreneurs with a higher competence in planning and organising will be better able to self-fund and not require external finance. Proposition: Partial Positive Indicator. Revised Proposition 5: Planning and Organising: Entrepreneurs with a higher competence in planning and organising will be better able to self-fund and not require external finance. Proposition: Partial Positive Indicator. Driving for Results is a strong trait for Capables, but not for Collaborators or Low Competence clusters. Emerging themes in the qualitative interviews included identifying growth and opportu- nity and using persistence and challenging behaviour. It is also a strong trait in successful applications. Driving for Results emerged as a stronger theme as the research programme progressed, particularly amongst equity seeking Capables. These entrepreneurs were able to meet challenges in the business and were able to indicate a more proactive approach. Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Revised Proposition 4: An entrepreneur who can problem solve is better able to select a self- funding strategy for the firm. Positive indicator. In the Capables cluster, Planning and Organising is a mid-level competence in terms of perfor- mance (5.82). It is at a lower competence for Collaborators and Low Competence group. Not as strong as Problem-Solving, but it is also a stronger competence in the Didn’t Apply group of entrepreneurs, again indicating a level of resourcefulness amongst entrepreneurs. Themes emer- ging from the data included producing business plans, cash management and using flexible staff management to increase working capital. Again, this is a stronger competence amongst non- applications and secured funders, where debt providers are more likely to require more formal management controls in place. Planning and Organising was a strong theme in the first phase of qualitative interviews. However, in the following 2 years when the study focused more on what the entrepreneur had actually “done” in the previous 12-month period, evidence of planning and organising was less prevalent. It remained a strong theme only in self-funded entrepreneurs. Higher competencies in Planning and Organising also seem more relevant to those entrepre- neurs choosing not to apply for finance (non-applications), but there is not the same evidence for this behaviour in the qualitative interviews, when this was a strong theme, but only in the first year of study. Therefore: Revised Proposition 3: Innovating and Creating: Innovating and creative competences gives entrepreneurs the opportunity to consider and access new forms of finance. Positive indicator. Finance is considered a disproportionately important problem for high-growth firms, compared to other businesses (NESTA, 2011), as the entrepreneurs seek ways of funding growth. Yet, the difficulties in solving these problems appears to be giving rise to an increase in the non-seeker of finance, as entrepreneurs describe the main barriers to an application and discouragement expec- tation of an unsuccessful outcome. The Problem-Solving competence itself is not one of the highest BCSs and both Capables (5.7) and Collaborators (5.2) have similar levels overall. It included developing the business model in order to attract finance and also using match funding. Where this competency became more significant was in the “Didn’t Apply” group of entrepreneurs, who had a higher level of competence in this competence (5.8) compared to either Successful (4.92) or Unsuccessful (4.3) entrepreneurs. Self-funded entrepreneurs also had the highest competency in Problem-Solving (6.0). It would appear, therefore, that this Problem-Solving competence indicates resourcefulness amongst self-funded, non-applying entrepreneurs. Interview data indicated evidence for this. Page 17 of 29 Page 17 of 29 Proposition 9: Coping with Pressure: Entrepreneurs who are better able to cope with pressure increase access to finance. No Positive Indicator. Proposition 9: Coping with Pressure: Entrepreneurs who are better able to cope with pressure increase access to finance. No Positive Indicator. Proposition 8: Leading Others: Competency in leadership increases access to finance. No Positive Indicator. The competence to cope with pressure was outlined by Dew and Wiltbank (2008), identifying entrepreneurs who excel in an ability to remain calm, composed and free from worry or anxiety at times of pressure. Starting and growing a business involves periods of dealing with problems and setbacks in a calm, positive way and higher competences will be critical to good decision-making. Often entrepreneurs are managing several activities at the same time, and managing a busy role with competing demands, without feeling undue pressure, will improve effective decision-making. Overall, it is not a strong competence either for Capables, or overall in all clusters, and there was very little evidence of this behaviour in the semi-structured interviews. Proposition 6: Driving for Results: An entrepreneur who is driven can access more funding opportunities. Positive Indicator. Within the study, Working with Customers was part of the collaborative competences that score most highly with Capables and Collaborators. In particular, some entrepreneurs were able to develop a relationship with customers which enabled more flexibility in payment terms, leveraging relationships with customers which increased working capital inflows into the business. Working with Customers was one of the strongest competences overall and both Capables and Collaborators were particularly strong in this behaviour. These entrepreneurs were able to utilise this working relationship with customers to leverage working capital, or in some cases, actually provide a service on behalf of the customer, and in doing so, making it easier to plan cash flow. Proposition 7: Working with Customers increases opportunities to access finance. Positive Indicator. The classic image of the entrepreneur as a “risk taker” or an “extrovert” may discourage some individuals from becoming entrepreneurs who would otherwise be successful at this pursuit. Page 18 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 In this research study, Leadership is not one of the strongest competences amongst the Capable cluster of entrepreneurs, and within the group as a whole, it is the weakest competence. However, there was very little evidence in the interview data of this behavioural competence. (In year 3 no data were coded to this competence). Proposition 8: Leading Others: Competency in leadership increases access to finance. No Positive Indicator. Citation information Bridge, S., O’Neill, K., & Cromie, S. (1998). Understanding enterprise entrepreneurship, and small business. London: MacMillan. Cite this article as: The role of behavioural competences in predicting entrepreneurial funding resource orchestration, Geoff Parkes, Mark Hart, John Rudd & Rebecca Liu, Cogent Business & Management (2018), 5: 1512833. Brockhaus, S. R. H. (1980). Risk taking propensity of entrepreneurs. Academy of Management Journal, 23(3), 509–520. Author details Geoff Parkes1 Begley, T. M., & Boyd, D. P. (1987). Psychological character- istics associated with performance in entrepreneurial firms and smaller businesses. Journal of Business Venturing, 2(1), 79. doi:10.1016/0883-9026(87)90020-6 p g ORCID ID: http://orcid.org/0000-0003-2126-6740 Mark Hart1 Bird, B., Schjoedt, L., & Baum, J. R. (2012). Entrepreneurs’ behavior: Elucidation and measurement introduction. Entrepreneurship: Theory & Practice, 36(5), 889–913. E-mail: mark.hart@aston.ac.uk John Rudd1 E-mail: John.Rudd@wbs.ac.uk Rebecca Liu1 Black, J. (1998). Entrepreneur or entrepreneurs? Justification for a range of definitions. Journal of Business and Entrepreneurship, 10, 45–65. E-mail: gordon.liu@open.ac.uk 1 1 Marketing & Strategy, Aston University, Birmingham, UK. 1 Marketing & Strategy, Aston University, Birmingham, UK. BOE. (2014). Trends In Lending. Relates to Published Lending Report from the Bank of England. Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 developed through analytical induction confirmed specific behaviours associated with finding finance. developed through analytical induction confirmed specific behaviours associated with finding finance. The study has implications for Financial Institutions, Business Angels and Accelerator Programmes using competency data to differentiate between entrepreneurs and aid decision- making in the allocation of funding and other support. The study also strengthens the argument for more longitudinal studies. Lumpkin and Dess (1996) report that around three quarters of the performance studies and all the intention studies which the authors reviewed were cross-sectional in nature, which raises a question, in their own words, of “the causal direction of our observed effects” (p.398). Through the domains of entrepreneurship and psychology, the study adds to existing literature through the novel application of “Big Five” and “Great Eight” theories of personality and competency. The three distinctive clusters were proven to have different characteristics in relation to funding outcomes, funding types and use of advisors, for example. It therefore follows that identification of an entrepreneur as belonging to one of the three groups has considerable predictive significance in relation to behavioural competences and how the entrepreneur accesses finance. For the practitioner, it provides a methodology which enables the identification of competences which overcome the difficulties caused by information asymmetry in the process of funding the firm. By both measuring behavioural competence and identifying the associated behaviour, the research makes a contribution through the use of psychometric testing to explain and predict how individuals finance the firm; what Wright and Stigliani (2013) describe as resource orchestration. Psychology, 89(4), 587–598. doi:10.1037/0021- 9010.89.4.587 8. Conclusion Since 2008, various Government initiatives have tried to improve the flow of funds to small firms, recognising this sector as key to economic growth. Despite these efforts, total stock of lending still remained lower in 2014 than at any point since 2008. New forms of funding have emerged including a variety of Peer-to-Peer funding models, business angel and asset finance has increased, but still today the environment for capital-raising remains a challenge for the small firm. Entrepreneurs are unable to influence supply side factors. This study therefore takes an alter- native position and looks at the demand side of market failure; so what can the individual entrepreneur do in order to successfully fund the firm (Mueller et al., 2012). This field presents a challenge to investors, as information asymmetry prevents the efficient flow of information about the firm. Prospective financiers, therefore, find it difficult to access the potential of new ventures (Baum & Silverman, 2004; Venkataraman, 1997). The lens through which this study examines entrepreneurship is behaviour, the “missing field of research” (O’Gorman et al., 2005, p.2), and specifically, behavioural competences. The study also seeks to introduce measurement validity (Bird et al., 2012) and therefore compare behavioural competence amongst a group of entrepreneurs. The study uses propositions developed through an analytical methodology in order to find explanation for the existence, or not, of observed phenom- enon. For construct validity, 60 cases are used, and all the data have been derived from coded interview data. For reliability, NVivo 10 was used to create a research database and literature was reviewed to inform the development of the research propositions. The results were also validated with a group of external practitioners including representatives from venture capital, business angels and clearing banks. With regard to limitations of the study, the work would benefit from consideration of other sectors, increased sample size and more in-depth interviews. In particular, the identification of three distinct groups in this longitudinal study means belong- ing to one of these groups predicts likely behaviour when searching for finance. Propositions Page 19 of 29 Funding Funding The authors received no direct funding for this research. Bazeley, P. (2007). Qualitative data analysis with NVivo. London: Sage. oi:10.1016/j.jebo.2006.10.008 O’Gorman, C., Bourke, S., & Murray, J. A. (2005). The nat- ure of managerial work in small growth-orientated businesses. Small Business Economics, 25(1), 1–16. doi:10.1007/s11187-005-4254-z Fraser, S. 2014. Back to borrowing? Perspectives on the ‘Arc of discouragement. White Paper No 8. Birmingha: Enterprise Research Centre. Pettigrew, A. M. (1990). Longitudinal field research on change: Theory and practice. Organization Science, 1 (3), 267–292. doi:10.1287/orsc.1.3.267 Frese, M., van Gelderen, M., & Ombach, M. (2000). How to plan as a small scale business owner: Psychological process characteristics of action strategies and suc- cess. Journal of Small Business Management, 38(2), 1–18. Rauch, A., & Frese, M. (2007). ‘Let’s put the person back into entrepreneurship research: A meta-analysis on the relationship between business owners’ personal- ity traits, business creation, and success. European Journal of Work & Organizational Psychology, 16(4), 353–385. doi:10.1080/13594320701595438 Gartner, W. B. (1989). Some suggestions for research on entrepreneurial traits and characteristics. Entrepreneurship: Theory & Practice, 14(1), 27–37. Entrepreneurship: Theory & Practice, 14(1), 27–37. Gartner, W. B., Bird, B. J., & Starr, J. A. (1992). Acting as if: Differentiating entrepreneurial from organizational behavior. Entrepreneurship: Theory & Practice, 16(3), 13–31. Rauch, R., van Doorn, R., & Hulsink, W. (2014). A qualita- tive approach to evidence-based entrepreneurship: Theoretical considerations and an example involving business clusters. Entrepreneurial Theory and Practice, 38, 333–368. doi:10.1111/etap.2014.38. issue-2 Gherardi, S. (2003). Feminist theory and organizational theory: A dialogue on new bases. The Oxford hand- book of organization theory. C. Knudsen and H. Tsoukas (pp. 210–237). Oxford: OUP. Sadler–Smith, E., Hampson, Y., Chaston, I., & Badger, B. (2003). Managerial behavior, entrepreneurial style, and small firm performance. Journal of Small Business Management, 41(1), 47. doi:10.1111/1540- 627X.00066 Gibb, A., & Davies, L. (1990). In pursuit of frameworks for the development of growth models of the small business. International Small Business Journal, 9(1), 15–31. doi:10.1177/026624269000900103 Goldberg, L. R. (1990). An alternative ‘Description of per- sonality’. The big five factor structure. Journal of Personality and Social Psychology, 59, 1216–1229. Sandberg, W. R., & Hofer, C. W. (1987). Improving new venture performance: The role of strategy, industry structure, and the entrepreneur. Journal of Business Venturing, 2(1), 5. doi:10.1016/0883-9026(87)90016-4 Granovetter, M. (1973). ‘The strength or weak ties. American Journal of Sociology, 78, 1360–1380. doi:10.1086/225469 Sarasvathy, S. D. (2001). Causation and effectuation: Toward a theoretical shift from economic inevitability to entrepreneurial contingency Academy of man- agement. The Academy of Management Review, 26 (2), 243–263. doi:10.5465/amr.2001.4378020 Gruglis, I. (1997). References Burgoyne, J. (1989). Creating the managerial portfolio: Building on competency approaches to management development,’ management education and develop- ment. Management Education and Development, 12 (1), 55–61. Ajzen, I. (1991). The theory of planned behavior. Organizational Behavior & Human Decision Processes, 50(2), 179. doi:10.1016/0749-5978(91)90020-T Bartram, D. (2005). The great eight competences: A cri- terion-centric approach to validation. Journal of Applied Psychology, 90, 1185–1203. doi:10.1037/ 0021-9010.90.6.1185 Burgoyne, J. (1993). The competence movement: Issues, stakeholders and prospects. Personnel Review, 22(6), 6–13. doi:10.1108/EUM0000000000812 Baum, J., & Silverman, B. (2004). Picking winners or building them? Alliance, intellectual, and human capital as selection criteria in venture ﬁnancing and performance of biotechnology startups. Journal of Business Venturing, 19, 411–436. doi:10.1016/S0883- 9026(03)00038-7 Bygrave, W., & Timmons, J. (1992). Venture capital at the cross roads. Boston: Harvard Business School Press. Caird, S. P. (1993). What do psychological tests suggest about entrepreneurs? Journal of Managerial Psychology, 8(6), 11. doi:10.1108/ 02683949310047428 Baum, J. R., & Locke, E. A. (2004). The relationship of entrepreneurial traits, skill, and motivation to sub- sequent venture growth. Journal of Applied Carter, S., Shaw, E., Lam, W., & Wilson, F. (2007). Gender, entrepreneurship, and bank lending: The criteria and processes used by bank loan officers in Page 20 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 assessing applications. Entrepreneurship: Theory & Practice, 31(3), 427–444. entrepreneurs. Entrepreneurship Theory and Practice, 15(2), 7–18. doi:10.1177/104225879101500203 Ciavarella, M. (2004). The Big Five and venture survival: Is there a linkage? Journal of Business Venturing, 19(4), 465. doi:10.1016/j.jbusvent.2003.03.001 McClelland, D. C. (1961). The achieving society. Princeton, NJ: Free Press. Moran, P. (1998). Personality characteristics and growth orientation of the small business owner. International Small Business Journal, 16(3), 17–38. doi:10.1177/0266242698163001 j j Collins, J., & Porras, J. (1994). Built to last: Successful habits of visionary companies. Harper: New York. Deakins, D., North, D., Baldock, R., & Whittam, G. (2008). SMEs access to finance: Is there still a debt gap? Belfast: ISBE. Mueller, S., Volery, T., & von Siemens, B. (2012). What do entrepreneurs actually do? An observational study of entrepreneurs’ Everyday behavior in the start-up and growth stages. Entrepreneurial Theory and Practice (September), 995–1017. doi:10.1111/j.1540- 6520.2012.00538.x Delmar, F., & Wiklund, J. (2008). ‘The effect of small business managers’ growth motivation on firm growth: A longitudinal study. Entrepreneurship: Theory & Practice, 32(3), 437–457. Dew, R. S., & Wiltbank. (2008). Outlines of a behavioral theory of the entrepreneurial firm. References Journal of Economic Behavior & Organization, 66(1). doi:10.1016/j.jebo.2006.10.008 Myers, S. C. (1984). The capital structure puzzle. The Journal of Finance, 39(3), 575. doi:10.2307/2327916 NESTA. (2011). The vital growth report research report. oi:10.1016/j.jebo.2006.10.008 The consequences of competence: A critical assessment of the management NVQ. Personnel Review, 26(6), 428–444. doi:10.1108/ 00483489710188865 Sarasvathy, S. D. (2004). Making it happen: Beyond the- ories of the firm to theories of firm. Wiley. Holton, E., & Naquin. (2000). Developing high-performance leadership competency: Advances in developing human resources. San Fransisco, CA: Berrett-Koehler. Schumpeter, J. (1934). The theory of economic develop- ment. Cambridge, Mass.: Harvard University Press. Jones, O., & Jayawarna, D. (2012). Are financial institu- tions failing UK SMEs. ISBE, 35th Annual Conference. Dublin. Shapero, A., & Sokol, L. (1982). The social dimension of entrepreneurship. The Encyclopedia of Entrepreneurship, 72–90. Locke, E., & Latham, G. (1990). A theory of goal setting and task performance. Englewood Cliffs, NY: Prentice Hall. SME Finance Monitor. (2012). BDRC Continental. London: BDRC SME Finance Monitor. (2013). BDRC Continental. London: BDRC Lumpkin, G. T., & Dess, G. G. (1996). Clarifying the entre- preneurial orientation construct and linking it to performance.’ Academy of management. The Academy of Management Review, 21, 1. doi:10.5465/ amr.1996.9602161568 SME Finance Monitor. (2014). BDRC Continental. London: BDRC Spence, M. (1973). Job market signalling. Journal of Quarterly Economics, 87, 355–379. doi:10.2307/ 1882010 Magnusson, D., & Endler, N. (1977). Personality at the crossroads: Current issues in interactional psychology. Hillsdale, NJ: Erlbaum. Venkataraman, S. (1997). The distinctive domain of entrepreneurship research: An editor’s perspective. Advances in Entrepreneurship, Firm Emergence, and Growth, J. K. Greenwich and B. R.3: 119–138. McCarthy, M., Krueger, D., & Schoenecker, T. (1990). Changes in the time allocation patterns of Page 21 of 29 Page 21 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Watson, T. (2013). ‘Entrepreneurial action and the EuroAmerican social science tradition: Pragmatism, realism and looking beyond ‘the entrepreneur’. Entrepreneurship & Regional Development, 25(1–2), 16–35. doi:10.1080/08985626.2012.754267 Wright, M., & Stigliani, I. (2013). Entrepreneurship and growth. International Small Business Journal, 31(1), 3–22. doi:10.1177/0266242612467359 Zhang, J., Souitaris, V., Soh, P.-H., & Wong, P.-K. (2008). A contingent model of network utilization in early Watson, T. (2013). ‘Entrepreneurial action and the EuroAmerican social science tradition: Pragmatism, realism and looking beyond ‘the entrepreneur’. Entrepreneurship & Regional Development, 25(1–2), 16–35. doi:10.1080/08985626.2012.754267 Wright, M., & Stigliani, I. (2013). Entrepreneurship and growth. International Small Business Journal, 31(1), 3–22. doi:10.1177/0266242612467359 Zhang, J., Souitaris, V., Soh, P.-H., & Wong, P.-K. (2008). A contingent model of network utilization in early Entrepreneurship & Regional Development, 25(1–2), 16–35. doi:10.1080/08985626.2012.754267 Wright, M., & Stigliani, I. (2013). oi:10.1016/j.jebo.2006.10.008 Entrepreneurship and growth. International Small Business Journal, 31(1), 3–22. doi:10.1177/0266242612467359 Zhang, J., Souitaris, V., Soh, P.-H., & Wong, P.-K. (2008). A contingent model of network utilization in early Zhao, S., & Lumpkin. (2010). The relationship of personality to entrepreneurial intentions and performance: A meta-analytic review. Journal of Management, 36(2), 381-404. Znaniecki, F. (1934). The method of sociology. New York, NY: Farrar & Rinehart. Appendix 1. Semi-Structured Interview Questionnaires Phase 1 Semi-Structured Interview Questionnaires (A) Introduction (1) Do you expect a requirement to raise finance in the next 3 years? (2) Have you raised any finance in the last 12 months? (3) When was the business established? (4) How many employees? (5) What is the most recent turnover? (6) What is the current funding structure? (7) How long have you spent in the industry? (8) What is your Functional Expertise? (9) What are your qualifications?(A) Funding (1) What is your experience of running an SME? (2) Have you any experience of family entrepreneurship? (3) How much do you think your knowledge of this industry helps you to solve funding problems? (4) Do your qualifications help you to access finance? (5) How have you planned your funding requirements? (6) How do you expect to plan your funding requirements differently in the future? (7) How have you solved funding problems in the past? (8) In what ways do you think you will solve funding problems in the future? (9) How have you presented and communicated your funding requirements? (10) Do you expect to change the way you communicate and present your funding requirements in the future? (11) Have you examples of how you have cooperated with other individuals or businesses in order to solve funding problems? Wright, M., & Stigliani, I. (2013). Entrepreneurship and growth. International Small Business Journal, 31(1), 3–22. doi:10.1177/0266242612467359 Zhang, J., Souitaris, V., Soh, P.-H., & Wong, P.-K. (2008). A contingent model of network utilization in early meta-analytic review. Journal of Management, 36(2), 381-404. Znaniecki, F. (1934). The method of sociology. New York, NY: Farrar & Rinehart. Appendix 1. Semi-Structured Interview Questionnaires Phase 1 Semi-Structured Interview Questionnaires (A) Introduction (1) Do you expect a requirement to raise finance in the next 3 years? (2) Have you raised any finance in the last 12 months? (3) When was the business established? (4) How many employees? (5) What is the most recent turnover? (6) What is the current funding structure? oi:10.1016/j.jebo.2006.10.008 (7) How long have you spent in the industry? (8) What is your Functional Expertise? (9) What are your qualifications?(A) Funding (1) What is your experience of running an SME? (2) Have you any experience of family entrepreneurship? (3) How much do you think your knowledge of this industry helps you to solve funding problems? (4) Do your qualifications help you to access finance? (5) How have you planned your funding requirements? (6) How do you expect to plan your funding requirements differently in the future? (7) How have you solved funding problems in the past? (8) In what ways do you think you will solve funding problems in the future? (9) How have you presented and communicated your funding requirements? (10) Do you expect to change the way you communicate and present your funding requirements in the future? (11) Have you examples of how you have cooperated with other individuals or businesses in order to solve funding problems? Page 22 of 29 Page 22 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 (12) Do you expect to change how you co-operate with other individuals or businesses to solve funding problems in the future? (13) Do you lead the funding process in your firm or is it the responsibility of others and you take a supporting role? (14) Can you give me any examples of how you could be innovative and creative in the way you fund your business? (15) How do you cope with pressure and managing uncertainty? (16) Do you think Driving for Results will be an important factor when trying to raise finance? (17) How do you develop relationships with customers? (18) Can you think how your own social skills could be important in raising funds for your business? (19) Do you think your own financial skills could help or hinder in funding applications? Phase 2 Semi-Structured Interview Questionnaires (A.) DBA: Entrepreneurial Behaviour and Finding Growth Finance (1) What new funding have you access to in the last 12 months? (i) Why now? (ii) Why did you select this? (iii) How did you do it? (iv) How much in relation to your current equity plus debt? (v) And how much have your employees grown by . . . from what to what (vi) How much time did it take (hours of your time) (vii) What were the stages in the process? oi:10.1016/j.jebo.2006.10.008 (time for each stage) (viii) Planning—inc cash flow (with who); what meetings (ix) Networking (x) Exit (xi) New Business Model (2) H b f l i f di li i h l 12 h ? (12) Do you expect to change how you co-operate with other individuals or businesses to solve funding problems in the future? (13) Do you lead the funding process in your firm or is it the responsibility of others and you take a supporting role? (14) Can you give me any examples of how you could be innovative and creative in the way you fund your business? (15) How do you cope with pressure and managing uncertainty? (ii) On reflection, why was the application unsuccessful? Page 23 of 29 Page 23 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 (iii) How much time did it take? (hours of your time) (iv) What were the stages in the process? (time for each stage) (v) Planning—inc cash flow (with who); meetings (vi) Networking (vii) New Business Model (3) Are you trying to access new funds currently? (i) Why now? (ii) Why did you select this type? (iii) How did you do it? (iv) How much time is it taking? (hours of your time) (v) What are the stages in the process? (time for each stage) (vi) Planning—inc cash flow (with who); meetings (vii) Networking (viii) New Business Model (4) Are you planning any new funds in the next 12 month? (i) Why (ii) Is it debt? (iii) How will you do this—Prompts (iv) How much time did it take? (hours of your time) (v) What were the stages in the process? (time for each stage) (vi) Planning inc cash flow (with who); meeting (vii) Or equity? (viii) How much time did it take? (hours of your time) (3) Are you trying to access new funds currently? Page 24 of 29 Page 24 of 29 (xii) New Business Model (5) Would you exchange equity for the opportunity to access growth finance in the future? (6) Are you considering any form of funding that you would consider being innovative? (i) Moving Premises (Clustering?) (ii) Through new income streams (iii) Learning about finance (iv) Customers or Suppliers (7) Is there someone you would describe as a formal Advisor (or Mentor) to the business—YES or NO (i) Who and why did you choose this person(s)? (ii) How often do you discuss issues with them? (iii) What issues; how much time? (8) Have you appointed formal Non-Exec to the Board? (i) Why? (ii) How often do you discuss issues with them? (iii) What issues; how much time? (9) Is it still your desire to grow? YES/NO/MAYBE (10) If yes, how much over the next 5 years? gement (2018), 5: 1512833 018.1512833 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 (xii) New Business Model (5) Would you exchange equity for the opportunity to access growth finance in the future? (6) Are you considering any form of funding that you would consider being innovative? (i) Moving Premises (Clustering?) (ii) Through new income streams (iii) Learning about finance (iv) Customers or Suppliers (7) Is there someone you would describe as a formal Advisor (or Mentor) to the business—YES or NO (i) Who and why did you choose this person(s)? (ii) How often do you discuss issues with them? (iii) What issues; how much time? (8) Have you appointed formal Non-Exec to the Board? (i) Why? (ii) How often do you discuss issues with them? (iii) What issues; how much time? (9) Is it still your desire to grow? YES/NO/MAYBE (10) If yes, how much over the next 5 years? (xii) New Business Model (xii) New Business Model (xii) New Business Model Page 25 of 29 Page 25 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Theme and code Description Sources References Communicating, Meeting, Presenting 23 36 Approaching International Investors Going outside the UK to see equity investors 1 1 Approaching Investors Evidence of entrepreneurs approaching individuals or organisations to make private equity investments 18 27 Entering Competitions Entering competitions for awards, recognition or prizes 0 0 Meeting Bankers, Brokers and Other Lenders Regular structured informed communication 7 8 Coping with Pressure 1 2 Personal Reflections Putting stress into context 1 2 Driving for Results 18 34 Consider acquisitions and mergers Entrepreneur considering mergers and acquisitions as a way of facilitating further funds into the business. 0 0 Identifying Growth and Opportunity Identification of growth opportunity is key at the outset of the funding process 16 27 Persistence and Challenging Evidence of the resilience of the entrepreneurs and an ability to cope with setbacks and adversity. 5 7 Innovating and Creating 25 65 Changing Bank Looking to switch lender as a way of potentially increasing funding. 0 0 Grant Applications Pursuing grants 13 19 Internal Funding Reinvesting profits including bootstrapping and other internally sourced funding 8 10 Peer-to-Peer Lenders Looking to use new peer-to-peer funding models 9 16 SME Educational Programme Joined a scheme to open funding sources 12 20 Leading Others 0 0 Planning and Organising 6 15 Cash Management This includes the preparation of Management Accounts and Cash Flow Forecasts 5 7 Using Flexible Staff Management Planning a flexible approach to labour management to help fund the business. (xii) New Business Model 4 8 Problem-Solving 12 20 Page 26 of Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Theme and code Description Sources References Developing a business model to attract funding Explaining how the business model is developing and therefore makes it possible to raise funds 6 8 Using Match Funding Evidence that the entrepreneur is willing to put match his own funds with external sources 9 12 Working with Customers 5 5 Creating Relationships Customers as potential investors 1 1 Flexible Payment Terms Getting flexible payment terms so it eves out cash flow and makes easier planning for investments 4 4 Working with Others 22 60 Advisors, Mentors, Non Execs Seeking and using third party assistance in the context of finding finance 14 29 Friends and family Looking to family or friends as a source of finance 1 1 JV’s Looking for a partner as a potential source of investment 12 23 Suppliers Seeking funding through arrangements with suppliers 5 7 Behavioural Difficulty 26 63 Bad Hiring Decisions Funding hindered by poor hiring decision 0 0 Bad Planning Where poor planning decisions have hindered the funding application process 3 3 Business Partner Problems Where problems cause problems in the funding process 2 2 Decision to consolidate Evidence of more cautious behaviour 7 7 Difficulty with Advisors and Mentors Problems taking advice 0 0 Difficulty with new funding sources Finding problems with more innovative forms of funding 5 7 Difficulty with SME Educational schemes Problem with application process’ 0 0 Doubts over personal skills 1 2 Given up Throwing in the towel type behaviour 0 0 Just focus on the family Evidence where a more family orientated focus prevents funding 0 0 (Continued) Page 27 o References Page 27 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Theme and code Description Sources References Keeping Control An emphasis on needing to keep control and as a result this closes funding streams 7 8 Problems with factoring See difficulties with debt factoring 0 0 Problems with Security Difficulties with arranging security 8 10 Problems with VC Process Just finding it difficult with VC Due Diligence process 3 3 Signs of Frustration Just losing ability to rationalise process 10 13 Struggling with the Bank Difficulties with lenders 4 6 Uncertainty Not being able to plan effectively due to uncertainty 2 2 code Description Sources References ntrol An emphasis on needing to keep control and as a result this closes funding streams 7 8 th factoring See difficulties with debt factoring 0 0 th Security Difficulties with arranging security 8 10 th VC Process Just finding it difficult with VC Due Diligence process 3 3 stration Just losing ability to rationalise process 10 13 with the Bank Difficulties with lenders 4 6 Not being able to plan effectively due to uncertainty 2 2 Page 28 of 29 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 Parkes et al., Cogent Business & Management (2018), 5: 1512833 https://doi.org/10.1080/23311975.2018.1512833 © 2018 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. You are free to: Share — copy and redistribute the material in any medium or format. Adapt — remix, transform, and build upon the material for any purpose, even commercially. The licensor cannot revoke these freedoms as long as you follow the license terms. Under the following terms: Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. No additional restrictions You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits. © 2018 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license py y Adapt — remix, transform, and build upon the material for any purpose, even commercially. References The licensor cannot revoke these freedoms as long as you follow the license terms. g Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. No additional restrictions You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits. Cogent Business & Management (ISSN: 2331-1975) is published by Cogent OA, part of Taylor & Francis Group. Publishing with Cogent OA ensures: • Immediate, universal access to your article on publication • High visibility and discoverability via the Cogent OA website as well as Taylor & Francis Online • Download and citation statistics for your article • Rapid online publication • Input from, and dialog with, expert editors and editorial boards • Retention of full copyright of your article • Guaranteed legacy preservation of your article • Discounts and waivers for authors in developing regions Submit your manuscript to a Cogent OA journal at www.CogentOA.com Page 29 of 29 Cogent Business & Management (ISSN: 2331-1975) is published by Cogent OA, part of Taylor & Francis Group. Publishing with Cogent OA ensures: • Immediate, universal access to your article on publication • High visibility and discoverability via the Cogent OA website as well as Taylor & Francis Online • Download and citation statistics for your article • Rapid online publication • Input from, and dialog with, expert editors and editorial boards • Retention of full copyright of your article • Guaranteed legacy preservation of your article • Discounts and waivers for authors in developing regions Submit your manuscript to a Cogent OA journal at www.CogentOA.com cis Online Page 29 of 29
https://openalex.org/W4309046273	https://ijcsm.springeropen.com/counter/pdf/10.1186/s40069-022-00547-3	English	null	Experimental and Numerical Investigation of the Tensile Performance and Failure Process of a Modified Portland Cement	International journal of concrete structures and materials	2,022	cc-by	9,910	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract A better understanding of the tensile performance and tensile failure mechanism of cement paste is significant in preventing rock reinforcement failure. Therefore, this paper aims to reveal the tensile performance and failure mecha- nism of a modified Portland cement: Stratabinder HS cement. To achieve this objective, the split tensile test was conducted on specimens followed by simulating the failure mechanism numerically. The results indicated that the water–cement rate significantly influenced the tensile performance of the cement paste. When the water–cement rate increased from 0.35 to 0.42, the tensile strength declined from 1.9 MPa to 1.5 MPa. It was also observed that verti- cal tensile failure constantly occurred regardless of the water–cement rate. During the testing process, tensile cracks and shear cracks occurred. The increasing rate in the number of specimen cracks was dependent on the tensile stress state. Before the tensile stress reached the peak, the crack quantity increased slightly. After the peak, the crack quan- tity increased dramatically. During the vertical loading process, horizontal tensile stress occurred in the specimen. This horizontal tensile stress zone showed a diamond shape. The higher the tensile stress is, the larger the area of the horizontal tensile stress zone. When the tensile strength was reached, horizontal tensile stress mainly concentrated at the vertical centre of the specimen. This finally led to tensile failure of the specimen. This paper indicated that the water–cement rate was the key factor in evaluating the tensile strength of the Stratabinder HS cement. Keywords: Cement paste, Tensile strength, Tensile failure, Fractures, Water–cement rate 2020; Wu et al., 2019). For example, Portland cement can be used to fill discontinuities in jointed rock masses around excavations to improve rock stability (Aili & Maruyama, 2020; Aziz et al., 2017). Experimental and Numerical Investigation of the Tensile Performance and Failure Process of a Modified Portland Cement Jianhang Chen1,2, Kangming Tao2, Banquan Zeng2, Lei Liu2, Hongbao Zhao1,2, Junwen Zhang1,2* and Danqi Li3* International Journal of Concrete Structures and Materials International Journal of Concrete Structures and Materials Open Access Chen et al. Int J Concr Struct Mater (2022) 16:61 https://doi.org/10.1186/s40069-022-00547-3 Chen et al. Int J Concr Struct Mater (2022) 16:61 https://doi.org/10.1186/s40069-022-00547-3 International Journal of Concrete Structures and Materials 1 Introduction Portland cement is an indispensable binding material in engineering projects. In civil engineering, Portland cement is used to fabricate concretes (Jung et al., 2018; Murmu & Singh, 2014). In mining engineering, Portland cement plays a paramount role in guaranteeing under- ground excavation safety (Chen & Li, 2022; Chen et al., A better understanding of the mechanical properties of Portland cement is beneficial for promoting its value in use. It is noteworthy that the majority of the atten- tion was given to Portland cement’s uniaxial compressive strength (UCS) (Rashad et al., 2014). Only limited work has been performed to analyse its tensile strength. Journal information: ISSN 1976-0485/eISSN 2234-1315 *Correspondence: zhangjw@cumtb.edu.cn; danqi.li@curtin.edu.au 1 State Key Laboratory for Geomechanics and Deep Underground Engineering, China University of Mining and Technology (Beijing), Beijing 100083, China 3 Energy and Chemical Engineering, WA School of Mines: Minerals, Curtin University, Kalgoorlie, WA 6430, Australia Full list of author information is available at the end of the article In fact, Portland cement’s tensile performance is cru- cial in engineering applications. In rock reinforcement, Portland cement fills the annulus between the rock tendon and rock mass (Li et al., 2021). After the rock tendon is loaded, radial dilation occurs at the tendon- to-grout interface (Fig. 1). This dilation leads to tensile Chen et al. Int J Concr Struct Mater (2022) 16:61 Page 2 of 14 Chen et al. Int J Concr Struct Mater interface dilation rock tendon surrounding rock mass cement-based grout tendon-to-grout interface tensile crack (a) (b) Fig. 1 Rock reinforcement system a cross-sectional view; b lateral view. et al. (2016) indicated that the tensile performance of the cement at the microscale was largely different from that at the macroscale.h (a) The above research provided valuable knowledge in revealing the tensile performance of cement. Recently, a modified Portland cement named the Stratabinder HS cement has been widely applied in civil and mining projects. However, little work has been reported on its tensile performance. Moreover, little research has been conducted to use the discrete element method (DEM) to evaluate the tensile failure of cement. Considering that failure of the material plays a significant role in engi- neering applications (Song & Zhang, 2022), this paper adopted experimental and numerical methods to analyse the tensile performance of Stratabinder HS cement. cracks in the cement-based grout (Chen et al., 2021). These tensile cracks may jeopardise the capacity of the rock reinforcement system (Chen et al., 2022; Skrzyp- kowski, 2021; Wu et al., 2018). The experimental program was first illustrated. Then, the experimental results and data analysis were pre- sented, followed by numerical simulation results. Finally, further studies were recommended. To evaluate the tensile performance of the cement, experimental tests were more widely used. Two differ- ent test methods can be used: the split tensile strength (STS) test or the direct uniaxial tensile strength (UTS) test (Bybee, 2004). With these two methods, research- ers compared the tensile performance of different cements (Paknahad et al., 2020). Bayindir et al. (2009) indicated that the cement type significantly influenced the tensile strength of the cement paste. Bybee (2004) found that fibre-reinforced cement had higher tensile strength than plain cement. In contrast, Syarif et al. (2018) indicated that the organic cement had a smaller strength than the normal Portland cement.l 2 Experimental Program 2.1 Materialsh This study aims to evaluate the tensile performance of the Stratabinder HS cement. To fulfil this purpose, fresh Stratabinder HS cement was ordered from the Minova Company.h The Stratabinder HS cement is generally composed of two parts. One is Portland cement, accounting for more than 60%. The other is the nonhazardous ingredients, accounting for the remaining proportion. The chemi- cal composition of the Stratabinder HS cement mainly includes calcium oxide, silica, alumina and iron oxide. Attention was also given to the influence of additives on the tensile performance of cement pastes. Mishra and Kantharia (2021) indicated that adding nanoz- inc oxide increased the tensile strength of the cement. Similarly, adding carbon nanotubes increased the ten- sile strength of the cement as well (Silvestro & Gleize, 2020). In situ observation shows that when this cement is used, a key factor is the water–cement rate. In this study, two different water–cement rates were used. One was 0.35, and the other was 0.42. The reason to use those two water–cement rates is that if the cement paste is too thick (water–cement rate < 0.3), cement mixing is a prob- lem without the water reducing agent added. Previous research indicated that once the water reducing agent was added, a solvation thin film was generated around the cement particles. This solvation thin film reduced the friction resistance of the cement particles. Therefore, with the water reducing agent added, the cement paste could be properly mixed even when the water–cement rate was smaller than 0.3 (Şahin et al., 2022). Additional attention was given to the influence of cement proportion (Fedrigo et al., 2018), porosity (Chen et al., 2013), curing time (Wang et al., 2020), temperature (Masoud et al., 2010), saturation condition (Gu et al., 2019), loading condition (Liu et al., 2019), specimen size (Xiong & Geng, 2020) and the use of aggregates (Bitouri et al., 2017) on the tensile perfor- mance of cement. The results showed that the tensile strength of the cement paste was critically influenced by various parameters. Therefore, to optimise the ten- sile strength of the cement paste, the correspond- ing test condition and environment should be strictly controlled. In contrast, if the cement paste is too thin (water– cement rate > 0.5), the strength of the cement paste may not meet the engineering quality requirement. 2.2 Experimental UCS Test In recent years, researchers have also studied the ten- sile performance of cement at the microscale. Němeček First, the UCS test was conducted to evaluate the com- pressive performance of the Stratabinder HS cement. Chen et al. Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater (2022) 16:61 Page 3 of 14 Chen et al. Int J Concr Struct Mater (2022) 16:61 During the experimental UCS test, cylinder specimens with a diameter of 54 mm and height of 142 mm were cast with cylindrical moulds. Two water–cement rates of 0.35 and 0.42 were used. After all specimens were fully cured for 28 days, a griding machine was used to polish two ending surfaces of the specimen. Then, those cylin- der specimens were placed in the MTS 815 machine to conduct UCS tests, as shown in Fig. 2. In contrast, the STS test method is relatively eas- ier. Therefore, in this paper, the STS test method was adopted. 2.3 Experimental Tensile Test After the Stratabinder HS cement was mixed with water, the cement paste was poured into the casting mould. Then, the casting mould was placed on a vibrator. The vibrator was switched on, and the casting mould was vibrated for 1 min. The purpose is to remove air voids in the cement paste. 2.3.1 Selection of the Tensile Test Method Although either the UTS or STS test method can be used to measure the tensile strength of cement, the UTS method is more difficult to conduct (Sun & Wu, 2021). This is because during the UTS test, specimens should be clamped by the gripping device, leading to the stress con- centration occurrence at the specimen’s corner. There- fore, it is possible that the specimen may fail due to stress concentration rather than tensile stress. After setting for 24 h, the specimen was demoulded. All specimens were immersed in fresh water for curing. After 28 days, they were removed from the water. Then, specimens were fixed on a lathe. An electric saw was used to cut the specimens into disks. Finally, all disk specimens had a diameter of approximately 42 mm and a thickness of approximately 25 mm. The height/diameter rate was approximately 0.59.i Fig. 2 Experimental UCS test. To confirm that the results are reliable, at least 5 speci- mens were prepared for each water–cement rate. The prepared specimens at a w/c rate of 0.35 are shown in Fig. 3. 2.3.2 Test Specimen Preparation b d h PVC tubes were used as the casting mould. They had a height of 110 mm and internal diameter of 42 mm. This casting mould was adhered to a wooden board to pre- vent cement leakage. Moreover, the internal surface of this casting mould was brushed with a thin oil. This is to avoid the cement paste sticking onto the casting mould after demoulding. The UCS test was used to calibrate and confirm the later numerical simulation. Specific information of the experimental UCS test can be found in Chen et al. (2014). 2.3.3 Experimental Test Process Consequently, the average tensile strength was 1.9 MPa. The tensile stress–displacement curves are shown in Fig. 5.h Since the data logger system automatically recorded the force–displacement curve, the tensile stress–dis- placement result can be calculated with Eq. (1). This shows that with increasing displacement, the ten- sile stress of the specimen increased nonlinearly before its peak. After the tensile stress reached its peak, the tensile stress of the specimen declined dramatically. The specimen may reach the initial peak when the displace- ment reaches approximately 0.2 mm, followed by a sharp decline in the bearing capacity. The specimen’s carrying capacity rose again until reaching its global peak value at approximately 0.4 mm. 2.3.3 Experimental Test Process Each specimen was installed in an MTS 815 testing machine, as shown in Fig. 4.h This machine was equipped with the servo controlling system. A displacement control can be applied in the full testing process. When conducting this STS test, a con- stant velocity of 3 μm/s was applied to the specimen. A load cell was set on the bottom plate to record the ver- tical force. In addition, the displacement transducer in Fig. 3 Prepared specimens. Fig. 3 Prepared specimens. g. 3 Prepared specimens. Fig. 3 Page 4 of 14 Chen et al. Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater Table 1 Experimental UCS test results. Fig. 4 Split tensile strength test. where E is the Young’s modulus, μ is the Poisson’s ratio Water–cement rate UCS (MPa) E (GPa) μ 0.35 63.1 11.8 0.23 0.42 54.3 9.9 0.21 Table 2.where SD is the standard deviation and SEM is the standard error of the mean. These two parameters were calculated with Eqs. (2) and (3): (2) SD = σti − −σt 2 n (3) SEM = S √n (2) SD = σti − −σt 2 n (2) (3) SEM = S √n (3) where σti is the peak tensile stress of specimens; −σt is the average tensile strength of specimens; and n is the num- ber of repetition tests.h The tensile strength of the specimens ranged between 1.3 MPa and 4.2 MPa. The SD was 1.03, and the SEM was 0.46. Considering that random results may occur, the data were further processed to guarantee the consist- ency of the testing results. The maximum value and mini- mum value were removed. The peak tensile stress of the remaining three specimens was averaged with Eq. (4) as the tensile strength: Fig. 4 Split tensile strength test. Fig. 4 Split tensile strength test. Fig. 4 Fig. 4 the testing machine automatically recorded the vertical displacement. Equation (1) was used to calculate the tensile stress in the specimen: (1) σt = 2F πDt (4) σtave = σt1 + σt2 + σt3 3 σt = 2F πDt (1) (4) where σtave is the average tensile strength of the remain- ing specimens. where σt is the tensile strength of the specimen; F is the vertical force that was applied on the specimen; D is the specimen diameter; and t is the specimen thickness. 3 Experimental Results 3.1 UCS Test Resultsh The experimental UCS tests showed that the specimen had a UCS of 63.1 MPa at a water–cement rate of 0.35. In contrast, the specimen showed a UCS of 54.3 MPa at a water–cement rate of 0.42 (Table 1). During testing, the deformation of the MTS 815 machine itself was ignored, as the loading frame is much stiffer than the test specimen. 3.2 Tensile Test Results f It is also noteworthy that additional methods were employed to minimise the gap between the test speci- men and loading frame before the test. The test specimen 3.2.1 Tensile Stress–Displacement Results After the STS test, the results for the cement paste with a water–cement rate of 0.35 are summarised in Chen et al. Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater Page 5 of 14 Page 5 of 14 Chen et al. Int J Concr Struct Mater (2022) 16:61 Table 2 STS test results when the water–cement rate was 0.35. No. t (mm) D (mm) t/D rate σt (MPa) SD SEM 1 24.4 42.3 0.58 1.6 1.03 0.46 2 25.6 41.0 0.63 2.1 3 24.8 41.3 0.6 4.2 4 24.3 41.5 0.59 1.9 5 24.3 41.7 0.58 1.3 0 0.1 0.2 0.3 0.4 0 0.5 1 1.5 2 Displacement (mm) ) a P M ( ss e rts elis n e T Sample 1 Sample 2 Sample 4 Fig. 5 Tensile stress–displacement curves when the water–cement rate was 0 35 Fig. 6 Failure state in the STS test. 1.5 MPa. The tensile stress–displacement curves of the Table 2 STS test results when the water–cement rate was 0.35. No. t (mm) D (mm) t/D rate σt (MPa) SD SEM 1 24.4 42.3 0.58 1.6 1.03 0.46 2 25.6 41.0 0.63 2.1 3 24.8 41.3 0.6 4.2 4 24.3 41.5 0.59 1.9 5 24.3 41.7 0.58 1.3 0 0.1 0.2 0.3 0.4 0 0.5 1 1.5 2 Displacement (mm) ) a P M ( ss e rts elis n e T Sample 1 Sample 2 Sample 4 Fig. 5 Tensile stress–displacement curves when the water–cement rate was 0.35. Fig. 6 Failure state in the STS test. Table 2 STS test results when the water–cement rate was 0.35. No. t (mm) D (mm) t/D rate σt (MPa) SD SEM 1 24.4 42.3 0.58 1.6 1.03 0.46 2 25.6 41.0 0.63 2.1 3 24.8 41.3 0.6 4.2 4 24.3 41.5 0.59 1.9 5 24.3 41.7 0.58 1.3 Table 2 STS test results when the water–cement rate was 0.35. 0 0.1 0.2 0.3 0.4 0 0.5 1 1.5 2 Displacement (mm) ) a P M ( ss e rts elis n e T Sample 1 Sample 2 Sample 4 Fig. 5 Tensile stress–displacement curves when the water–cement rate was 0.35. 0 0.1 0.2 0.3 0.4 0 0.5 1 1.5 2 Displacement (mm) ) a P M ( ss e rts elis n e T Sample 1 Sample 2 Sample 4 Fig. 3.2.1 Tensile Stress–Displacement Results 5 Tensile stress–displacement curves when the water–cement rate was 0.35. Fig. 6 Failure state in the STS test. Fig. 6 Failure state in the STS test. Fig. 6 Fig. 5 Tensile stress–displacement curves when the water–cement rate was 0.35. 1.5 MPa. The tensile stress–displacement curves of the remaining specimens are plotted in Fig. 7. Apparently, for the water–cement rate of 0.42, the ten- sile stress–displacement curves were smoother. With increasing displacement, the tensile stress of the speci- men continuously increased. After the specimen reached the tensile strength, the carrying capacity of the speci- mens declined dramatically. was placed underneath the loading plate in the machine. The bottom plate was lifted until the smallest visible gap formed. Then, the bottom plate was further lifted with the help of the micro-displacement control in the computer interface so that a very small positive load was applied to the specimen. By doing this, tight but not excessive con- tact between the specimen and loading frame could be formed. This would guarantee the accuracy of the testing results by ensuring that any displacement in the speci- men would lead to a change in the stress. 3.2.2 The Impact of the Water–Cement Rate In PFC, properties of the particles, including the effective modulus and stiffness rate, should be input. These properties can be acquired by conducting numerical UCS tests. First, a rectangular domain with an edge length of 100 mm was created. Then, the numerical UCS speci- men was created in this domain, as shown in Fig. 9. This numerical specimen had a height of 142 mm and Table 3 STS test results when the water–cement rate was 0.42. No. t (mm) D (mm) t/D rate σt (MPa) SD SEM 1 25.0 42.1 0.59 1.8 0.43 0.19 2 25.4 41.8 0.61 0.7 3 24.8 42.2 0.59 1.9 4 24.9 41.9 0.59 1.3 5 25.1 42.1 0.6 1.4 0 0.05 0.1 0.15 0.2 0.25 0 0.5 1 1.5 2 Displacement (mm) ) a P M ( ss e rts elis n e T Sample 1 Sample 4 Sample 5 Fig. 7 Tensile stress–displacement curves when the water–cement rate was 0.42. 0.3 0.4 0.5 0 1 2 3 4 water-cement rate ) a P M ( h t g n e rts elis n e T Fig. 8 Impact of the water–cement rate on the tensile strength of the Stratabinder HS cement. v=0.1m/s Fig. 9 Numerical simulation of the UCS test. Chen et al. Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater Page 6 of 14 Table 3 STS test results when the water–cement rate was 0.42. 0 0.05 0.1 0.15 0.2 0.25 0 0.5 1 1.5 2 Displacement (mm) ) a P M ( ss e rts elis n e T Sample 1 Sample 4 Sample 5 v=0.1m/s Fig. 9 Numerical simulation of the UCS test. Fig. 7 Tensile stress–displacement curves when the water–cement rate was 0.42. Fig. 7 Tensile stress–displacement curves when the water–cement rate was 0.42. Fig. 9 Numerical simulation of the UCS test. 0.3 0.4 0.5 0 1 2 3 4 water-cement rate ) a P M ( h t g n e rts elis n e T Fig. 8 Impact of the water–cement rate on the tensile strength of the Stratabinder HS cement. addition, numerical simulation was able to reproduce the tensile failure process of the cement paste. 3.4 Selection of the Numerical Code Among all numerical simulation methods, the DEM was adopted. The reason is that the DEM assumes that the numerical zones are discrete. Moreover, the bond between numerical zones is allowed to break when sub- jected to loading. As such, it is believed that tensile fail- ure of the cement paste can be more closely simulated with DEM. For the software, the Particle Flow Code (PFC) was used. 3.5 Simulation Process Fig. 8 Impact of the water–cement rate on the tensile strength of the Stratabinder HS cement. Initially, the cement paste with a water–cement rate of 0.35 was simulated. In PFC, properties of the particles, including the effective modulus and stiffness rate, should be input. These properties can be acquired by conducting numerical UCS tests. 3.2.2 The Impact of the Water–Cement Rate Previous research has been conducted to evaluate the impact of the water–cement rate on the tensile perfor- mance of normal Portland cement. It showed that there was an adverse impact of the water–cement rate on the tensile strength (Hyett et al., 1992). After the STS test, a typical failure mode is shown in Fig. 6. Apparently, vertical failure was generated at the specimen centre. The two sides of the specimen tended to separate along the horizontal direction. Following similar procedures, the impact of the water– cement rate on the tensile performance of the Strat- abinder HS cement was analysed. As shown in Fig. 8, there was a strong relationship between the water– cement rate and the tensile strength of the Stratabinder HS cement. As the water–cement rate increased, the ten- sile strength of the cement decreased. This result agreed well with the in situ observation results. Specifically, for a high water–cement rate used in rock reinforcement, ten- sile failure of the cement grout annulus was more likely to occur. Further tests were conducted on specimens with a water–cement rate of 0.42. The results are summarised in Table 3. For the water–cement rate of 0.42, the specimen’s tensile strength ranged between 0.7 MPa and 1.9 MPa. The SD was 0.43, and the SEM was 0.19. To remove random results, the minimum values and maximum values were eliminated. Then, the tensile strength of the cement paste with a water–cement rate of 0.42 was Page 6 of 14 Chen et al. Int J Concr Struct Mater (2022) 16:61 3.3 Numerical Simulation Scheme Numerical simulation was conducted, because it is pow- erful in revealing the microscopic failure behaviour of materials (Chen et al., 2019; Wang et al., 2021). In addition, numerical simulation was able to reproduce the tensile failure process of the cement paste. 3.4 Selection of the Numerical Code Among all numerical simulation methods, the DEM was adopted. The reason is that the DEM assumes that the numerical zones are discrete. Moreover, the bond between numerical zones is allowed to break when sub- jected to loading. As such, it is believed that tensile fail- ure of the cement paste can be more closely simulated with DEM. For the software, the Particle Flow Code (PFC) was used. 3.5 Simulation Process Initially, the cement paste with a water–cement rate of 0.35 was simulated. 3.3 Numerical Simulation Scheme The input parameter of tensile strength was cali- brated until the numerical tensile stress–displacement curve fit well with the experimental curve. εa = H H (6) where εa is the compressive strain or axial strain; ΔH is the variation of the specimen height; and H is the speci- men height. The tensile strain or diametric strain of the specimen can be calculated with Eq. (7): (7) εd = W W 3.3 Numerical Simulation Scheme First, a rectangular domain with an edge length of 100 mm was created. Then, the numerical UCS speci- men was created in this domain, as shown in Fig. 9. This numerical specimen had a height of 142 mm and Numerical simulation was conducted, because it is pow- erful in revealing the microscopic failure behaviour of materials (Chen et al., 2019; Wang et al., 2021). In Chen et al. Int J Concr Struct Mater (2022) 16:61 Page 7 of 14 a width of 54 mm. Moreover, its dimension was con- sistent with the size used in laboratory tests. This numerical specimen was composed of 5188 balls. The ball radius ranged between 0.6 mm and 0.7 mm. The porosity of this numerical specimen was 0.1. After this numerical specimen was created, this model was solved automatically until the averaged unbalanced force ratio decreased to 1 × 10–5 to eliminate the overlap between balls. v=0.1m/s Fig. 10 Numerical STS test created in PFC. The bottom boundary of the specimen was fixed to support the numerical specimen. Meanwhile, a con- stant compressive velocity of 0.1 m/s was applied on the top boundary of the specimen. During the test, the applied force and vertical deformation were recorded. In addition, the increment of the specimen’s width at the middle section was recorded. friction coefficient, cohesion and friction angle, can be determined.h Based on the recorded information, the axial stress of the specimen can be calculated with Eq. (5): Then, the numerical STS test was performed. Spe- cifically, a disk geometry was created (Fig. 10). In this simulation, the specimen diameter was 42 mm. This was the same as the geometric size in the experimental STS test. (5) σc = F Wt (5) where σc is the compressive stress of the specimen; W is the specimen width; t is the specimen thickness, equal- ling 1 in this two-dimensional simulation.h The bottom boundary in the numerical STS test was fixed. Then, a constant compressive velocity of 0.1 m/s was applied on the top boundary. During loading, the vertical force and displacement were monitored. Then, Eq. (1) was used to calculate the tensile stress.hf The compressive strain or the axial strain of the spec- imen can be calculated with Eq. (6): (6) εa = H H The input parameters, including the stiffness rate, surface gap, friction coefficient, cohesion and friction angle, were directly acquired from the numerical UCS test. 4 Numerical Simulation Results and Analysis Calibration was conducted on the input parameter of the tensile strength. When the input parameter of tensile strength equalled 2.38 MPa, a satis- factory match between the numerical and experimental results was acquired (Fig. 12). At the end of this numerical STS test, the specimen failure mode was checked (Fig. 13). There was an apparent vertical tensile failure at the middle of this specimen. Comparing it with the experi- mental failure mode, there was a close match between them. This further confirmed the accuracy of this numer- ical STS test. Table 4 Input factors for the cement paste. Water–cement rate Effective modulus (GPa) Stiffness rate Surface gap Friction coefficient Cohesion (MPa) Friction angle (°) 0.35 6.5 1.18 1 × 10–4 0.3 26 10 0.42 4.8 1.13 1 × 10–4 0.3 18 8 -0.5 -0.3 -0.1 0.1 0.3 0.5 0.7 0.9 0 15 30 45 60 Strain (%) ) a P M ( ss e rts l ai x A Simulation Experiment (a) specimen failure (b) (c) particle failure Fig. 11 Comparison between the numerical and experimental UCS results when the water–cement rate was 0.35: a stress–strain curves; b experimental failure mode; c numerical failure mode. 0 0.1 0.2 0.3 0 0.5 1 1.5 Displacement (mm) ) a P M ( ss e rts elis n e T Numerical result Experimental result Fig. 12 Tensile stress–displacement curve comparison when the water–cement rate was 0.35. Table 4 Input factors for the cement paste. Water–cement rate Effective modulus (GPa) Stiffness rate Surface gap Friction coefficient Cohesion (MPa) Friction angle (°) 0.35 6.5 1.18 1 × 10–4 0.3 26 10 0.42 4.8 1.13 1 × 10–4 0.3 18 8 -0.5 -0.3 -0.1 0.1 0.3 0.5 0.7 0.9 0 15 30 45 60 Strain (%) ) a P M ( ss e rts l ai x A Simulation Experiment (a) specimen failure (b) (c) particle failure Fig. 11 Comparison between the numerical and experimental UCS results when the water–cement rate was 0.35: a stress–strain curves; b experimental failure mode; c numerical failure mode. -0.5 -0.3 -0.1 0.1 0.3 0.5 0.7 0.9 0 15 30 45 60 Strain (%) ) a P M ( ss e rts l ai x A Simulation Experiment (a) Fig. 11 Comparison between the numerical and experimental UCS results when the water–cement rate was 0.35: a stress–strain curves; b experimental failure mode; c numerical failure mode. 4 Numerical Simulation Results and Analysis 0 0.1 0.2 0.3 0 0.5 1 1.5 Displacement (mm) ) a P M ( ss e rts elis n e T Numerical result Experimental result Fig. 12 Tensile stress–displacement curve comparison when the water–cement rate was 0.35. 0 0.1 0.2 0.3 0 0.5 1 1.5 Displacement (mm) ) a P M ( ss e rts elis n e T Numerical result Experimental result Fig. 12 Tensile stress–displacement curve comparison when the water–cement rate was 0.35. Then, the axial stress–strain curves of the specimen can be acquired. When the water–cement rate was 0.35, the axial stress–strain curve of the cement paste is plotted in Fig. 11a.h The numerical simulation showed that the UCS, Young’s modulus and Poisson’s ratio were 64.5 MPa, 12.3 GPa and 0.22, respectively. The relative differences between the numerical simulation and experimental test were 2.2%, 4.2% and 4.3%. There was a close match between them. This validated that the input parameters were effective in analysing the compressive performance of the cement paste. Moreover, the UCS failure mode comparison when the water–cement rate was 0.35 is shown in Fig. 11b, c. In the experiment, part of the cement paste was ejected from the specimen. This phenomenon also occurred in the numerical simulation. Apparently, in the numerical simu- lation, some of the particles detached from the numeri- cal specimen. Therefore, there was a general consistency between the experimental and numerical failure modes.h Displacement (mm) Fig. 12 Tensile stress–displacement curve comparison when the water–cement rate was 0.35. At the end of this numerical STS test, the specimen failure mode was checked (Fig. 13). There was an apparent vertical tensile failure at the middle of this specimen. Comparing it with the experi- mental failure mode, there was a close match between them. This further confirmed the accuracy of this numer- ical STS test. At the end of this numerical STS test, the specimen failure mode was checked (Fig. 13).h Then, those input parameters were substituted into the numerical STS test. Calibration was conducted on the input parameter of the tensile strength. When the input parameter of tensile strength equalled 2.38 MPa, a satis- factory match between the numerical and experimental results was acquired (Fig. 12). There was an apparent vertical tensile failure at the middle of this specimen. Comparing it with the experi- mental failure mode, there was a close match between them. 4 Numerical Simulation Results and Analysis (7) Following the above simulation procedures, input factors for two different water–cement rates were obtained, as summarised in Table 4. For the numerical test, there was an apparent difference in the mechanical properties of the specimen.f where εd is the diametric strain; ΔW is the variation of the specimen width.h Then, Poisson’s ratio of the specimen can be calcu- lated with Eq. (8): Among the above parameters, the stiffness rate rep- resents the relative rate between the normal stiffness and shear stiffness. The surface gap represents the maximum gap interval to bond the numerical balls. The friction coefficient represents the factor that is used to calculate the friction resistance between numerical balls in the postfailure stage. The cohesion represents the cohesive strength between numerical balls before shear bond breaks. The friction angle represents the factor that is used to calculate the friction resistance between numerical balls before shear bond breaks. (8) µ = −εd εa µ = −εd εa (8) where μ is the Poisson’s ratio.hl The flat joint model was used to simulate the parti- cles’ contact behaviour. Then, the input parameters were calibrated until the numerical results fit well with the experimental results. By conducting numerical UCS tests, input param- eters, including the stiffness rate, surface gap, Chen et al. Int J Concr Struct Mater (2022) 16:61 Page 8 of 14 Then, the axial stress–strain curves of the specimen can be acquired. When the water–cement rate was 0.35, the axial stress–strain curve of the cement paste is plotted in Fig. 11a. The numerical simulation showed that the UCS, Young’s modulus and Poisson’s ratio were 64.5 MPa, 12.3 GPa and 0.22, respectively. The relative differences between the numerical simulation and experimental test were 2.2%, 4.2% and 4.3%. There was a close match between them. This validated that the input parameters were effective in analysing the compressive performance of the cement paste. Moreover, the UCS failure mode comparison when the water–cement rate was 0.35 is shown in Fig. 11b, c. In the experiment, part of the cement paste was ejected from the specimen. This phenomenon also occurred in the numerical simulation. Apparently, in the numerical simu- lation, some of the particles detached from the numeri- cal specimen. Therefore, there was a general consistency between the experimental and numerical failure modes. Then, those input parameters were substituted into the numerical STS test. 4 Numerical Simulation Results and Analysis This further confirmed the accuracy of this numer- ical STS test. There was an apparent vertical tensile failure at the middle of this specimen. Comparing it with the experi- mental failure mode, there was a close match between them. This further confirmed the accuracy of this numer- ical STS test. Chen et al. Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater Page 9 of 14 tensile cracks Fig. 13 Failure state when the water–cement rate was 0.35. 2 3 4 5 6 x 10 4 0 1000 2000 3000 4000 Timestep y tit n a u q e r u tc a r F Fig. 15 Increasing process of the fracture quantity in the specimen. Fig. 13 Failure state when the water–cement rate was 0.35. An advantage of the numerical simulation is that it can reveal the microscopic failure behaviour of the material. Since the created numerical model has been successfully validated with experimental tests, it was further used to reveal the microscopic failure of the cement paste. The fractures in the specimen were recorded. At the end of this numerical STS test, those fractures are plotted in Fig. 14. Apparently, along the vertical direction, a number of cracks were generated in the central part of the speci- men. Tensile cracks occurred at the top, middle and bot- tom sections of the specimen. More interestingly, there were also several shear cracks. This indicated that during the STS test, shear failure of the particles may also occur.h shown in Fig. 16a. Before the tensile stress reached the peak, the horizontal tensile stress showed a diamond shape. With increasing tensile stress, the area of this dia- mond shape increased, as shown in Fig. 16b. When the tensile stress reached the peak, the diamond shape of the horizontal tensile stress disappeared. Moreover, horizon- tal tensile stress mainly appeared at the central section of the specimen, as shown in Fig. 16c. With further loading, apparent tensile failure was generated at the middle sec- tion of the specimen. The majority of the specimen lost the ability to bear the tensile stress, as shown in Fig. 16d. The variation in the fracture quantity was also checked (Fig. 15). At the initial loading stage, the number of frac- tures in the specimen increased slightly. 4 Numerical Simulation Results and Analysis During this period, the bearing capacity of the specimens could only increase gently. Consequently, the initial stiffness of the specimens in the experiment was small. However, after the air voids in the specimens fully closed, the speci- mens became dense. Then, the bearing capacity of the specimens started increasing dramatically. In contrast, the numerical specimen is composed of h the experimental specimen cannot be simulated with the PFC program. Then, at the initial loading stage of the numerical specimen, the bearing capacity increased dramatically. Consequently, the initial stiffness of the experimental specimen was much smaller than that of the numerical specimen. Next, a STS test was created in the PFC. The geom- etry and specimen diameter were consistent with the experimental test. Calibration was conducted on the Fig. 16 Horizontal stress state when the water–cement rate was 0.35: a 50% tensile strength; b 80% tensile strength; c 100% tensile strength; d after tensile strength. -0.5 0 0.5 0 10 20 30 40 50 Strain (%) ) a P M ( ss e rts l ai x A Simulation Experiment (b) (c) (a) specimen failure particle failure Fig. 17 Comparison between the numerical and experimental UCS results when the water–cement rate was 0.42: a stress–strain curves; b experimental failure mode; c numerical failure mode. Fig. 16 Horizontal stress state when the water–cement rate was 0.35: a 50% tensile strength; b 80% tensile strength; c 100% tensile strength; d after tensile strength. Fig. 16 Horizontal stress state when the water–cement rate was 0.35: a 50% tensile strength; b 80% tensile strength; c 100% tensile strength; d after tensile strength. -0.5 0 0.5 0 10 20 30 40 50 Strain (%) ) a P M ( ss e rts l ai x A Simulation Experiment (b) (c) (a) specimen failure particle failure Fig. 17 Comparison between the numerical and experimental UCS results when the water–cement rate was 0.42: a stress–strain curves; b experimental failure mode; c numerical failure mode. (c) (b) specimen failure specimen failure Fig. 17 Comparison between the numerical and experimental UCS results when the water–cement rate was 0.42: a stress–strain curves; b experimental failure mode; c numerical failure mode. After the experimental UCS test was conducted, these air voids in the specimens were compressed to close. During this period, the bearing capacity of the specimens could only increase gently. 4 Numerical Simulation Results and Analysis When the time step increased to 53,410, the tensile stress of the speci- men reached the tensile strength. Meanwhile, the quan- tity of fractures arrived at 624, which was still a small value. After that, the quantity of fractures in the speci- men increased dramatically. This indicated that the sig- nificant increase in the fracture quantity started after the specimen reached the tensile strength.h Further simulation work was continued to evaluate the tensile performance of the cement paste when the water– cement rate was 0.42. Table 4 summarises the calibrated input parameters. Then, the specimen’s axial stress–strain curve can be acquired. The simulation showed that the UCS, Young’s modulus and Poisson’s ratio were 54.4 MPa, 10 GPa and 0.22. The relative differences between the numerical and experimental tests were 0.2%, 1.0% and 4.8%. There was a close match between them, as shown in Fig. 17a. The other advantage of numerical modelling is that the stress state in the specimen can be exported dynamically. For example, in this case, the horizontal stress state in the specimen when the tensile stress reached 50%, 80% and 100% tensile strength was exported (Fig. 16).i The experimental and numerical UCS failure modes are shown in Fig. 17b, c. In the experiment, part of the cement paste was ejected from the specimen. This was also monitored in the numerical simulation. Specifi- cally, a number of particles detached from the numeri- cal specimen. Therefore, these results were in reasonable agreement. In this figure, positive values represent tensile stress. Although vertical force was applied on the specimen, apparent horizontal tensile stress was generated, as Fig. 14 Crack state when the water–cement rate was 0.5. It is interesting to see that when comparing the experi- mental and numerical UCS curves, there is an apparent difference in the initial stiffness. Specifically, the initial stiffness obtained from the experiment was much smaller than that obtained from the simulation. The reason is that in the experiment, after the specimens were fully cured, there were still certain air voids in the specimens. Fig. 14 Crack state when the water–cement rate was 0.5. Chen et al. Int J Concr Struct Mater (2022) 16:61 Page 10 of 14 Chen et al. Int J Concr Struct Mater After the experimental UCS test was conducted, these air voids in the specimens were compressed to close. 4 Numerical Simulation Results and Analysis Consequently, the initial stiffness of the specimens in the experiment was small. However, after the air voids in the specimens fully closed, the speci- mens became dense. Then, the bearing capacity of the specimens started increasing dramatically. the experimental specimen cannot be simulated with the PFC program. Then, at the initial loading stage of the numerical specimen, the bearing capacity increased dramatically. Consequently, the initial stiffness of the experimental specimen was much smaller than that of the numerical specimen. Next, a STS test was created in the PFC. The geom- etry and specimen diameter were consistent with the experimental test. Calibration was conducted on the input parameter of tensile strength. When the input In contrast, the numerical specimen is composed of numerous dense particles. Therefore, the air voids in Chen et al. Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater Page 11 of 14 0 0.1 0.2 0 0.5 1 1.5 Displacement (mm) ) a P M ( ss e rts elis n e T Numerical result Experimental result Fig. 18 Numerical and experimental result comparison when the water–cement rate was 0.42. Fig. 20 Horizontal stress state when the water–cement rate was 0.42. 0 0.1 0.2 0 0.5 1 1.5 Displacement (mm) ) a P M ( ss e rts elis n e T Numerical result Experimental result Fig. 18 Numerical and experimental result comparison when the water–cement rate was 0.42. Stratabinder HS cement, when the water–cement rate was 0.35, the tensile strength was 1.9 MPa. Moreover, when the water–cement rate changed, the variation trend of the Stratabinder HS cement’s tensile strength agreed well with that of normal Port- land cement. It is known that as the water–cement rate increases, the tensile strength of normal Portland cement decreases (Hyett et al., 1992). This phenom- enon was also monitored when testing the Stratabinder HS cement. parameter of tensile strength equalled 1.98 MPa, there was a reasonable match between the physical and numerical tests (Fig. 18).h In addition, Hyett et al. (1992) indicated that when normal Portland cement was used, a low water–cement rate was likely to result in a scatter of the tensile strength results. This was also true for the Stratabinder HS cement. When the water–cement rate was 0.42, the standard error of the mean was 0.19. When the water– cement rate was 0.35, the standard error of the mean was 0.46. 4 Numerical Simulation Results and Analysis This indicated that when the water–cement rate decreased, the tensile strength of the Stratabinder HS cement became scattered. The failure mode and crack state of the specimen were also checked (Fig. 19). After the numerical STS test, apparent tensile failure was generated in the central part of the specimen. Moreover, in the specimen, both tensile and shear cracks occurred. In the simulation, at the end of the STS test, the hori- zontal stress state in the specimen was exported (Fig. 20). Since tensile failure was generated in the specimen, the majority of the specimen lost the ability to bear the ten- sile stress. It is interesting to see that when conducting the STS test on the Stratabinder HS cement, the speci- men showed different performance when two different water–cement rates were used. For the water–cement rate of 0.35, the bearing capacity of the specimen reached the final peak when the displacement was approximately 0.4 mm. Before that, an initial peak in the bearing capacity was reached. In contrast, when the water–cement rate was 0.42, only one peak in the bear- ing capacity occurred. Abbreviations l DEM: Discrete element method;; PFC: Particle flow code;; SD: Standard deviation;; SEM: Standard error of the mean;; STS: Split tensile strength;; UCS: Uniaxial compressive strength;; UTS: Uniaxial tensile strength. DEM: Discrete element method;; PFC: Particle flow code;; SD: Standard deviation;; SEM: Standard error of the mean;; STS: Split tensile strength;; UCS: Uniaxial compressive strength;; UTS: Uniaxial tensile strength. Authors’ Information Jianhang Chen—Associate professor, State Key Laboratory for Geomechanics and Deep Underground Engineering, China University of Mining and Technol- ogy (Beijing), Beijing, 100083, China. Kangming Tao—Graduate student, School of Energy and Mining Engineering, China University of Mining and Technology (Beijing), Beijing, 100083, China. Banquan Zeng—Graduate student, School of Energy and Mining Engineering, China University of Mining and Technology (Beijing), Beijing, 100083, China. Lei Liu—Graduate student, School of Energy and Mining Engineering, China University of Mining and Technology (Beijing), Beijing, 100083, China. Hongbao Zhao—Professor, School of Energy and Mining Engineering, China University of Mining and Technology (Beijing), Beijing, 100083, China. Junwen Zhang—Professor, School of Energy and Mining Engineering, China University of Mining and Technology (Beijing), Beijing, 100083, China. Danqi Li—Lecturer, WA School of Mines: Minerals, Energy and Chemical Engi- neering, Curtin University, Kalgoorlie, WA, 6430, Australia. (1) The water–cement rate significantly influenced the tensile performance of the cement paste. When the water–cement rate increased, the tensile strength of the cement paste apparently declined. (2) During the STS test, the specimens consistently failed by generating vertical cracks at the mid- dle section of the specimen. This failure mode was independent of the water–cement rate.hf (3) The DEM method was effective in simulating the failure process of the cement paste. Based on cali- bration, the stress–displacement curve and failure mode of the specimen agreed well with the experi- mental test. According to the numerical simulation, during the STS test, not only tensile cracks but also shear cracks occurred in the specimen. 7 Recommendations for Further Work 7 Recommendations for Further Work Further studies will continue to develop an effective method to conduct a direct UTS test on cement paste. Numerical simulation of the UTS test will also be con- ducted as a supplementary method to evaluate the failure behaviour and stress state of the cement paste. 6 Conclusions In this study, experimental and numerical mechanical tests were carried out on a modified Portland cement: Stratabinder HS cement. The stress–displacement, fail- ure modes and stress state of the tested specimens were recorded and compared. The following conclusions were drawn: Author Contributions Conceptualisation: JZ and DL; writing: JC, KT, BZ, LL, and HZ. All authors read and approved the final manuscript. Conceptualisation: JZ and DL; writing: JC, KT, BZ, LL, and HZ. All authors read and approved the final manuscript. 5 Discussionh The experimental STS results showed that the tensile performance of the Stratabinder HS cement generally agreed with that of normal Portland cement. For exam- ple, Rocha and Ludvig (2017) conducted STS tests on normal Portland cement. They indicated that when the water–cement rate was 0.33, the normal Portland cement showed a tensile strength of 2.13 MPa. For the Fig. 19 Failure mode and crack state when the water–cement rate was 0.42: a failure mode; b crack state. Fig. 19 Failure mode and crack state when the water–cement rate was 0.42: a failure mode; b crack state. Page 12 of 14 Page 12 of 14 Chen et al. Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater (2022) 16:61 (4) In the STS test, before the tensile stress reached the peak, the number of fractures increased slightly. However, after the peak, the crack quantity increased dramatically. During the STS test, frac- tures mainly developed in the postpeak stage. For this reason, the authors assumed that when the dis- placement reached approximately 0.2 mm, micro tensile cracks were generated along the vertical direction. This led to a sudden decrease in the bearing capacity. How- ever, since the water–cement rate was 0.35, the pieces of the specimen had relatively high strengths. Therefore, these pieces could still join together to support the exter- nal loading force. Then, as the displacement increased, the bearing capacity of the specimen rose again. (5) During the vertical STS test, apparent horizontal tensile stress occurred in the specimen. Before the tensile stress reached the tensile strength, the hori- zontal tensile stress zone showed a diamond shape. The higher the tensile stress is, the larger the area of the diamond-shaped tensile stress zone. When the tensile stress reached the peak, the horizontal ten- sile stress was mainly concentrated in the vertical centre of the specimen. After tensile strength test- ing, apparent vertical cracks occurred. Then, parti- cles in the cement paste lost the ability to bear the tensile stress. (5) During the vertical STS test, apparent horizontal tensile stress occurred in the specimen. Before the tensile stress reached the tensile strength, the hori- zontal tensile stress zone showed a diamond shape. The higher the tensile stress is, the larger the area of the diamond-shaped tensile stress zone. 5 Discussionh When the tensile stress reached the peak, the horizontal ten- sile stress was mainly concentrated in the vertical centre of the specimen. After tensile strength test- ing, apparent vertical cracks occurred. Then, parti- cles in the cement paste lost the ability to bear the tensile stress. Meanwhile, the micro tensile cracks continuously developed. Finally, when the displacement reached approximately 0.4 mm, these micro tensile cracks were connected. Consequently, macro tensile cracks were generated in the specimen. Moreover, the specimen was separated into two main parts. Before those two main parts separated thoroughly, the specimen bearing capac- ity reached the final peak. After that, the bearing capacity of the specimen was lost. In contrast, for the water–cement rate of 0.42, when the displacement reached approximately 0.2 mm, micro tensile cracks were generated in the specimen along the vertical direction. This led to a decrease in the speci- men bearing capacity. More importantly, since the water–cement rate was a high value of 0.42, the pieces of the specimen had a relatively low strength. There- fore, these pieces of the specimen were not able to join. Consequently, the specimen bearing capacity dropped continuously. References Aili, A., & Maruyama, I. (2020). Review of several experimental methods for characterization of micro- and nano-scale pores in cement-based mate- rial. International Journal of Concrete Structures and Materials, 14(1), 1–18. https://doi.org/10.1186/s40069-020-00431-y Němeček, J., Králíka, V., Šmilauera, V., Polívkab, L., & Jägerb, A. (2016). Tensile strength of hydrated cement paste phases assessed by micro-bending tests and nanoindentation. Cement and Concrete Composites, 73, 164–173. https://doi.org/10.1016/j.cemconcomp.2016.07.010 Aziz, N., Majoor, D., & Mirzaghorbanali, A. (2017). Strength properties of grout for strata reinforcement. Procedia Engineering, 191(2017), 1178–1184. https://doi.org/10.1016/j.proeng.2017.05.293 Paknahad, A., Kucko, N. W., Leeuwenburgh, S. C. G., & Sluys, L. J. (2020). Experi- mental and numerical analysis on bending and tensile failure behavior of calcium phosphate cements. Journal of the Mechanical Behavior of Biomedical Materials, 103, 1–16. https://doi.org/10.1016/j.jmbbm.2019. 103565 Bayindir, F., Akyil, M., & Bayindir, Y. Z. (2009). Effect of storage pH on compres- sive and diametral tensile strength of adhesive luting cements. Materials Research Innovations, 13(2), 124–128. https://doi.org/10.1179/143307509X 435673 Rashad, A. M., Seleem, H.E.-D.H., & Shaheen, A. F. (2014). Effect of Silica Fume and Slag on Compressive Strength and Abrasion Resistance of HVFA Concrete. International Journal of Concrete Structures and Materials, 8(1), 69–81. https://doi.org/10.1007/s40069-013-0051-2 Bitouri, Y. E., Jamin, F., & Youssoufi, M. S. E. (2017). Tensile and shear bond strength between cement paste and aggregate subjected to high temperature. Materials and Structures, 50(6), 1–9. https://doi.org/10.1617/ s11527-017-1105-8 Şahin, H. G., Biricik, Ö., & Mardani-Aghabaglou, A. (2022). Polycarboxylate- based water reducing admixture - clay compatibility; literature review. Journal of Polymer Research, 29(33), 1–19. https://doi.org/10.1007/ s10965-021-02884-5 Bybee, K. (2004). Analysis of tensile-strength test methods for oilfield cement. Journal of Petroleum Technology, 56(8), 62–64. https://doi.org/10.2118/ 0804-0062-JPT Chen, J., & Li, D. (2022). Numerical simulation of fully encapsulated rock bolts with a tri-linear constitutive relation. Tunnelling and Underground Space Technology, 120, 1–13. https://doi.org/10.1016/j.tust.2021.104265l Silvestro, L., & Gleize, P. J. P. (2020). Effect of carbon nanotubes on compres- sive, flexural and tensile strengths of Portland cement-based materials: A systematic literature review. Construction and Building Materials, 264, 1–12. https://doi.org/10.1016/j.conbuildmat.2020.120237 Chen, X., Wu, S., & Zhou, J. (2013). Influence of porosity on compressive and tensile strength of cement mortar. Construction and Building Materials, 40, 869–874. https://doi.org/10.1016/j.conbuildmat.2012.11.072 Skrzypkowski, K. (2021). An experimental investigation into the stress-strain characteristic under static and quasi-static loading for partially embed- ded rock bolts. Energies, 14(5), 1–17. https://doi.org/10.3390/en14051483 Chen, Y., Zuo, J., Liu, D., & Wang, Z. (2019). Funding National Natural Science Foundation of China (52034009, 51904302, 52174093), Opening funding by State Key Laboratory for Geomechanics and Deep Underground Engineering, CUMTB (SKLGDUEK2122), Yue Qi Distin- guished Scholar Project (800015Z1179) and Fundamental Research Funds for the Central Universities (2022YQNY08). Page 13 of 14 Chen et al. Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater Earth and Environmental Science, 267, 1–7. https://doi.org/10.1088/1755- 1315/267/2/022003 Availability of Data and Materials Earth and Environmental Science, 267, 1–7. https://doi.org/10.1088/1755- 1315/267/2/022003 All data, models, and codes generated or used during the study appear in the submitted article. Jung, S., Saraswathy, V., Karthik, S., Kathirvel, P., & Kwon, S.-J. (2018). Microstruc- ture characteristics of fly ash concrete with rice husk ash and lime stone powder. International Journal of Concrete Structures and Materials, 12(2), 279–287. https://doi.org/10.1186/s40069-018-0257-4 Author details Liu, P., Zhou, X., Qian, Q., Berto, F., & Zhou, L. (2019). Dynamic splitting tensile properties of concrete and cement mortar. Fatigue and Fracture of Engi- neering Materials and Structures, 43(4), 757–770. https://doi.org/10.1111/ ffe.13162 1 State Key Laboratory for Geomechanics and Deep Underground Engineer- ing, China University of Mining and Technology (Beijing), Beijing 100083, China. 2 School of Energy and Mining Engineering, China University of Mining and Technology (Beijing), Beijing 100083, China. 3 Energy and Chemical Engineering, WA School of Mines: Minerals, Curtin University, Kalgoorlie, WA 6430, Australia. Masoud, G., Ali, B., & Mostafa, K. (2010). Residual mechanical properties of high- strength concretes after exposure to elevated temperatures. Journal of Materials in Civil Engineering, 22(1), 59–64. https://doi.org/10.1061/(ASCE) 0899-1561(2010)22:1(59) Received: 20 February 2022 Accepted: 4 July 2022 Received: 20 February 2022 Accepted: 4 July 2022 Received: 20 February 2022 Accepted: 4 July 2022 Mishra, P. K., & Kantharia, M. (2021). Tensile strength investigation of cement mortar using Nano zinc oxide (ZnO). Materials Today: Proceedings, 47, 7178–7180. https://doi.org/10.1016/j.matpr.2021.06.402 Murmu, M., & Singh, S. P. (2014). Hydration products, morphology and microstructure of activated slag cement. International Journal of Concrete Structures and Materials, 8(1), 61–68. https://doi.org/10.1007/ s40069-013-0056-x Declarations 279–287. https://doi.org/10.1186/s40069-018-0257-4 Li, D., Masoumi, H., & Ming, C. (2021). A constitutive model for modified cable bolts exhibiting cone shaped failure mode. International Journal of Rock Mechanics and Mining Science, 145, 104855. https://doi.org/10.1016/j. ijrmms.2021.104855 References Int J Concr Struct Mater (2022) 16:61 Chen et al. Int J Concr Struct Mater (2022) 16:61 Wu, S., Ramandi, H. L., Chen, H., Crosky, A., Hagan, P. C., & Saydam, S. (2019). Mineralogically influenced stress corrosion cracking of rockbolts and cable bolts in underground mines. International Journal of Rock Mechanics and Mining Sciences, 119, 109–116. https://doi.org/10.1016/j.ijrmms.2019. 04.011 Xiong, L., & Geng, D. (2020). Scale effect of cement mortar specimens sub- jected to high temperatures using uniaxial compressive and splitting tensile tests. Archives of Civil Engineering, LXVI(3), 139–155. https://doi.org/ 10.24425/ace.2020.134389 Chen, J., Hagan, P. C., & Saydam, S. (2014). Mechanical properties of cementi- tious grout serving in fully grouted cable bolting systems. AusRock 2014: Third Australasian ground control in mining conference, Sydney, Australia, pp. 269–275. Hyett, A. J., Bawden, W. F., & Coulson, A. L. (1992). Physical and mechanical properties of normal Portland cement pertaining to fully grouted cable bolts. In: Proceedings of the International symposium on rock support, Sudbury, Canada, pp. 341–348. Rocha, V. V., & Ludvig, P. (2017). Characterization of Portland cement compos- ites prepared by a dispersion of carbon nanotubes on cement particles. In: 12th central european congress on concrete engineering, Tokaj, Hungary, pp. 195–201. References Deformation failure characteristics of coal-rock combined body under uniaxial compression: Experimental and numerical investigations. Bulletin of Engineering Geology and the Environ- ment, 78, 3449–3464. https://doi.org/10.1007/s10064-018-1336-0 Song, Z., & Zhang, J. (2022). Research on the progressive failure process and fracture mechanism of rocks with the structural evolution perspective. Journal of Structural Geology, 154, 1–12. https://doi.org/10.1016/j.jsg.2021. 104484 Chen, Y., Teng, J., Bin Sadiq, R. A., & Zhang, K. (2020). Experimental study of bolt-anchoring mechanism for bedded rock mass. International Journal of Geomechanics, 20(4), 1–12. https://doi.org/10.1061/(ASCE)GM.1943-5622. 0001561 Sun, W., & Wu, S. (2021). A study of crack initiation and source mechanism in the Brazilian test based on moment tensor. Engineering Fracture Mechan- ics, 246, 1–23. https://doi.org/10.1016/j.engfracmech.2021.107622 Chen, J., Zhao, Y., Zhao, H., Zhang, J., Zhang, C., & Li, D. (2021). Analytic study on the force transfer of full encapsulating rockbolts subjected to tensile force. International Journal of Applied Mechanics, 13(9), 1–13. https://doi. org/10.1142/S1758825121500976 Syarif, M., Sampebulu, V., & Tjaronge, M. W. (2018). Characteristic of compres- sive and tensile strength using the organic cement compare with portland cement. Case Studies in Construction Materials, 9, 1–9. https://doi. org/10.1016/j.cscm.2018.e00172 Chen, J., Liu, P., Liu, L., Zeng, B., Zhao, H., Zhang, C., Zhang, J., & Li, D. (2022). Anchorage performance of a modified cable anchor subjected to dif- ferent joint opening conditions. Construction and Building Materials, 336, 1–12. https://doi.org/10.1016/j.conbuildmat.2022.127558 Wang, S., Chen, F., Xue, Q., & Zhang, P. (2020). Splitting tensile strength of cement soil reinforced with basalt fibers. Materials, 13(14), 1–15. https:// doi.org/10.3390/ma13143110 Wang, Z., Wang, T., Wu, S., & Hao, Y. (2021). Investigation of microcracking behaviors in brittle rock using polygonal grain-based distinct method. International Journal for Numerical and Analytical Methods in Geomechan- ics, 45(13), 1871–1899. https://doi.org/10.1002/nag.3246 Fedrigo, W., Núñez, W. P., López, M. A. C., Kleinert, T. R., & Ceratti, J. A. P. (2018). A study on the resilient modulus of cement-treated mixtures of RAP and aggregates using indirect tensile, triaxial and flexural tests. Construction and Building Materials, 171, 161–169. https://doi.org/10.1016/j.conbu ildmat.2018.03.119 Wu, S., Chen, H., Ramandi, F. L., Hagan, P. C., Hebblewhite, B., Crosky, A., & Say- dam, S. (2018). Investigation of cable bolts for stress corrosion cracking failure. Construction and Building Materials, 187, 1224–1231. https://doi. org/10.1016/j.conbuildmat.2018.08.066 Gu, C., Xu, J. Y., & Meng, B. X. (2019). Effect of saturation on mechanical behav- ior of cement mortar based on 8-type tensile test. IOP Conference Series: Page 14 of 14 Chen et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W2288010236	https://europepmc.org/articles/pmc4813144?pdf=render	English	null	MicroRNAs as Biomarkers for Liver Disease and Hepatocellular Carcinoma	International journal of molecular sciences	2,016	cc-by	11,364	C. Nelson Hayes 1,2 and Kazuaki Chayama 1,2,3,* Keywords: microRNA; non-coding RNA; viral hepatitis; inﬂammation; occult HBV; hepatocellular carcinoma; ﬁbrosis; HBe antigen; α-fetoprotein; biomarker International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences C. Nelson Hayes 1,2 and Kazuaki Chayama 1,2,3,* C. Nelson Hayes 1,2 and Kazuaki Chayama 1,2,3,* 1 Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan; nelsonhayes@hiroshima-u.ac.jp 1 Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan; nelsonhayes@hiroshima-u.ac.jp 1 Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical an Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan; nelsonhayes@hiroshima-u.ac.jp 1 Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan; nelsonhayes@hiroshima-u.ac.jp 2 Liver Research Project Center, Hiroshima University, Hiroshima 734-8551, Japan 3 2 Liver Research Project Center, Hiroshima University, Hiroshima 734-8551, Japan 3 Laboratory for Digestive Diseases, Center for Genomic Medicine, RIKEN, Hiroshima 734-8551, Japan * Correspondence: chayama@hiroshima-u.ac.jp; Tel.: +81-82-257-5190; Fax: +81-82-255-6220 3 Laboratory for Digestive Diseases, Center for Genomic Medicine, RIKEN, Hirosh * Correspondence: chayama@hiroshima-u.ac.jp; Tel.: +81-82-257-5190; Fax: +81-82- Academic Editors: Nalini Santanam and William Chi-shing Cho Received: 19 January 2016; Accepted: 19 February 2016; Published: 24 February 2016 Academic Editors: Nalini Santanam and William Chi-shing Cho Received: 19 January 2016; Accepted: 19 February 2016; Published: 24 February 2016 Abstract: Serum levels of liver enzymes, such as alanine transaminase, aspartate transaminase, and α-fetoprotein, provide insight into liver function and are used during treatment of liver disease, but such information is limited. In the case of hepatocellular carcinoma (HCC), which is often not detected until an advanced stage, more sensitive biomarkers may help to achieve earlier detection. Serum also contains microRNAs, a class of small non-coding RNAs that play an important role in regulating gene expression. miR-122 is speciﬁc to the liver and correlates strongly with liver enzyme levels and necroinﬂammatory activity, and other microRNAs are correlated with the degree of ﬁbrosis. miR-122 has also been found to be required for hepatitis C virus (HCV) infection, whereas other microRNAs have been shown to play antiviral roles. miR-125a-5p and miR-1231 have been shown to directly target hepatitis B virus (HBV) transcripts, and others are up- or down-regulated in infected individuals. MicroRNA proﬁles also differ in the case of HBV and HCV infection as well as between HBeAg-positive and negative patients, and in patients with occult versus active HBV infection. In such patients, monitoring of changes in microRNA proﬁles might provide earlier warning of neoplastic changes preceding HCC. MicroRNAs as Biomarkers for Liver Disease and Hepatocellular Carcinoma C. Nelson Hayes 1,2 and Kazuaki Chayama 1,2,3,* www.mdpi.com/journal/ijms 1. Introduction Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancers and is the ﬁfth most common cancer worldwide, as well as the third most common cause of cancer-related death [1,2]. Progression of chronic hepatitis to cirrhosis or HCC may occur slowly over several decades, but by the time the cancer is detected, prospects for successful treatment are often poor. Viral hepatitis is responsible for most cases of HCC, with a worldwide incidence of about 54% for hepatitis B virus (HBV) infection and 31% for hepatitis C virus (HCV) [3]. Direct acting antiviral (DAA) therapy, with agents such as sofosbuvir and ledipasvir, is expected to substantially reduce mortality and morbidity among the 185 million people throughout the world chronically infected with HCV, although, the long-term risk of HCC remains high in cirrhotic patients even after achieving sustained virological response (SVR) [4]. No comparable success has been achieved for the 360 million people with chronic HBV infection. Although an effective vaccine has been available since 1986 and most adult infections are successfully resolved during the acute phase, chronic HBV infection remains a serious public health threat requiring long term antiviral therapy with a low probability of complete clearance of the virus Int. J. Mol. Sci. 2016, 17, 280; doi:10.3390/ijms17030280 www.mdpi.com/journal/ijms 2 of 17 2 of 17 Int. J. Mol. Sci. 2016, 17, 280 due to the long-term stability of viral covalently closed circular DNA (cccDNA) in the nucleus. In these patients, gradual changes and deterioration in liver function must be monitored over long periods. While serum levels of liver enzymes, such as alanine transaminase (ALT), aspartate transaminase (AST), and α-fetoprotein (AFP), provide insight into liver function, enzyme levels convey limited information, and HCC is often not detected until an advanced stage. Therefore, more sensitive biomarkers may help to achieve earlier detection in patients with chronic hepatitis, and serum microRNAs represent a promising approach to monitoring liver function. 2. MicroRNAs MicroRNAs are short, non-coding RNAs consisting of 18–25 nucleotides that are partially complementary to regulatory regions in the 31 or, less commonly, in the 51 untranslated region (UTR) of target messenger RNAs. MicroRNA binding suppresses translation of target mRNAs or promotes mRNA degradation, providing a rapid and sensitive mechanism to ﬁne tune gene expression. MicroRNAs serve as guides to position the RNA-induced silencing complex (RISC), a molecular scaffold that facilitates interaction of a microRNA with its target sequence and mediates the inhibitory effect on gene expression [5]. MicroRNAs form complex post-transcriptional regulatory networks that regulate numerous cellular processes [6]. Although not discovered until 1993, microRNAs are now known to affect the expression of at least 30% of human genes, making them the most abundant regulators of gene expression [7]. Around 60% of messenger RNAs (mRNAs) contain a predicted binding site in the 31 UTR, and many mRNAs contain multiple potential binding sites. However, relatively few of the computationally-predicted microRNA–mRNA interactions have been experimentally validated. 2588 mature human microRNAs and 1881 precursors are currently registered in the miRBase database (Release 21; June 2014). A single microRNA is likely to regulate multiple genes, and conversely a single gene might be regulated by multiple microRNAs. MicroRNA expression may also be tissue- or organ-speciﬁc. Since even a small change in microRNA expression may affect expression of hundreds of target genes and signiﬁcantly alter the transcriptome [8], disruption of microRNA regulatory networks has been implicated in a number of diseases [9–11]. MicroRNA involvement in cancer was ﬁrst reported in 2002 for its role in leukemia [12]. While the predictive utility might vary among individual microRNAs, expression proﬁles involving multiple microRNAs are expected to play a useful role in tumor classiﬁcation, diagnosis, and prognosis. 3. Production of MicroRNAs MicroRNA genes, often located in the introns of protein-coding genes, are transcribed by RNA polymerase II into long, capped, polyadenylated pri-microRNAs and subsequently processed by Drosha into pre-microRNAs [13] (Figure 1). Pre-microRNA hairpins are exported from the nucleus by exportin-5 and processed into double-stranded mature microRNAs by Dicer. When the microRNA has been incorporated into the RISC complex, the unused complementary strand is degraded. The microRNA serves as a guide to orient the RISC complex in position at regulatory sequences in target genes. While argonaute 2 (AGO2) can directly cleave messenger RNA, particularly in the case of perfect complementarity, argonaute proteins normally facilitate translational inhibition by reducing RNA stability through uncapping and de-adenylation. 3 of 17 Int. J. Mol. Sci. 2016, 17, 280 Figure 1. MicroRNA synthesis and function. MicroRNA genes are transcribed by RNA polymerase II to produce primary microRNAs (pri-microRNAs) and cleaved by Drosha/DGCR8 into pre-microRNAs. Pre-microRNAs are exported from the nucleus by exportin-5 and Ran-GTP, and then the hairpin is cleaved by Dicer/TRBP in the cytoplasm. The microRNA duplex is unwound and one strand is complexed with argonuate 2 (AGO2) to form the RNA-induced silencing complex (RISC), and the unused passenger strand (miRNA) is degraded. The microRNA guides the RISC to a partially complementary target region in the 31 untranslated regions of one or more genes, resulting in translational repression or target cleavage. Distinct proteins or protein complexes are depicted as colored ovals. Red and blue lines indicate RNA. Figure 1. MicroRNA synthesis and function. MicroRNA genes are transcribed by RNA polymerase II to produce primary microRNAs (pri-microRNAs) and cleaved by Drosha/DGCR8 into pre-microRNAs. Pre-microRNAs are exported from the nucleus by exportin-5 and Ran-GTP, and then the hairpin is cleaved by Dicer/TRBP in the cytoplasm. The microRNA duplex is unwound and one strand is complexed with argonuate 2 (AGO2) to form the RNA-induced silencing complex (RISC), and the unused passenger strand (miRNA) is degraded. The microRNA guides the RISC to a partially complementary target region in the 31 untranslated regions of one or more genes, resulting in translational repression or target cleavage. Distinct proteins or protein complexes are depicted as colored ovals. Red and blue lines indicate RNA. 4. Serum MicroRNAs Enzymes such as ALT and γ-glutamyl transpeptidase (GGT) are considered liver-speciﬁc (although ALT may be elevated with kidney or muscle damage), but other enzymes commonly used in liver function tests such as AST and alkaline phosphatase are also expressed in muscle and bone, respectively, limiting their speciﬁcity and requiring more complex interpretation. Conversely, many microRNAs are expressed in a tissue- or organ-speciﬁc manner, suggesting that microRNA biomarkers are more likely to have high speciﬁcity. The presence of microRNAs in serum and their potential use as biomarkers was ﬁrst reported by Lawrie et al. [14] in 2008 with regard to their role in diffuse large B-cell lymphoma, and the idea has since been pursued in a number of studies. Analysis of differential microRNA expression in liver tissues has identiﬁed microRNAs associated with different stages of liver disease, as well as speciﬁc microRNA subsets associated with HCV versus HBV infection [15–19]. However, direct measurement of tissue microRNAs is intrusive and inconvenient as a biomarker. On the other hand, measurement of serum microRNAs is much less intrusive, and microRNA levels in the liver have been found to be correlated with serum levels for a number of microRNAs [20,21]. MicroRNAs from the liver may enter the serum passively through apoptosis and necrosis or actively through secretion of exosomes and viral particles [22]. Therefore, microRNA levels in the serum might provide a way to estimate microRNA activity in the liver. Comparison of serum microRNA levels before and after tumor resection suggests that circulating microRNAs such as miR-15b, miR-21, miR-130b, and miR-183 originate in tumor cells [23]. Fortunately, microRNA 4 of 17 Int. J. Mol. Sci. 2016, 17, 280 is detectable and relatively stable in serum, plasma, urine, saliva, and cerebrospinal ﬂuid [24], and frozen samples can be stored without substantial degradation. Free RNA is quickly degraded by RNases and, typically, has a short half-life. Conversely, mature microRNAs are much more stable and are normally complexed with AGO2 or other argonaute proteins [5]. Circulating microRNAs may exist as vesicle-free ribonucleoprotein complexes or may be transported within HBV surface antigen (HBsAg) particles or contained within exosomes/microvesicles [5,22,25], although serum microRNAs are typically found in exosomes [26]. 5. Exosomes Exosomes are ubiquitous cell-derived vesicles ranging from 30 to 100 nm that have been shown to affect gene expression in recipient cells. Exosomes contain characteristic RNA transcripts, including microRNAs, transfer RNAs, and other types of non-coding RNAs [27], which may vary by cell type but do not necessarily mirror the RNA proﬁle of the parent cell due to selective sorting and response to cellular conditions [27]. miR-99a, miR-128, miR-124, miR-22, and miR-99b account for 49% of identiﬁed exosome-associated microRNAs [27]. Hepatocyte-derived exosomes are enriched for gene products involved in lipoprotein metabolism and xenobiotic processing and might, therefore, prove useful as a diagnostic tool by reﬂecting hepatic changes linked to disease [28]. Although interferon normally acts on cells directly by binding to receptors and triggering expression of numerous interferon-stimulated genes sharing a common response element, viruses, such as HBV and HCV, frequently interfere with interferon signaling and suppress intracellular innate immune responses. An alternative antiviral mechanism was recently described in which interferon stimulates release of exosomes that contain antiviral products, which are then internalized by HBV-infected hepatocytes, bypassing viral interference [29]. The role of exosomes in disease pathogenesis and cancer metastasis has yet to be fully elucidated. 6. Measurement of Serum MicroRNAs To serve as a reliable biomarker, standardized methods for serum microRNA measurement are needed. Many methods exist for isolating RNA from serum but accurate measurement of serum microRNAs is challenging due to the low quantity, short length, and high sequence variability of microRNAs. Initially, RNA is extracted, and the small RNA fraction is enriched. Bulk measurement of microRNA expression can be performed using microarray analysis or next generation RNA sequencing, but validation and absolute quantiﬁcation of individual microRNAs is typically performed using quantitative real-time polymerase chain reaction [30]. However, microRNA analysis requires additional quality control and normalization steps that remain unstandardized [31]. Selection of an appropriate internal control is difﬁcult due to high variability of small RNAs such as RNU4, RNU6b, and RNU48 among healthy controls [32] or confounding with disease state, such as down-regulation of RNU6b during ﬁbrosis [33]. Similarly microRNAs such miR-15b and miR-16 are sensitive to hemolysis [34]. While the choice of internal control may depend on the nature of the study, miR-24, miR-126, and miR-484 have been suggested for use in normalization [32]. Differentiating among variable-length isomiRs and closely-related microRNAs in the same family as well as between mature microRNAs and pre-/pri-microRNAs poses an additional challenge. In some cases, the status of a non-coding RNA as a microRNA has been called into question. While miR-720 has recently been reported to target the oncogene twist family BHLH transcription factor 1 (TWIST1) involved in tumor metastasis in breast cancer [35], the microRNA entry for miR-720 was removed from miRBase after Schopman et al. [36] proposed that the microRNA was actually a mis-annotated tRNA fragment. Examination of miRBase using next generation sequencing data has revealed numerous other such potential conﬂicts, frequently involving mis-annotation of small nucleolar RNAs (snoRNAs) [37]. Although these RNAs are not microRNAs, recent studies have shown that tRNA fragments and other small non-coding RNA species may serve secondary regulatory or signaling functions and that their presence in serum may vary depending on physiological state [38,39]. Selitsky et al. [40] showed that tRNA fragments are more 5 of 17 Int. J. Mol. Sci. 2016, 17, 280 abundant than microRNAs in liver tissue and were signiﬁcantly elevated in patients with chronic HBV or HCV compared to healthy subjects, suggesting a potential role for other types of non-coding RNA for use as biomarkers. 8.1. Autoimmune Liver Diseases Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are chronic autoimmune diseases characterized by immune-directed damage to hepatocytes and cholangiocytes, respectively. Diagnosis of autoimmune liver diseases requires differential diagnosis involving clinical and histological ﬁndings and exclusion of other factors. A recent study by Migita et al. [47] found elevated serum levels of miR-21 miR-122 in patients with AIH compared to heathy controls or patients with HCV, but levels of these microRNAs decreased in patients with cirrhosis. Tan et al. [48] showed that a panel including miR-122-5p, miR-141-3p, and miR-26b-5p was diagnostic for PBC with higher sensitivity and speciﬁcity than traditional biomarkers, such as alkaline phosphatase (ALP) and antinuclear antibody (ANA). Ideally, microRNA panels such as these will lead to earlier and more accurate diagnosis in patients with autoimmune diseases. 7. Baseline MicroRNA Expression in the Liver Baseline microRNA levels in hepatocytes and liver tissue have been established using deep sequencing methods [41–43]. While miR-122 accounts for 70% of the total microRNA in the liver [44], a set of 9 microRNAs including miR-192, miR-199a/b, miR-101, miR-99a, and let-7a/b/c/f accounts for 88% of total microRNA [42]. Genome-wide expression proﬁling identiﬁed a total of 277 microRNAs expressed in the liver, 166 of which were expressed in all samples, including miR-16, miR-27b, miR-30d, miR-126, and several members of the let-7 family [43]. Baseline miR-122 expression is also strongly affected by single nucleotide polymorphisms (SNPs) (rs2999200 and rs6551952), and other SNPs have been shown to affect expression of miR-938, miR-200c, and miR-10b [43]. Some microRNAs show age-dependent differences in expression. 114 microRNAs were up-regulated and 72 were down-regulated in fetal versus pediatric liver tissues, whereas only two microRNAs were up-regulated and three were down-regulated in pediatric versus adult tissue. Sex-speciﬁc differences in microRNA expression have also been observed [45]. miR-29a and miR-29b are induced by estrogen and up-regulated in females [46]. These microRNAs suppress collagen deposition in the extracellular matrix, potentially providing better protection against ﬁbrosis in females. Consideration of factors affecting baseline microRNA expression in the liver may be important in the interpretation of microRNA biomarkers. Validation studies should consider stratifying by age and/or sex and might consider genotyping SNPs of large effect. 8. The Role of MicroRNAs in Liver Injury and Disease MicroRNAs are involved in regulation of numerous metabolic pathways, and changes in serum microRNA proﬁles might reﬂect underlying liver injury or inﬂammation. 6. Measurement of Serum MicroRNAs While these will likely remain open issues until the non-coding RNA transcriptome is more fully characterized, the methodology for serum microRNA measurement should standardize once effective microRNA panels have been validated and interest in their use in clinical practice increases in the same way that protocols for performing and reporting genome-wide association studies (GWAS) have become more standardized. 8.3. Inﬂammatory Liver Damage In contrast, a different pattern was observed for inﬂammatory liver damage resulting from alcoholic liver disease (ALD) and Toll-like receptor (TLR) activation [50]. miR-122 and miR-155 were up-regulated in serum of alcohol-fed mice, and miR-122, miR-155, and miR-146a were up-regulated following inﬂammation induced by TLR4/TLR9 ligand administration. Bala et al. [50] reported that circulating microRNAs in the exosome-rich fraction versus the protein-rich fraction could discriminate between liver injury and inﬂammation. 8.2. Drug-Induced Liver Injury Drug-induced liver injury (DILI) results from drug overdose or the hepatotoxic effects of a drug metabolite. Early diagnosis of DILI is important, especially in the case of idiopathic DILI where the underlying cause is difﬁcult to determine, but standard serum biomarkers such as ALT, AST, bilirubin, and alkaline phosphatase have poor sensitivity and speciﬁcity, and more accurate biomarkers are needed [49]. In mouse models of acetaminophen-induced liver injury, miR-122 was found to be up-regulated in liver tissue, and miR-122, miR-192, miR-155, miR-125b, and miR-146a Int. J. Mol. Sci. 2016, 17, 280 6 of 17 were up-regulated in serum or plasma [16,50,51]. However, Wang et al. [51] reported an inverse correlation between microRNAs levels in the liver versus the plasma, in which elevated plasma levels corresponded with lower levels in liver tissue, and vice versa. Fukushima et al. [52] reported that miR-298 and miR-370 were down-regulated in rats within six hours after exposure to acetaminophen or carbon tetrachloride and showed that altered microRNA expression levels were associated with mitochondrial damage. While the number of human microRNA studies of DILI is limited, Starkey Lewis et al. [53] found that patients who experienced acetaminophen-related DILI had higher plasma levels of miR-122 and miR-192, and Ding et al. [54] found that serum miR-122 levels were up-regulated in subjects exposed to paraquat, a toxic herbicide. 8.4. Alcoholic Liver Disease Aside from the liver, alcohol abuse damages multiple organs, including the pancreas, intestinal epithelium, heart, brain, and muscle tissue, but microRNA dysregulation is thought to play a key role in alcoholic liver disease. Mouse models of ALD have shown increased tissue expression of miR-320, miR-486, miR-705, and miR-1224, and decreased expression of miR-27b, miR-214, miR-199a-3p, miR-182, miR-183, miR-200a, and miR-322 [55]. Rat models have shown elevated serum levels of miR-122 and miR-155 in exosomes following alcohol consumption [50]. Due to the systemic effects of alcohol, however, circulating microRNA biomarkers might not originate exclusively from the liver [31]. Chen et al. [56] found up- or down-regulation of 32 serum microRNAs in rats, but expression levels between the liver and serum were correlated only for miR-185, miR-199a-3p, miR-214, and miR-490. 8.5. Non-Alcoholic Fatty Liver Disease Excess fat accumulation in the liver also induces inﬂammation and can result in non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). NAFLD is becoming increasingly common, affecting up to 40% of the population, but because proper diagnosis requires a biopsy, incidence is likely to be under-reported and, therefore, less invasive biomarkers are needed for earlier detection [57,58]. In mouse models of obesity, miR-122, miR-34a, miR-31, miR-103, miR-107, miR-194, miR-334-5p, miR-221, and miR-200a were up-regulated, and miR-29c, miR-451, and miR-21 were down-regulated in ob/ob mice [59,60]. miR-146, miR-152, and miR-200 were up-regulated in rats fed a high-fat diet [61]. In human studies, miR-21 was found to be up-regulated in patients with steatohepatitis [62], and 23 microRNAs, including miR-122, were up-regulated and 23 were down-regulated in patients with NASH, with predicted effects on apoptosis, inﬂammation, oxidative stress, and lipid metabolism [62]. In serum, levels of miR-122, miR-34a, and miR-16 were up-regulated in patients with NAFLD compared to healthy controls [63]. Using global microRNA proﬁling, Pirola et al. [64] analyzed 84 serum microRNAs and developed a proﬁle based on up-regulation of miR-122, miR-192, miR-19a, miR-19b, miR-125b, and miR-375. A similar study by Tan et al. [48] using Illumina sequencing identiﬁed a diagnostic panel of microRNAs associated with NAFLD that included miR-122-5p, miR-1290, miR-27b-3p, and miR-192-5p. 9.1. Hepatitis B Virus Zhang et al. [70] used antisense screening in HepG2 to identify host microRNAs involved in HBV replication and found that miR-199a-3p suppressed HBV replication by directly binding to the S protein coding region, whereas miR-210 inhibited replication by binding to the pre-S1 region. Potenza et al. [71] similarly found that miR-125a-5p inhibits translation of the S transcript. Additional microRNAs, including let-7, miR-196b, miR-433, miR-511, miR-205, and miR-345 were predicted to recognize targets in the HBV genome [72]. MiR-1231 has high homology with the core and X regions of the HBV genome and is predicted to hybridize with this region [73]. Overexpression of miR-1231 signiﬁcantly suppressed HBV replication but did not affect expression of interferon-stimulated genes [73]. The highly-expressed liver microRNA miR-122 was found to directly target the viral genome in the region of the HBV polymerase overlapping the 31 UTR of the core protein [74]. This key microRNA strongly suppresses HBV replication both through direct binding to HBV RNA, as well as indirectly through cyclin G1-modulated p53 activity [74–78]. Other microRNAs that affect HBV replication indirectly through regulation of host proteins include miR-99a, which acts as a tumor suppressor that targets insulin-like growth factor 1 receptor (IGF-1R) and induces cell cycle arrest [27,79]. MiR-99a also suppresses activity of nuclear factor κB (NF-κB), a transcription factor associated with inﬂammation and tumorigenesis [80]. miR-22 acts as a tumor suppressor [27] and induces cellular senescence by regulating cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase 6 (CDK6), sirtuin 1 (SIRT1), and speciﬁcity protein 1 (Sp1) [81,82]. miR-22 has been shown to be down-regulated in HBV-related HCC [82]. MiR-141 down-regulates peroxisome proliferator-activated receptor α (PPARα), a liver-enriched nuclear receptor required for efﬁcient transcription of the HBV genome [83]. Conversely, other microRNAs promote HBV replication. miR-1 increases expression of farnesoid X receptor α (FXRα), another nuclear receptor involved in HBV transcription [84]. miR-501 down-regulates hepatitis B virus X interacting protein (HBXIP), which acts as an HBV inhibitor [85]. While several viruses encode their own microRNAs, no virally-encoded microRNAs have yet been conﬁrmed for RNA although, interestingly, Jin et al. [86] identiﬁed a potential pre-microRNA sequence present in the HBV genome for which the only predicted target was the HBV genome itself. A number of studies have shown characteristic serum microRNA proﬁles in patients with chronic HBV infection compared to healthy control subjects. Using Solexa screening followed by validation with quantitative reverse transcription PCR, Li et al. 9. The Role of MicroRNAs in Viral Hepatitis MicroRNAs play important roles in HBV and HCV infection both through regulation of host genes as well as by direct targeting of viral transcripts. Both viruses also disrupt gene expression in the host cell, including microRNA regulatory networks, in order to establish a more permissive environment for viral replication [69]. Several microRNAs might also serve a therapeutic role. 8.6. Fibrosis Liver ﬁbrosis is a consequence of repeated cycles of liver damage and repair, resulting in excessive deposition of extracellular matrix proteins [65,66]. While various factors, such as inﬂammation, Int. J. Mol. Sci. 2016, 17, 280 7 of 17 oxidative stress, and apoptosis can activate hepatic stellate cells, ﬁbrosis underlies most types of chronic liver disease and is a precursor to cirrhosis and HCC [67]. Within the liver, several microRNAs including miR-21, miR-221/222, and miR-181b promote liver ﬁbrosis through the TGF-β or NF-κB pathways, whereas miR-29b, miR-101, miR-122, and miR-214-3p prevent ﬁbrosis by inhibiting collagen synthesis or suppressing activation of the TGF-β pathway [67]. Serum miR-34a was found to be up-regulated in patients with ﬁbrosis in a stage-dependent manner [63], and miR-571 and miR-513-3p were elevated in patients with cirrhosis [46]. miR-29a was down-regulated in patients with ﬁbrosis in an inversely stage-dependent manner [68]. Due to the central role of ﬁbrosis in liver disease, better understanding of microRNA regulation of inﬂammation and hepatic stellate cell activation might lead to new therapeutic approaches. Along these lines, anti-ﬁbrotic microRNA therapy involving activation of miR-29 or miR-101 or inhibition of miR-21 has been considered [65]. 9.1. Hepatitis B Virus [87] identiﬁed a set of 13 microRNAs that were up-regulated at least 3-fold in serum of patients with HBV infection, including miR-375, miR-92a, miR-10a, miR-223, miR-423, miR-23b/a, miR-342-3p, miR-99a, miR-122a, miR-125b, miR-150, and Int. J. Mol. Sci. 2016, 17, 280 8 of 17 let-7c. This set was also able to discriminate between HBV and HCV cases and HBV and HBV-HCC cases. Serum miR-122 levels have been shown to correlate with ALT levels as well as serum levels of miR-22, HBV DNA, and HBsAg [50,88]. Other studies have reported up-regulation of miR-99a and miR-125b in HBV-infected patients [20,89,90], and serum microRNA levels have been shown to be up-regulated in HBeAg-positive patients compared HBeAg-negative patients [89]. p g g p p p g g p The relationship between microRNAs and HBV is complex. HBV down-regulates expression of Drosha, part of the microprocessor complex responsible for converting pri-microRNA into pre-microRNA hairpins during the ﬁrst stage of microRNA processing. Down-regulation of Drosha might, therefore, have a globally suppressive effect on microRNA expression levels [91]. Novellino et al. [22] demonstrated through immunoprecipitation that microRNAs are contained within HBsAg subviral particles, which are produced in excess in infected hepatocytes. As several microRNAs are known to target HBV, knockdown of AGO2 might be expected to relieve inhibition of HBV replication. Instead, however, HBV DNA and HBs antigen levels decreased following siRNA-mediated AGO2 knockdown [20]. HBcAg and AGO2 were found to physically interact and co-localize in the endoplasmic reticulum, and HBs and AGO2 were found to co-localize in several subcellular compartments. These results suggest that AGO2 might play a role in the HBV life cycle. 9.2. Hepatitis C Virus HCV infection disrupts microRNA regulation of multiple pathways, including immune response, antigen presentation, cell cycle, proteasome, and lipid metabolism [19]. At least nine gene targets (peroxisome proliferator-activated receptor γ (PPARG), signal transducer and activator of transcription 3 (STAT3), interferon regulatory factor 1 (IRF1), insulin-like growth factor 1 receptor (IGF1R), ﬁbronectin 1 (FN1), stearoyl-CoA desaturase (SCD), and cAMP responsive element binding protein 1 (CREB1)), regulated by at least 11 microRNAs (miR-130a/b, miR-200, miR34a, miR-23b, miR-24, miR-146a, miR-381, miR-25, miR-200a, and miR-371-5p) were altered as a result of HCV infection [92]. Similarly, Peng et al. [69] used a graph theoretical approach to identify 38 microRNA-mRNA regulatory modules associated with HCV infection. The host immune response employs microRNAs both to control expression of target genes as well as to directly target the HCV genome [8,69,93]. HCV replication is highly dependent on miR-122, which binds to and helps to stabilize the 51 UTR (S1 and S2) and the 31 UTR (S3) of the HCV RNA genome [94,95]. Sequestration of this microRNA dramatically reduces HCV RNA abundance [94,95]. Miravirsen, a 15 nucleotide locked nucleic acid (LNA) that binds to miR-122 and inhibits its function is undergoing clinical trials as an anti-HCV therapy [96]. MiR-141, miR-192, miR-215, and miR-491 also promote HCV replication. miR-141 promotes HCV replication by down-regulating tumor suppressor deleted in liver cancer 1 (DLC-1) [96], and miR-491 facilitates HCV entry through the PI3 kinase/Akt pathway [97]. While these microRNAs are required or exploited by HCV for replication, other microRNAs including miR-199a, miR-196, miR-29, let-7b, miR-130a, and miR-27a show antiviral activity against HCV [98–103]. Murakami et al. [98] demonstrated that miR-199a inhibits viral replication by binding to a complementary target sequence within domain II of the internal ribosomal entry site (IRES). While interferon is normally associated with up-regulation of interferon-stimulated genes, Pederson et al. [93] showed that interferon-β induces expression of several microRNAs with antiviral activity against HCV, including miR-196, miR296, miR-351, miR-431, and miR-448, of which miR-196 and miR-448 may directly target HCV RNA. 10. Hepatocellular Carcinoma In addition to being the most common form of primary liver cancer, HCC is the ﬁfth most common cancer worldwide and the third most common cause of cancer-related death, responsible for 500,000 deaths per year [1]. Incidence is highest in East Asia and other regions where hepatitis B virus is endemic. Multiple risk factors have been implicated, including chronic HBV or HCV infection, gender, age, alcohol, and aﬂatoxin B exposure. Given these diverse etiologies, HCC is, accordingly, a complex disease characterized by stepwise accumulation of genetic and epigenetic changes, including mutations, Int. J. Mol. Sci. 2016, 17, 280 9 of 17 translocations, ampliﬁcations or deletions, chromatin remodeling, changes in DNA methylation, and dysregulation of non-coding RNAs [104–106]. The ﬁve year survival rate is less than 10%, due in part to resistance to chemotherapy and a high recurrence rate. Resection offers the best chance of curative therapy for HCC, but only 10% of patients are eligible at the time of detection. However, the survival rate following resection approaches 70% when the tumor is single and smaller than 2 cm. Therefore, earlier diagnosis is essential in order to improve prognosis. AFP (>400 ng/mL) is the most commonly used biomarker for HCC, but has only modest sensitivity and accuracy and fails to detect HCC in half of patients. Another marker, des-γ-carboxy prothrombin (DCP), also known as prothrombin induced by vitamin K absence-II (PIVKA-II), is an abnormal, nonfunctional form of prothrombin that does not undergo N-terminal carboxylation prior to secretion [107]. The carboxylase responsible is frequently lacking in HCC cells. In combination with AFP and AFP-L3, these biomarkers are predictive of progression of HCC in patients with chronic HBV or HCV, particularly with respect to portal vein invasion or intrahepatic metastasis [107,108]. However, DCP elevation may be due to other causes, and normal DCP does not preclude HCC. Therefore, additional biomarkers are needed, especially those associated with early changes to improve prognosis. MicroRNA dysregulation is involved in all stages of hepatocarcinogenesis, and microRNA profiles have the potential to discriminate patients with HCC from healthy subjects as well as those with other liver diseases [27,109]. MicroRNA profiles differ between benign and malignant tissue and may vary by malignant subtype [7]. Detectable in liver tumor tissue, serum, plasma, and urine, microRNAs might also provide a minimally invasive way to monitor response to therapy and establish prognosis. A number of studies have reported microRNAs associated with HCC. 10. Hepatocellular Carcinoma 2016, 17, 280 10 of 17 samples [116]. Using serum samples, Li et al. [87] reported that the combination of miR-25, mR-375, and let-7f could discriminate HBV-associated HCC samples from healthy controls, whereas the combination of miR-16, miR-195, and miR-199a could discriminate HBV-associated HCC from chronic HBV infection. In a retrospective study, Liu et al. [23] reported that miR-15b, miR-21, miR-130b, and miR-183 were up-regulated in HCC tumor tissue relative to adjacent non-tumor tissue. Levels of these microRNAs were detectable in serum and cell culture supernatant, and serum levels declined signiﬁcantly following surgical resection. In a validation study, they found that the combination of miR-15b and miR-130b was strongly predictive of HCC (AUC 0.98) and could detect HCC earlier than AFP [23]. The combination of miR-15b and miR-130 could also discriminate HCC from healthy samples with high sensitivity and speciﬁcity. Similarly, Lin et al. [117] identiﬁed a set of 19 microRNAs that were up-regulated in serum of patients with HBV-related HCC compared to patients with chronic HBV. Using a training cohort and two independent validation cohorts, they developed and validated a seven-microRNA panel including miR-29a, miR-29c, miR-133a, miR-143, miR-145, miR-192, and miR-505. This panel was more sensitive than AFP and could detect small AFP-negative HCC samples, providing hope for earlier detection of HCC. While microRNAs have great potential as a biomarker for HCC, there is no consensus yet on optimal microRNA panels or detection methods [7]. A potential limitation of studies to date involves the initial screening of candidate microRNAs using microRNA microarrays, which typically contain only a limited set of probes that may not include recently-identiﬁed microRNAs nor adequately prevent cross-hybridization with unrepresented microRNAs [57]. The increasing use of global transcriptome sequencing should offer a more unbiased approach to identiﬁcation of candidate RNAs. In addition to their potential roles in diagnosis and classiﬁcation, microRNAs might also provide a way to monitor response to therapy as well as serve as drug targets. For example, low tumor miR-26 levels is associated with better response to interferon therapy, but is also associated with poorer survival [118], and miR-29a-5p is associated with early post-resection recurrence of HCC in patients with HBV [119]. MicroRNAs themselves might also serve as therapeutic agents. In an interesting study, Kourtidis et al. [120] note that microRNAs such as miR-30b normally suppress cell growth when cells come into contact but lose control when adhesion is disrupted in cancer. 10. Hepatocellular Carcinoma They showed that, while adhesion proteins normally interact with components of the microprocessor complex in the cytoplasm, this structure was lost in most of the tumors they examined. However, restoration of miR-30b levels reversed the abnormal cell growth. While targeted delivery and expression of therapeutic microRNAs will likely present a challenge, greater understanding of the roles of microRNAs and other non-coding RNAs may lead to more effective and better tolerated treatments for HCC and other cancers. 10. Hepatocellular Carcinoma miR-17-92, miR-21, miR-221, miR-222, miR-224 are frequently up-regulated in HCC tumors [109,110], whereas let-7, miR-200, miR-29, miR-122, miR-123, miR-199a, miR-199b, are often down-regulated [7,109,111]. While miR-122 is down-regulated in primary HCC tumors, serum miR-122 is up-regulated in patients with HCC [112], possibly due to miR-122 release from tumors into circulation. miR-199 is highly expressed in normal liver tissue but is consistently down-regulated in HCC [42]. Since miR-199a/b-3p suppresses HCC in part by inhibiting the p21-activated kinase 4 (PAK4)/Raf/MEK/ERL pathway, down-regulation of miR-199a/b is associated with poor survival. Similarly, miR-99a down-regulation is associated with poor prognosis [79]. Conversely, miR-224 has been reported to be up-regulated in HCC [113] and was recently shown to reflect tumor stage and liver function, with elevated levels associated with reduced survival [114]. Etiology-related differences in microRNA expression may complicate efforts to develop effective biomarker panels due to geographic differences in the underlying causes of HCC. In a recent study, microRNA expression proﬁling of liver tissue was used to identify dysregulated HBV- or HCV-HCC-associated microRNAs [115]. Of the 40 differentially expressed microRNAs, 10 were strongly dysregulated. Of these, six were validated in tissue samples, but only miR-126 and miR-142-3p were elevated in plasma in HBV patients with HCC compared to patients without HCC. Neither performed better than AFP alone, but the combination of either microRNA with AFP improved the AUC to 0.92. No difference in miR-126 levels in patients with HCV-related HCC or non-viral HCC was detected, suggesting that differences in the underlying etiology of HCC may affect the predictive power of microRNA biomarkers. While HBV is the most common cause of HCC in Asia and other HBV-endemic regions, HCV-related HCC is more common in Japan, and the world-wide incidence of HBV is gradually declining due to vaccination, whereas incidence of non-viral, non-alcoholic HCC due to non-alcoholic steatohepatitis and other causes is increasing [74]. Therefore, biomarkers developed and validated in one region should be validated in regions in with different underlying etiologies. The predictive effects of various microRNAs in combination have been evaluated in a number of studies, although so far there has been little overlap among panels, partly due to differences in the source and measurement of microRNAs, making comparison difﬁcult. The combination of miR-126 and miR-141 measured in tumor samples was reported to be discriminative between HCC and metastatic adenocarcinoma of the liver, making them potentially useful diagnostic markers for tissue Int. J. Mol. Sci. 11. Mechanisms of MicroRNA Dysregulation MicroRNAs are often located at fragile sites and breakpoint regions associated with cancer development [121]. Changes in microRNA expression in HCC partially reﬂects underlying genomic instability affecting microRNA gene expression through mutations, translocations, copy number changes, deletions, DNA methylation, and histone modiﬁcations [11,122–124]. For example, histone deacetylases, which regulate gene expression through chromatin remodeling, are frequently up-regulated in HCC which, in turn, reduces expression of miR-449, a c-Met inhibitor [124]. Increased c-Met expression prevents apoptosis and promotes cell proliferation. He et al. [125] reported that miR-191 expression is 59% higher in HCC tumor versus adjacent non-tumor tissue and is associated with poor prognosis. Using methylation-speciﬁc PCR and bisulﬁte sequencing PCR, they showed that hypomethylation was correlated with elevated miR-191 expression levels, which induced a mesenchyme-like transition characterized by loss of adhesion, change from epithelial to mesenchymal cell markers, and increased invasiveness, whereas down-regulation of miR-191 had the opposite effect. Understanding the mechanism behind microRNA dysregulation is not necessary to develop biomarkers, but elucidating the cellular and metabolic changes underlying disruption of Int. J. Mol. Sci. 2016, 17, 280 11 of 17 microRNA expression patterns might provide greater insight into pathogenesis and reveal potential therapeutic targets. 12. Conclusions Development of microRNA panels, either alone or in combination with classical biomarkers, might eventually be used to classify samples with respect to liver function and progression to HCC, helping to inform treatment decisions and establish prognosis. However, several obstacles remain, including standardization of quantiﬁcation protocols and validation of microRNA panels. Differences in microRNA expression should be examined with respect to age, sex, genetic background, and underlying etiology, and potential mis-annotation or cross-hybridization of potential microRNA biomarkers should be conﬁrmed. Use of global RNA sequencing to identify candidate microRNAs and selection of appropriate internal controls should improve reproducibility of results among studies and overcome limitations inherent in earlier microarray studies. Monitoring of changes in microRNA proﬁles might provide earlier warning of changes in liver function preceding appearance of HCC, resulting in more effective treatment and improved survival. Such improvements are urgently needed, especially among the aging chronic HBV patient population. Acknowledgments: This work was supported in part by Grants-in-Aid for scientiﬁc research and development from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare, Government of Japan. Author Contributions: Both authors contributed to the literature review and preparation of the manuscript Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. ﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Jemal, A.; Bray, F.; Center, M.M.; Ferlay, J.; Ward, E.; Forman, D. Global cancer statistics. CA Cancer J. Clin. 2011, 61, 69–90. [CrossRef] [PubMed] 1. Jemal, A.; Bray, F.; Center, M.M.; Ferlay, J.; Ward, E.; Forman, D. Global cancer statistics. CA Cancer J. Clin. 2011, 61, 69–90. [CrossRef] [PubMed] . Fattovich, G.; Stroffolini, T.; Zagni, I.; Donato, F. Hepatocellular carcinoma in cirrhosis: Incidence and factors. Gastroenterology 2004, 127, S35–S50. [CrossRef] [PubMed] 3. Parkin, D.M.; Bray, F.; Ferlay, J.; Pisani, P. Global cancer statistics, 2002. CA Cancer J. Clin. 2005, 55, [CrossRef] [PubMed] 4. Aleman, S.; Rahbin, N.; Weiland, O.; Davidsdottir, L.; Hedenstierna, M.; Rose, N.; Verbaan, H.; Stal, P.; Carlsson, T.; Norrgren, H.; et al. A risk for hepatocellular carcinoma persists long-term after sustained virologic response in patients with hepatitis C-associated liver cirrhosis. Clin. Infect. Dis. 2013, 57, 230–236. [CrossRef] [PubMed] 5. Turchinovich, A.; Weiz, L.; Langheinz, A.; Burwinkel, B. Characterization of extracellular circulating microrna. Nucleic Acids Res. 2011, 39, 7223–7233. [CrossRef] [PubMed] 5. Turchinovich, A.; Weiz, L.; Langheinz, A.; Burwinkel, B. Characterization of extracellular circulating microrna. Nucleic Acids Res. 2011, 39, 7223–7233. [CrossRef] [PubMed] 6. Lewis, B.P.; Burge, C.B.; Bartel, D.P. Conserved seed pairing, often ﬂanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005, 120, 15–20. [CrossRef] [PubMed] 6. Lewis, B.P.; Burge, C.B.; Bartel, D.P. Conserved seed pairing, often ﬂanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005, 120, 15–20. [CrossRef] [PubMed] 7. Anwar, S.L.; Lehmann, U. Micrornas: Emerging novel clinical biomarkers for hepatocellular carcinomas. J. Clin. Med. 2015, 4, 1631–1650. [CrossRef] [PubMed] 8. Kerr, T.A.; Korenblat, K.M.; Davidson, N.O. MicroRNAs and liver disease. Transl. Res. 2011, 157, 241–252. [CrossRef] [PubMed] 9. Li, H.; Yang, R.; Fan, X.; Gu, T.; Zhao, Z.; Chang, D.; Wang, W.; Wang, C. MicroRNA array analysis of microRNAs related to systemic scleroderma. Rheumatol. Int. 2012, 32, 307–313. [CrossRef] [PubMed] 10. Te, J.L.; Dozmorov, I.M.; Guthridge, J.M.; Nguyen, K.L.; Cavett, J.W.; Kelly, J.A.; Bruner, G.R.; Harley, J.B.; Ojwang, J.O. Identiﬁcation of unique microRNA signature associated with lupus nephritis. PLoS ONE 2010, 5, e10344. [CrossRef] [PubMed] 11. Croce, C.M.; Calin, G.A. MiRNAs, cancer, and stem cell division. Cell 2005, 122, 6–7. [CrossRef] [PubMed 12. Calin, G.A.; Dumitru, C.D.; Shimizu, M.; Bichi, R.; Zupo, S.; Noch, E.; Aldler, H.; Rattan, S.; Keating, M.; Rai, K.; et al. References Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 2002, 99, 15524–15529. [CrossRef] [PubMed] 12 of 17 Int. J. Mol. Sci. 2016, 17, 280 13. Cai, X.; Hagedorn, C.H.; Cullen, B.R. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 2004, 10, 1957–1966. [CrossRef] [PubMed] 14. Lawrie, C.H.; Gal, S.; Dunlop, H.M.; Pushkaran, B.; Liggins, A.P.; Pulford, K.; Banham, A.H.; Pezzella, F.; Boultwood, J.; Wainscoat, J.S.; et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br. J. Haematol. 2008, 141, 672–675. [CrossRef] [PubMed] 15. Liu, A.M.; Zhang, C.; Burchard, J.; Fan, S.T.; Wong, K.F.; Dai, H.; Poon, R.T.; Luk, J.M. Global regulation on microRNA in hepatitis B virus-associated hepatocellular carcinoma. Omics 2011, 15, 187–191. [CrossRef] [PubMed] 16. Bala, S.; Marcos, M.; Szabo, G. Emerging role of microRNAs in liver diseases. World J. Gastroenterol. 2009, 15, 5633–5640. [CrossRef] [PubMed] 17. Ji, F.; Yang, B.; Peng, X.; Ding, H.; You, H.; Tien, P. Circulating microRNAs in hepatitis B virus-infected patients. J. Viral Hepat. 2011, 18, e242–e251. [CrossRef] [PubMed] 18. Mitchell, P.S.; Parkin, R.K.; Kroh, E.M.; Fritz, B.R.; Wyman, S.K.; Pogosova-Agadjanyan, E.L.; Peterson, A.; Noteboom, J.; O’Briant, K.C.; Allen, A.; et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc. Natl. Acad. Sci. USA 2008, 105, 10513–10518. [CrossRef] [PubMed] 19. Ura, S.; Honda, M.; Yamashita, T.; Ueda, T.; Takatori, H.; Nishino, R.; Sunakozaka, H.; Sakai, Y.; Horimoto, K.; Kaneko, S. Differential microRNA expression between hepatitis B and hepatitis C leading disease progression to hepatocellular carcinoma. Hepatology 2009, 49, 1098–1112. [CrossRef] [PubMed] 20. Hayes, C.N.; Akamatsu, S.; Tsuge, M.; Miki, D.; Akiyama, R.; Abe, H.; Ochi, H.; Hiraga, N.; Imamura, M.; Takahashi, S.; et al. Hepatitis B virus-speciﬁc miRNAs and argonaute2 play a role in the viral life cycle. PLoS ONE 2012, 7, e47490. [CrossRef] [PubMed] 21. Shwetha, S.; Gouthamchandra, K.; Chandra, M.; Ravishankar, B.; Khaja, M.N.; Das, S. Circulating miRNA proﬁle in HCV infected serum: Novel insight into pathogenesis. Sci. Rep. 2013, 3, 1555. [CrossRef] [PubMed] 22. Novellino, L.; Rossi, R.L.; Bonino, F.; Cavallone, D.; Abrignani, S.; Pagani, M.; Brunetto, M.R. Circulating hepatitis b surface antigen particles carry hepatocellular microRNAs. PLoS ONE 2012, 7, e31952. [CrossRef] [PubMed] 23. References Liu, A.M.; Yao, T.J.; Wang, W.; Wong, K.F.; Lee, N.P.; Fan, S.T.; Poon, R.T.; Gao, C.; Luk, J.M. Circulating miR-15b and miR-130b in serum as potential markers for detecting hepatocellular carcinoma: A retrospective cohort study. BMJ Open 2012, 2, e000825. [CrossRef] [PubMed] y p 24. Cortez, M.A.; Bueso-Ramos, C.; Ferdin, J.; Lopez-Berestein, G.; Sood, A.K.; Calin, G.A. MicroRNAs in body ﬂuids—The mix of hormones and biomarkers. Nat. Rev. Clin. Oncol. 2011, 8, 467–477. [CrossRef] [PubMed] 25. Arroyo, J.D.; Chevillet, J.R.; Kroh, E.M.; Ruf, I.K.; Pritchard, C.C.; Gibson, D.F.; Mitchell, P.S.; Bennett, C.F.; Pogosova-Agadjanyan, E.L.; Stirewalt, D.L.; et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc. Natl. Acad. Sci. USA 2011, 108, 5003–5008. [CrossRef] [PubMed] 26. Gallo, A.; Tandon, M.; Alevizos, I.; Illei, G.G. The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PLoS ONE 2012, 7, e30679. [CrossRef] [PubMed] 27. Huang, X.; Yuan, T.; Tschannen, M.; Sun, Z.; Jacob, H.; Du, M.; Liang, M.; Dittmar, R.L.; Liu, Y.; Kohli, M.; et al. Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genom. 2013, 14, 319. [CrossRef] [PubMed] 28. Conde-Vancells, J.; Rodriguez-Suarez, E.; Embade, N.; Gil, D.; Matthiesen, R.; Valle, M.; Elortza, F.; Lu, S.C.; Mato, J.M.; Falcon-Perez, J.M. Characterization and comprehensive proteome proﬁling of exosomes secreted by hepatocytes. J. Proteome Res. 2008, 7, 5157–5166. [CrossRef] [PubMed] 29. Li, J.; Liu, K.; Liu, Y.; Xu, Y.; Zhang, F.; Yang, H.; Liu, J.; Pan, T.; Chen, J.; Wu, M.; et al. Exosomes mediate the cell-to-cell transmission of IFN-α-induced antiviral activity. Nat. Immunol. 2013, 14, 793–803. [CrossRef] [PubMed] 30. Taylor, C.J.; Satoor, S.N.; Ranjan, A.K.; Pereira e Cotta, M.V.; Joglekar, M.V. A protocol for measurement of noncoding RNA in human serum. Exp. Diabetes Res. 2012, 2012, 168368. [CrossRef] [PubMed] 31. Natarajan, S.K.; Pachunka, J.M.; Mott, J.L. Role of microRNAs in alcohol-induced multi-organ injury. Biomolecules 2015, 5, 3309–3338. [CrossRef] [PubMed] 32. Marabita, F.; de Candia, P.; Torri, A.; Tegner, J.; Abrignani, S.; Rossi, R.L. Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR. Brief. Bioinform. 2015. [CrossRef] [PubMed] 13 of 17 Int. J. Mol. Sci. 2016, 17, 280 33. Benz, F.; Roderburg, C.; Vargas Cardenas, D.; Vucur, M.; Gautheron, J.; Koch, A.; Zimmermann, H.; Janssen, J.; Nieuwenhuijsen, L.; Luedde, M.; et al. U6 is unsuitable for normalization of serum miRNA levels in patients with sepsis or liver ﬁbrosis. Exp. References Mol. Med. 2013, 45, e42. [CrossRef] [PubMed] 4. McDonald, J.S.; Milosevic, D.; Reddi, H.V.; Grebe, S.K.; Algeciras-Schimnich, A. Analysis of circula microRNA: Preanalytical and analytical challenges. Clin. Chem. 2011, 57, 833–840. [CrossRef] [PubMed 35. Li, L.Z.; Zhang, C.Z.; Liu, L.L.; Yi, C.; Lu, S.X.; Zhou, X.; Zhang, Z.J.; Peng, Y.H.; Yang, Y.Z.; Yun, J.P. MiR-720 inhibits tumor invasion and migration in breast cancer by targeting twist1. Carcinogenesis 2014, 35, 469–478. [CrossRef] [PubMed] 36. Schopman, N.C.; Heynen, S.; Haasnoot, J.; Berkhout, B. A miRNA-tRNA mix-up: TRNA origin of proposed miRNA. RNA Biol. 2010, 7, 573–576. [CrossRef] [PubMed] 37. Langenberger, D.; Bartschat, S.; Hertel, J.; Hoffmann, S.; Tafer, H.; Stadler, P. MicroRNA or not microRNA? In Advances in Bioinformatics and Computational Biology; Norberto de Souza, O., Telles, G., Palakal, M., Eds.; Springer: Berlin Heidelberg, Germany, 2011; Volume 6832, pp. 1–9. 38. Dhahbi, J.M.; Spindler, S.R.; Atamna, H.; Yamakawa, A.; Boffelli, D.; Mote, P.; Martin, D.I. 51 tRNA halves are present as abundant complexes in serum, concentrated in blood cells, and modulated by aging and calorie restriction. BMC Genom. 2013, 14, 298. [CrossRef] [PubMed] 9. Anderson, P.; Ivanov, P. TRNA fragments in human health and disease. FEBS Lett. 2014, 588, 4297–4 [CrossRef] [PubMed] 40. Selitsky, S.R.; Baran-Gale, J.; Honda, M.; Yamane, D.; Masaki, T.; Fannin, E.E.; Guerra, B.; Shirasaki, T.; Shimakami, T.; Kaneko, S.; et al. Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C. Sci. Rep. 2015, 5, 7675. [CrossRef] [PubMed] 41. Law, P.T.; Qin, H.; Ching, A.K.; Lai, K.P.; Co, N.N.; He, M.; Lung, R.W.; Chan, A.W.; Chan, T.F.; Wong, N. Deep sequencing of small RNA transcriptome reveals novel non-coding RNAs in hepatocellular carcinoma. J. Hepatol. 2013, 58, 1165–1173. [CrossRef] [PubMed] 42. Hou, J.; Lin, L.; Zhou, W.; Wang, Z.; Ding, G.; Dong, Q.; Qin, L.; Wu, X.; Zheng, Y.; Yang, Y.; et al. Identiﬁcation of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma. Cancer Cell 2011, 19, 232–243. [CrossRef] [PubMed] 43. Gamazon, E.R.; Innocenti, F.; Wei, R.; Wang, L.; Zhang, M.; Mirkov, S.; Ramirez, J.; Huang, R.S.; Cox, N.J.; Ratain, M.J.; et al. A genome-wide integrative study of microRNAs in human liver. BMC Genom. 2013, 14, 395. [CrossRef] [PubMed] 44. Jopling, C. Liver-speciﬁc microRNA-122: Biogenesis and function. RNA Biol. 2012, 9, 137–142. [CrossRef] [PubMed] 45. Sharma, S.; Eghbali, M. References Ding, X.; Ding, J.; Ning, J.; Yi, F.; Chen, J.; Zhao, D.; Zheng, J.; Liang, Z.; Hu, Z.; Du, Q. Circulating microRNA-122 as a potential biomarker for liver injury. Mol. Med. Rep. 2012, 5, 1428–1432. [CrossRef] [PubMed] 55. Dolganiuc, A.; Petrasek, J.; Kodys, K.; Catalano, D.; Mandrekar, P.; Velayudham, A.; Szabo, G. MicroRNA expression proﬁle in lieber-decarli diet-induced alcoholic and methionine choline deﬁcient diet-induced nonalcoholic steatohepatitis models in mice. Alcohol. Clin. Exp. Res. 2009, 33, 1704–1710. [CrossRef] [PubMed] 56. Chen, Y.P.; Jin, X.; Xiang, Z.; Chen, S.H.; Li, Y.M. Circulating microRNAs as potential biomarkers for alcoholic steatohepatitis. Liver Int. 2013, 33, 1257–1265. [CrossRef] [PubMed] 57. Baffy, G. MicroRNAs in nonalcoholic fatty liver disease. J. Clin. Med. 2015, 4, 1977–1988. [CrossRef] [PubMed] 58. Zarfeshani, A.; Ngo, S.; Sheppard, A.M. MicroRNA expression relating to dietary-induced liver steato nash. J. Clin. Med. 2015, 4, 1938–1950. [CrossRef] [PubMed] 58. Zarfeshani, A.; Ngo, S.; Sheppard, A.M. MicroRNA expression relating to dietary-induced liver steatosis and nash. J. Clin. Med. 2015, 4, 1938–1950. [CrossRef] [PubMed] 59 Ji X Y YF Ch S H Y C H Li J Li YM Mi RNA i tt i diff t t f 59. Jin, X.; Ye, Y.F.; Chen, S.H.; Yu, C.H.; Liu, J.; Li, Y.M. MicroRNA expression pattern in different s nonalcoholic fatty liver disease. Dig. Liver Dis. 2009, 41, 289–297. [CrossRef] [PubMed] 60. Li, S.; Chen, X.; Zhang, H.; Liang, X.; Xiang, Y.; Yu, C.; Zen, K.; Li, Y.; Zhang, C.Y. Differential expression of microRNAs in mouse liver under aberrant energy metabolic status. J. Lipid Res. 2009, 50, 1756–1765. [CrossRef] [PubMed] 61. Feng, Y.Y.; Xu, X.Q.; Ji, C.B.; Shi, C.M.; Guo, X.R.; Fu, J.F. Aberrant hepatic microRNA expression in nonalcoholic fatty liver disease. Cell. Physiol. Biochem. 2014, 34, 1983–1997. [CrossRef] [PubMed] 62. Cheung, O.; Puri, P.; Eicken, C.; Contos, M.J.; Mirshahi, F.; Maher, J.W.; Kellum, J.M.; Min, H.; Luketic, V.A.; Sanyal, A.J. Nonalcoholic steatohepatitis is associated with altered hepatic microRNA expression. Hepatology 2008, 48, 1810–1820. [CrossRef] [PubMed] 63. Cermelli, S.; Ruggieri, A.; Marrero, J.A.; Ioannou, G.N.; Beretta, L. Circulating microRNAs in patients with chronic hepatitis c and non-alcoholic fatty liver disease. PLoS ONE 2011, 6, e23937. [CrossRef] [PubMed] 64. Pirola, C.J.; Fernandez Gianotti, T.; Castano, G.O.; Mallardi, P.; San Martino, J.; Mora Gonzalez Lopez Ledesma, M.; Flichman, D.; Mirshahi, F.; Sanyal, A.J.; Sookoian, S. References Inﬂuence of sex differences on microRNA gene regulation in disease. Biol. Sex Differ. 2014, 5, 3. [CrossRef] [PubMed] 46. Roderburg, C.; Urban, G.W.; Bettermann, K.; Vucur, M.; Zimmermann, H.; Schmidt, S.; Janssen, J.; Koppe, C.; Knolle, P.; Castoldi, M.; et al. Micro-RNA proﬁling reveals a role for miR-29 in human and murine liver ﬁbrosis. Hepatology 2011, 53, 209–218. [CrossRef] [PubMed] 47. Migita, K.; Komori, A.; Kozuru, H.; Jiuchi, Y.; Nakamura, M.; Yasunami, M.; Furukawa, H.; Abiru, S.; Yamasaki, K.; Nagaoka, S.; et al. Circulating microRNA proﬁles in patients with type-1 autoimmune hepatitis. PLoS ONE 2015, 10, e0136908. [CrossRef] [PubMed] 48. Tan, Y.; Ge, G.; Pan, T.; Wen, D.; Gan, J. A pilot study of serum microRNAs panel as potential biomar diagnosis of nonalcoholic fatty liver disease. PLoS ONE 2014, 9, e105192. [CrossRef] [PubMed] 48. Tan, Y.; Ge, G.; Pan, T.; Wen, D.; Gan, J. A pilot study of serum microRNAs panel as potential biomarkers for diagnosis of nonalcoholic fatty liver disease. PLoS ONE 2014, 9, e105192. [CrossRef] [PubMed] 49. Li, L.M.; Wang, D.; Zen, K. MicroRNAs in drug-induced liver injury. J. Clin. Transl. Hepatol. 2014, 2, 162–169. [CrossRef] [PubMed] g y , , [ ] [ ] 49. Li, L.M.; Wang, D.; Zen, K. MicroRNAs in drug-induced liver injury. J. Clin. Transl. Hepatol. 2014, 2, 162–169. [CrossRef] [PubMed] 50. Bala, S.; Petrasek, J.; Mundkur, S.; Catalano, D.; Levin, I.; Ward, J.; Alao, H.; Kodys, K.; Szabo, G. Circulating microRNAs in exosomes indicate hepatocyte injury and inﬂammation in alcoholic, drug-induced, and inﬂammatory liver diseases. Hepatology 2012, 56, 1946–1957. [CrossRef] [PubMed] 51. Wang, K.; Zhang, S.; Marzolf, B.; Troisch, P.; Brightman, A.; Hu, Z.; Hood, L.E.; Galas, D.J. Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc. Natl. Acad. Sci. USA 2009, 106, 4402–4407. [CrossRef] [PubMed] 52. Fukushima, T.; Hamada, Y.; Yamada, H.; Horii, I. Changes of micro-RNA expression in rat liver treated by acetaminophen or carbon tetrachloride—Regulating role of micro-RNA for RNA expression. J. Toxicol. Sci. 2007, 32, 401–409. [CrossRef] [PubMed] 14 of 17 Int. J. Mol. Sci. 2016, 17, 280 14 of 17 53. Starkey Lewis, P.J.; Dear, J.; Platt, V.; Simpson, K.J.; Craig, D.G.; Antoine, D.J.; French, N.S.; Dhaun, N.; Webb, D.J.; Costello, E.M.; et al. Circulating microRNAs as potential markers of human drug-induced liver injury. Hepatology 2011, 54, 1767–1776. [CrossRef] [PubMed] 54. References Circulating microRNA signature in non-alcoholic fatty liver disease: From serum non-coding RNAs to liver histology and disease pathogenesis. Gut 2015, 64, 800–812. [CrossRef] [PubMed] 65. Teng, K.Y.; Ghoshal, K. Role of noncoding RNAs as biomarker and therapeutic targets for liver ﬁbrosis. Gene Expr. 2015, 16, 155–162. [CrossRef] [PubMed] 66. Friedman, S.L.; Roll, F.J.; Boyles, J.; Bissell, D.M. Hepatic lipocytes: The principal collagen-producing cells of normal rat liver. Proc. Natl. Acad. Sci. USA 1985, 82, 8681–8685. [CrossRef] [PubMed] 67. Bataller, R.; Brenner, D.A. Liver ﬁbrosis. J. Clin. Investig. 2005, 115, 209–218. [CrossRef] [PubMed] 68. Zhang, Y.; Wu, L.; Wang, Y.; Zhang, M.; Li, L.; Zhu, D.; Li, X.; Gu, H.; Zhang, C.Y.; Zen, K. Protective role of estrogen-induced miRNA-29 expression in carbon tetrachloride-induced mouse liver injury. J. Biol. Chem. 2012, 287, 14851–14862. [CrossRef] [PubMed] 69. Peng, X.; Li, Y.; Walters, K.A.; Rosenzweig, E.R.; Lederer, S.L.; Aicher, L.D.; Proll, S.; Katze, M.G. Computational identiﬁcation of hepatitis C virus associated microRNA-mRNA regulatory modules in human livers. BMC Genom. 2009, 10, 373. [CrossRef] [PubMed] 70. Zhang, G.L.; Li, Y.X.; Zheng, S.Q.; Liu, M.; Li, X.; Tang, H. Suppression of hepatitis b virus replication by microRNA-199a-3p and microRNA-210. Antivir. Res. 2010, 88, 169–175. [CrossRef] [PubMed] 71. Potenza, N.; Papa, U.; Mosca, N.; Zerbini, F.; Nobile, V.; Russo, A. Human microRNA hsa-miR-125a-5p interferes with expression of hepatitis B virus surface antigen. Nucleic Acids Res. 2011, 39, 5157–5163. [CrossRef] [PubMed] 72. Wu, F.L.; Jin, W.B.; Li, J.H.; Guo, A.G. Targets for human encoded microRNAs in hbv genes. Virus Genes 2011, 42, 157–161. [CrossRef] [PubMed] 73. Kohno, T.; Tsuge, M.; Murakami, E.; Hiraga, N.; Abe, H.; Miki, D.; Imamura, M.; Ochi, H.; Hayes, C.N.; Chayama, K. Human microRNA hsa-miR-1231 suppresses hepatitis B virus replication by targeting core mRNA. J. Viral Hepat. 2014, 21, e89–e97. [CrossRef] [PubMed] Int. J. Mol. Sci. 2016, 17, 280 15 of 17 74. Chen, Y.; Shen, A.; Rider, P.J.; Yu, Y.; Wu, K.; Mu, Y.; Hao, Q.; Liu, Y.; Gong, H.; Zhu, Y.; et al. A liver-speciﬁc microRNA binds to a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication. FASEB J. 2011, 25, 4511–4521. [CrossRef] [PubMed] 75. Wang, S.; Qiu, L.; Yan, X.; Jin, W.; Wang, Y.; Chen, L.; Wu, E.; Ye, X.; Gao, G.F.; Wang, F.; et al. References Loss of miR-122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G1 modulated P53 activity. Hepatology 2012, 55, 730–741. [CrossRef] [PubMed] 76. Hu, J.; Xu, Y.; Hao, J.; Wang, S.; Li, C.; Meng, S. MiR-122 in hepatic function and liver diseases. Protein Cell 2012, 3, 364–371. [CrossRef] [PubMed] 77. Chang, J.; Nicolas, E.; Marks, D.; Sander, C.; Lerro, A.; Buendia, M.A.; Xu, C.; Mason, W.S.; Moloshok, T.; Bort, R.; et al. MiR-122, a mammalian liver-speciﬁc microRNA, is processed from hcr mRNA and may downregulate the high afﬁnity cationic amino acid transporter CAT-1. RNA Biol. 2004, 1, 106–113. [CrossRef] [PubMed] 78. Qiu, L.; Fan, H.; Jin, W.; Zhao, B.; Wang, Y.; Ju, Y.; Chen, L.; Chen, Y.; Duan, Z.; Meng, S. MiR-122-induced down-regulation of HO-1 negatively affects miR-122-mediated suppression of HBV. Biochem. Biophys. Res. Commun. 2010, 398, 771–777. [CrossRef] [PubMed] 79. Li, D.; Liu, X.; Lin, L.; Hou, J.; Li, N.; Wang, C.; Wang, P.; Zhang, Q.; Zhang, P.; Zhou, W.; et al. MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma. J. Biol. Chem. 2011, 286, 36677–36685. [CrossRef] [PubMed] 80. Takata, A.; Otsuka, M.; Kojima, K.; Yoshikawa, T.; Kishikawa, T.; Yoshida, H.; Koike, K. MicroRNA-22 and microRNA-140 suppress NF-κB activity by regulating the expression of NF-κB coactivators. Biochem. Biophys. Res. Commun. 2011, 411, 826–831. [CrossRef] [PubMed] 81. Xu, D.; Takeshita, F.; Hino, Y.; Fukunaga, S.; Kudo, Y.; Tamaki, A.; Matsunaga, J.; Takahashi, R.U.; Takata, T.; Shimamoto, A.; et al. MiR-22 represses cancer progression by inducing cellular senescence. J. Cell Biol. 2011, 193, 409–424. [CrossRef] 82. Shi, C.; Xu, X. MicroRNA-22 is down-regulated in hepatitis B virus-related hepatocellular carcinoma. Biomed. Pharmacother. 2013, 67, 375–380. [CrossRef] [PubMed] 83. Hu, W.; Wang, X.; Ding, X.; Li, Y.; Zhang, X.; Xie, P.; Yang, J.; Wang, S. MicroRNA-141 represses HBV replication by targeting PPARA. PLoS ONE 2012, 7, e34165. [CrossRef] [PubMed] 84. Zhang, X.; Zhang, E.; Ma, Z.; Pei, R.; Jiang, M.; Schlaak, J.F.; Roggendorf, M.; Lu, M. Modulation of hepatitis b virus replication and hepatocyte differentiation by microRNA-1. Hepatology 2011, 53, 1476–1485. [CrossRef] [PubMed] 85. Jin, J.; Tang, S.; Xia, L.; Du, R.; Xie, H.; Song, J.; Fan, R.; Bi, Q.; Chen, Z.; Yang, G.; et al. MicroRNA-501 promotes hbv replication by targeting HBXIP. Biochem. Biophys. Res. Commun. 2013, 430, 1228–1233. [CrossRef] [PubMed] 86. Jin, W.B.; Wu, F.L.; Kong, D.; Guo, A.G. References Hbv-encoded microRNA candidate and its target. Comput. Biol. Chem. 2007, 31, 124–126. [CrossRef] [PubMed] 87. Li, L.M.; Hu, Z.B.; Zhou, Z.X.; Chen, X.; Liu, F.Y.; Zhang, J.F.; Shen, H.B.; Zhang, C.Y.; Zen, K. Serum microRNA proﬁles serve as novel biomarkers for HBV infection and diagnosis of HBV-positive hepatocarcinoma. Cancer Res. 2010, 70, 9798–9807. [CrossRef] [PubMed] p 88. Arataki, K.; Hayes, C.N.; Akamatsu, S.; Akiyama, R.; Abe, H.; Tsuge, M.; Miki, D.; Ochi, H.; Hiraga, N.; Imamura, M.; et al. Circulating microRNA-22 correlates with microRNA-122 and represents viral replication and liver injury in patients with chronic hepatitis B. J. Med. Virol. 2013, 85, 789–798. [CrossRef] [PubMed] 89. Akamatsu, S.; Hayes, C.N.; Tsuge, M.; Miki, D.; Akiyama, R.; Abe, H.; Ochi, H.; Hiraga, N.; Imamura, M.; Takahashi, S.; et al. Differences in serum microRNA proﬁles in hepatitis B and C virus infection. J. Infect. 2015, 70, 273–287. [CrossRef] [PubMed] 90. Giray, B.G.; Emekdas, G.; Tezcan, S.; Ulger, M.; Serin, M.S.; Sezgin, O.; Altintas, E.; Tiftik, E.N. Proﬁles of serum microRNAs; miR-125b-5p and miR223–3p serve as novel biomarkers for HBV-positive hepatocellular carcinoma. Mol. Biol. Rep. 2014, 41, 4513–4519. [CrossRef] [PubMed] 91. Ren, M.; Qin, D.; Li, K.; Qu, J.; Wang, L.; Wang, Z.; Huang, A.; Tang, H. Correlation between hepatitis B virus protein and microRNA processor drosha in cells expressing HBV. Antivir. Res. 2012, 94, 225–231. [CrossRef] [PubMed] Int. J. Mol. Sci. 2016, 17, 280 16 of 17 92. Steuerwald, N.M.; Parsons, J.C.; Bennett, K.; Bates, T.C.; Bonkovsky, H.L. Parallel microRNA and mRNA expression proﬁling of (genotype 1b) human hepatoma cells expressing hepatitis C virus. Liver Int. 2010, 30, 1490–1504. [CrossRef] [PubMed] 3. Pedersen, I.M.; Cheng, G.; Wieland, S.; Volinia, S.; Croce, C.M.; Chisari, F.V.; David, M. Interferon modula of cellular microRNAs as an antiviral mechanism. Nature 2007, 449, 919–922. [CrossRef] [PubMed] 94. Jopling, C.L.; Yi, M.; Lancaster, A.M.; Lemon, S.M.; Sarnow, P. Modulation of hepatitis C virus RNA abundance by a liver-speciﬁc microRNA. Science 2005, 309, 1577–1581. [CrossRef] [PubMed] 95. Lanford, R.E.; Hildebrandt-Eriksen, E.S.; Petri, A.; Persson, R.; Lindow, M.; Munk, M.E.; Kauppinen, S.; Orum, H. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 2010, 327, 198–201. [CrossRef] [PubMed] 96. Banaudha, K.; Kaliszewski, M.; Korolnek, T.; Florea, L.; Yeung, M.L.; Jeang, K.T.; Kumar, A. MicroRNA silencing of tumor suppressor DLC-1 promotes efﬁcient hepatitis C virus replication in primary human hepatocytes. Hepatology 2011, 53, 53–61. [CrossRef] [PubMed] 97. References Ishida, H.; Tatsumi, T.; Hosui, A.; Nawa, T.; Kodama, T.; Shimizu, S.; Hikita, H.; Hiramatsu, N.; Kanto, T.; Hayashi, N.; et al. Alterations in microRNA expression proﬁle in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/akt pathway. Biochem. Biophys. Res. Commun. 2011, 412, 92–97. [CrossRef] [PubMed] 98. Murakami, Y.; Aly, H.H.; Tajima, A.; Inoue, I.; Shimotohno, K. Regulation of the hepatitis C virus genome replication by miR-199a. J. Hepatol. 2009, 50, 453–460. [CrossRef] [PubMed] 99. Hou, W.; Tian, Q.; Zheng, J.; Bonkovsky, H.L. MicroRNA-196 represses Bach1 protein and hepatitis C virus gene expression in human hepatoma cells expressing hepatitis C viral proteins. Hepatology 2010, 51, 1494–1504. [CrossRef] [PubMed] 100. Bandyopadhyay, S.; Friedman, R.C.; Marquez, R.T.; Keck, K.; Kong, B.; Icardi, M.S.; Brown, K.E.; Burge, C.B.; Schmidt, W.N.; Wang, Y.; et al. Hepatitis C virus infection and hepatic stellate cell activation downregulate miR-29: MiR-29 overexpression reduces hepatitis C viral abundance in culture. J. Infect. Dis. 2011, 203, 1753–1762. [CrossRef] [PubMed] 101. Cheng, J.C.; Yeh, Y.J.; Tseng, C.P.; Hsu, S.D.; Chang, Y.L.; Sakamoto, N.; Huang, H.D. Let-7b is a novel regulator of hepatitis C virus replication. Cell. Mol. Life Sci. 2012, 69, 2621–2633. [CrossRef] [PubMed] 102. Bhanja Chowdhury, J.; Shrivastava, S.; Steele, R.; Di Bisceglie, A.M.; Ray, R.; Ray, R.B. Hepatitis C virus infection modulates expression of interferon stimulatory gene IFITM1 by upregulating miR-130a. J. Virol. 2012, 86, 10221–10225. [CrossRef] [PubMed] 103. Shirasaki, T.; Honda, M.; Shimakami, T.; Horii, R.; Yamashita, T.; Sakai, Y.; Sakai, A.; Okada, H.; Watanabe, R.; Murakami, S.; et al. MicroRNA-27a regulates lipid metabolism and inhibits hepatitis C virus replication in human hepatoma cells. J. Virol. 2013, 87, 5270–5286. [CrossRef] [PubMed] 104. You, J.S.; Jones, P.A. Cancer genetics and epigenetics: Two sides of the same coin? Cancer Cell 2012, 22, 9–20. [CrossRef] [PubMed] 105. Ashworth, A.; Lord, C.J.; Reis-Filho, J.S. Genetic interactions in cancer progression and treatment. Cell 2011, 145, 30–38. [CrossRef] [PubMed] 106. Pogribny, I.P.; Rusyn, I. Role of epigenetic aberrations in the development and progression of human hepatocellular carcinoma. Cancer Lett. 2014, 342, 223–230. [CrossRef] [PubMed] 07. Bertino, G.; Ardiri, A.M.; Calvagno, G.S.; Bertino, N.; Boemi, P.M. Prognostic and diagnostic valu des-γ-carboxy prothrombin in liver cancer. Drug News Perspect. 2010, 23, 498–508. [PubMed] 108. Durazo, F.A.; Blatt, L.M.; Corey, W.G.; Lin, J.H.; Han, S.; Saab, S.; Busuttil, R.W.; Tong, M.J. References Des-γ-carboxyprothrombin, α-fetoprotein and AFP-L3 in patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma. J. Gastroenterol. Hepatol. 2008, 23, 1541–1548. [CrossRef] [PubMed] 109. Borel, F.; Konstantinova, P.; Jansen, P.L. Diagnostic and therapeutic potential of miRNA signatures in patients with hepatocellular carcinoma. J. Hepatol. 2012, 56, 1371–1383. [CrossRef] [PubMed] 110. Ladeiro, Y.; Couchy, G.; Balabaud, C.; Bioulac-Sage, P.; Pelletier, L.; Rebouissou, S.; Zucman-Rossi, J. MicroRNA proﬁling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology 2008, 47, 1955–1963. [CrossRef] [PubMed] 111. Huang, S.; He, X. The role of microRNAs in liver cancer progression. Br. J. Cancer 2011, 104, 235–240. [CrossRef] [PubMed] Int. J. Mol. Sci. 2016, 17, 280 17 of 17 17 of 17 112. Qi, P.; Cheng, S.Q.; Wang, H.; Li, N.; Chen, Y.F.; Gao, C.F. Serum microRNAs as biomarkers for hepatocellular carcinoma in chinese patients with chronic hepatitis B virus infection. PLoS ONE 2011, 6, e28486. [CrossRef] [PubMed] 113. Wang, Y.; Lee, A.T.; Ma, J.Z.; Wang, J.; Ren, J.; Yang, Y.; Tantoso, E.; Li, K.B.; Ooi, L.L.; Tan, P.; et al. Proﬁling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-speciﬁc target. J. Biol. Chem. 2008, 283, 13205–13215. [CrossRef] [PubMed] 114. Zhuang, L.P.; Meng, Z.Q. Serum miR-224 reﬂects stage of hepatocellular carcinoma and predicts survival. Biomed. Res. Int. 2015, 2015, 731781. [CrossRef] [PubMed] 115. Ghosh, A.; Ghosh, A.; Datta, S.; Dasgupta, D.; Das, S.; Ray, S.; Gupta, S.; Datta, S.; Chowdhury, A.; Chatterjee, R.; et al. Hepatic miR-126 is a potential plasma biomarker for detection of hepatitis B virus infected hepatocellular carcinoma. Int. J. Cancer 2016. [CrossRef] [PubMed] 116. Barshack, I.; Meiri, E.; Rosenwald, S.; Lebanony, D.; Bronfeld, M.; Aviel-Ronen, S.; Rosenblatt, K.; Polak-Charcon, S.; Leizerman, I.; Ezagouri, M.; et al. Differential diagnosis of hepatocellular carcinoma from metastatic tumors in the liver using microRNA expression. Int. J. Biochem. Cell Biol. 2010, 42, 1355–1362. [CrossRef] [PubMed] 117. Lin, X.J.; Chong, Y.; Guo, Z.W.; Xie, C.; Yang, X.J.; Zhang, Q.; Li, S.P.; Xiong, Y.; Yuan, Y.; Min, J.; et al. A serum microRNA classifier for early detection of hepatocellular carcinoma: A multicentre, retrospective, longitudinal biomarker identification study with a nested case-control study. Lancet Oncol. 2015, 16, 804–815. [CrossRef] 118. Ji, J.; Shi, J.; Budhu, A.; Yu, Z.; Forgues, M.; Roessler, S.; Ambs, S.; Chen, Y.; Meltzer, P.S.; Croce, C.M.; et al. MicroRNA expression, survival, and response to interferon in liver cancer. N. References Engl. J. Med. 2009, 361, 1437–1447. [CrossRef] [PubMed] 119. Zhu, H.T.; Dong, Q.Z.; Sheng, Y.Y.; Wei, J.W.; Wang, G.; Zhou, H.J.; Ren, N.; Jia, H.L.; Ye, Q.H.; Qin, L.X. MicroRNA-29a-5p is a novel predictor for early recurrence of hepatitis b virus-related hepatocellular carcinoma after surgical resection. PLoS ONE 2012, 7, e52393. [CrossRef] [PubMed] 120. Kourtidis, A.; Ngok, S.P.; Pulimeno, P.; Feathers, R.W.; Carpio, L.R.; Baker, T.R.; Carr, J.M.; Yan, I.K.; Borges, S.; Perez, E.A.; et al. Distinct E-cadherin-based complexes regulate cell behaviour through miRNA processing or Src and p120 catenin activity. Nat. Cell Biol. 2015, 17, 1145–1157. [CrossRef] [PubMed] 121. Calin, G.A.; Sevignani, C.; Dumitru, C.D.; Hyslop, T.; Noch, E.; Yendamuri, S.; Shimizu, M.; Rattan, S.; Bullrich, F.; Negrini, M.; et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. USA 2004, 101, 2999–3004. [CrossRef] [PubMed] 122. Anwar, S.L.; Albat, C.; Krech, T.; Hasemeier, B.; Schipper, E.; Schweitzer, N.; Vogel, A.; Kreipe, H.; Lehmann, U. Concordant hypermethylation of intergenic microRNA genes in human hepatocellular carcinoma as new diagnostic and prognostic marker. Int. J. Cancer 2013, 133, 660–670. [CrossRef] [PubMed] Lehmann, U. Concordant hypermethylation of intergenic microRNA genes in human hepatocellular carcinoma as new diagnostic and prognostic marker. Int. J. Cancer 2013, 133, 660–670. [CrossRef] [PubMed] 123. Anwar, S.L.; Lehmann, U. DNA methylation, microRNAs, and their crosstalk as potential biomarkers in hepatocellular carcinoma. World J. Gastroenterol. 2014, 20, 7894–7913. [CrossRef] [PubMed] 123. Anwar, S.L.; Lehmann, U. DNA methylation, microRNAs, and their crosstalk as potential biomarkers in hepatocellular carcinoma. World J. Gastroenterol. 2014, 20, 7894–7913. [CrossRef] [PubMed] 124. Buurman, R.; Gurlevik, E.; Schaffer, V.; Eilers, M.; Sandbothe, M.; Kreipe, H.; Wilkens, L.; Schlegelberger, B.; Kuhnel, F.; Skawran, B. Histone deacetylases activate hepatocyte growth factor signaling by repressing microRNA-449 in hepatocellular carcinoma cells. Gastroenterology 2012, 143, 811–820. [CrossRef] [PubMed] 124. Buurman, R.; Gurlevik, E.; Schaffer, V.; Eilers, M.; Sandbothe, M.; Kreipe, H.; Wilkens, L.; Schlegelberger, B.; Kuhnel, F.; Skawran, B. Histone deacetylases activate hepatocyte growth factor signaling by repressing microRNA-449 in hepatocellular carcinoma cells. Gastroenterology 2012, 143, 811–820. [CrossRef] [PubMed] 125. He, Y.; Cui, Y.; Wang, W.; Gu, J.; Guo, S.; Ma, K.; Luo, X. Hypomethylation of the hsa-miR-191 locus causes high expression of hsa-miR-191 and promotes the epithelial-to-mesenchymal transition in hepatocellular carcinoma. Neoplasia 2011, 13, 841–853. [CrossRef] [PubMed] 125. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). References He, Y.; Cui, Y.; Wang, W.; Gu, J.; Guo, S.; Ma, K.; Luo, X. Hypomethylation of the hsa-miR-191 locus causes high expression of hsa-miR-191 and promotes the epithelial-to-mesenchymal transition in hepatocellular carcinoma. Neoplasia 2011, 13, 841–853. [CrossRef] [PubMed] © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4237837032	https://newprairiepress.org/cgi/viewcontent.cgi?article=1219&context=kaesrr	English	null	Balanced Nutrition and Crop Production Practices for Closing Sorghum Yield Gaps	Kansas Agricultural Experiment Station research reports	2,016	cc-by	2,523	Balanced Nutrition and Crop Production Practices for Closing Balanced Nutrition and Crop Production Practices for Closing Sorghum Yield Gaps Sorghum Yield Gaps See next page for additional authors Follow this and additional works at: https://newprairiepress.org/kaesrr Follow this and additional works at: https://newprairiepress.org/kaesrr Part of the Agronomy and Crop Sciences Commons Part of the Agronomy and Crop Sciences Commons Recommended Citation Recommended Citation Kansas Agricultural Experiment Station Research Reports Kansas Agricultural Experiment Station Research Reports Volume 2 Issue 5 Kansas Field Research Article 2 January 2016 Balanced Nutrition and Crop Production Practices for Closing Balanced Nutrition and Crop Production Practices for Closing Sorghum Yield Gaps Sorghum Yield Gaps B. McHenry Kansas State University, baileymc@ksu.edu Eric Adee Kansas State University, eadee@ksu.edu J. Kimball Kansas State University, jkimball@ksu.edu See next page for additional authors This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 2016 the Author(s). Contents of this publication may be freely reproduced for educational purposes. All other rights reserved. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State R h d E t i i l t it id d Follow this and additional works at: https://newprairiepress.org/kaesrr Part of the Agronomy and Crop Sciences Commons Recommended Citation Recommended Citation McHenry, B.; Adee, Eric; Kimball, J.; Prasad, P. V. Vara; and Ciampitti, I. A. (2016) "Balanced Nutrition and Crop Production Practices for Closing Sorghum Yield Gaps," Kansas Agricultural Experiment Station Research Reports: Vol. 2: Iss. 5. https://doi.org/10.4148/2378-5977.1219 ansas Agricultural Experiment Station Research Reports ansas Agricultural Experiment Station Research Reports Volume 2 Issue 5 Kansas Field Research Article 2 This east central kansas experiment field is available in Kansas Agricultural Experiment Station Research Reports: https://newprairiepress.org/kaesrr/vol2/iss5/2 Recommended Citation Recommended Citation McHenry, B.; Adee, Eric; Kimball, J.; Prasad, P. V. Vara; and Ciampitti, I. A. (2016) "Balanced Nutrition and Crop Production Practices for Closing Sorghum Yield Gaps," Kansas Agricultural Experiment Station Research Reports: Vol. 2: Iss. 5. https://doi.org/10.4148/2378-5977.1219 This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 2016 the Author(s). Contents of this publication may be freely reproduced for educational purposes. All other rights reserved. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State Research and Extension is an equal opportunity provider and employer. Balanced Nutrition and Crop Production Practices for Closing Sorghum Yield Balanced Nutrition and Crop Production Practices for Closing Sorghum Yield Gaps Gaps Authors Authors B. McHenry, Eric Adee, J. Kimball, P. V. Vara Prasad, and I. A. Ciampitti Summary y In order to study how diverse cropping system approaches influence grain sorghum productivity, field experiments were conducted in Topeka, KS at the Kansas River Val ley Experiment Field; and in Ottawa, KS at the East Central Kansas Experiment Field. The primary objective of this study was to understand how to close yield gaps between the current on-farm yields and the maximum attainable yields. The factors that were tested include narrow row spacing; high and low plant population; balanced nutrition practices, including various timings of nitrogen, phosphorus, and potassium (N-P-K); micronutrient applications of iron and zinc (Fe and Zn); crop protection with fungi cide and insecticide applications; plant growth regulator effects; and the use of precision agricultural technology for maximizing yields, including a GreenSeeker meter (Trimble Navigation, Westminster, CO) for more precisely determining N needs for sorghum. Grain sorghum yields ranged from 149 to 166 bu/a in Topeka, KS under irrigation, and from 78 to 100 bu/a in Ottawa, KS, under dryland conditions. At Ottawa, yield poten tial was limited by precipitation, 10.8 inch. Still, sorghum yield gap between the highest (treatment #2, “kitchen sink” but with low seeding rate) and lowest (treatment #10, “standard practice”) was 22 bushels per acre. The production practices that produced the highest yields varied between the two locations. Balanced Nutrition and Crop Production Practices for Closing Sorghum Yield Gaps McHenry, E.A. Adee, J. Kimball, P.V. Vara Prasad, and I.A. Ciampitti Introduction Low productivity is one of the biggest problems in grain sorghum production in Kansas. This productivity issue stems from a combination of management practices, genetics, and varied environmental conditions. Understanding best management practices and using better genotypes are essential to closing yield gaps between current on-farm yields and maximum attainable yield. This project seeks to take into account the multitude of factors that influence farmers’ decisions in an effort to achieve higher yields through best management practices. Kansas State University Agricultural Experiment Station and Cooperative Extension Service Soil Characterization and Phenological Information for Both Sitesh Soil Characterization and Phenological Information for Both Sites Soil samples were collected prior to planting and fertilization. The Ottawa site had low values of P but a slightly higher organic matter content than Topeka. Both sites were planted the same date with hybrids of similar maturity groups. Soil samples were collected prior to planting and fertilization. The Ottawa site had low values of P but a slightly higher organic matter content than Topeka. Both sites were planted the same date with hybrids of similar maturity groups. Stand Countsh The plant population after emergence was measured for all the plots at both sites. The target population for the treatments at “Normal” seeding rate was 45,000 plants per acre (commonly used by producers). For the “Optimum” seeding rate, the target popu lation was 90,000 plants per acre. 2015 Seasonal Precipitation and Irrigation for All Sites At the Kansas River Valley Experiment Field in Topeka, KS the seasonal rainfall totaled 16.2 inches with an additional 4.2 inches of irrigation applied. At the East Central Kan sas Experiment Field in Ottawa, KS, there was a total of 10.9 inches of rainfall during the growing season. Kansas Field Research 2016 Kansas Field Research 2016 included: plant population stand counts, leaf area index (LAI) at V5 and flowering, chlorophyll (SPAD) readings at V5 and flowering, aboveground biomass and nutrient concentrations at these growth stages, and grain yield and its components (grain num ber and seed weight). Kansas State University Agricultural Experiment Station and Cooperative Extension Service Procedures At both locations, Topeka, KS (irrigated); and Ottawa, KS (dryland); the plots were set up in a randomized complete block design with 5 replications and 11 treatments in each replication (Table 1). In Topeka, KS, the plots were 10 × 70 ft. In Ottawa, KS the plots were 10 × 50 ft, or 0.01 acres. The hybrids used were DKS 53-67 for Topeka, and DKS 44-20 for Ottawa, these were chosen based on the Kansas Performance Tests for their suitability to each specific site. Measurements for plant characterization were taken at the V5 growth stage, at flowering, and at harvest. The measurements taken Kansas State University Agricultural Experiment Station and Cooperative Extension Service 1 CV = Coefficient of Variation (%). CV = Coefficient of Variation (%). Results Grain sorghum yields portrayed a contrasting picture at the evaluated sites. In Topeka under irrigation, sorghum yields ranged from 149 to 166 bu/a (Figure 2). At Ottawa under dryland, sorghum yields ranged from 78 to 100 bu/a (Figure 3). In Topeka, the higher yield potentials were related to the irrigation scheduling system, with the high est yields being achieved from treatments 4 and 10. The yield gaps between the highest yielding and the lowest was 17 bu/a, and the two highest yielding treatments were sig nificantly different from the rest. This yield difference can be partially explained by the fertilizer N program employed during the 2015 growing season. With the GreenSeeker technology and in-season N application there was damage done to the green leaves on the plants, which was counter-productive by inhibiting photosynthesis and limiting yields (producing rapid senescence). The yield gaps across all the other treatments were minimized with the addition of adequate irrigation. The yields in Ottawa were quite different with the greatest yield gap being 22 bu/a between the highest, treatment #2, and the lowest, treatment #10 (Figure 3). The yields were limited overall by the low precipitation of only 10.8 inches (Figure 1) during the growing season. Treatment #2 included a high input, narrow row spacing, except at a low seeding rate; and treatment #10 was a low input treatment with traditional 30 inch row spacing and low seeding rate. This site demonstrated how narrowing the row spacing and utilization of improved management practices can maximize the yields even in a low-yielding, water-limiting environment. Regardless of the treatment evalu ated in both sites, the yield per plant was highly related to the grain number per head (Figure 4). By focusing on this yield component, such as how management factors affect the grain number per head, the variation in yields across environments can be better explained. 2 2 Treatments 5 6 7 8 m Optimum Optimum Optimum Optimu 15 in. 15 in. 15 in. 15 in. rd GS GS GS GS No Yes Yes Yes n Fe, Zn None Fe, Zn Fe, Zn Yes Yes No Yes Zn NPKSZn NPKSZn NPKSZn NP Yes Yes Yes Yes No No No No w spacing; 30 in. = wide row spacing; GS = GreenSeeker meter Iron; Zn = Zinc; N = Nitrogen; P = Phosphorous; K = Potassi Kansas Field Research 2016 Kansas State University Agricultural Experiment Station and Cooperative Extension Service Kansas Field Research 2016 Table 2. Soil characterization before planting Topeka Ottawa Soil parameters 0-6” 6-24” 0-6” 6-24” pH (units) 6.9 6.9 6.3 6.5 Mehlich P (ppm) 67.1 40.2 12.1 4.6 K (ppm) 395 287.9 128.1 248.9 CEC (meq/100 g) 17.9 19.4 20.5 28.4 OM (%) 2.86 2.26 3.15 2.71 Table 2. Soil characterization before planting Table 3. Grain sorghum phenology during the 2015 growing season at Ottawa and Topeka sites Plant phenology Topeka Ottawa Planting date June 9 June 9 V5 Growth stage July 7 July 7 Flowering August 10 August 12 Harvest September 30 October 12 Table 3. Grain sorghum phenology during the 2015 growing season at Ottawa and Topeka sites Table 4. Stand counts at each treatment and for each site Treatments Topeka Ottawa ----------------- plants in 17.5 ft row-length ----------------- 1 92 88 2 48 46 3 88 90 4 92 90 5 95 90 6 92 89 7 91 90 8 92 91 9 92 91 10 49 47 11 92 88 CV 3.1 3.7 CV = Coefficient of Variation (%). Kansas State University Agricultural Experiment Station and Cooperative Extension Service ansas State University Agricultural Experiment Station and Cooperative Extension Service Kansas State University Agricultural Experiment Station and Cooperative Extension Service 4 Kansas Field Research 2016 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Precipitation, in Days since planting 42 35 21 7 49 98 91 77 63 112 84 105 70 56 28 14 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Precipitation, in Days since planting 42 35 21 7 49 98 91 77 63 119 84 105 70 56 28 14 Irrigation Rainfall 112 a b Figure 1. (a) 2015 seasonal precipitation in Topeka, KS, (b) 2015 seasonal precipitation in Ottawa, KS. Kansas Field Research 2016 Kansas Field Research 2016 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Precipitation, in Days since planting 42 35 21 7 49 98 91 77 63 112 84 105 70 56 28 14 Irrigation Rainfall a 3.0 2.5 2.0 1.5 1.0 0.5 0 Precipitation, in Days since planting 42 35 21 7 49 98 91 77 63 112 84 105 70 56 28 14 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Precipitation, in Days since planting 42 35 21 7 49 98 91 77 63 119 84 105 70 56 28 14 112 b Figure 1. Kansas Field Research 2016 (a) 2015 seasonal precipitation in Topeka, KS, (b) 2015 seasonal precipitation in Ottawa, KS. Days since planting 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Precipitation, in Days since planting 42 35 21 7 49 98 91 77 63 119 84 105 70 56 28 14 112 b Figure 1. (a) 2015 seasonal precipitation in Topeka, KS, (b) 2015 seasonal precipitation in Ottawa, KS. Kansas State University Agricultural Experiment Station and Cooperative Extension Service 6 a 180 170 160 150 140 130 120 110 100 90 80 70 60 50 Yield, bu/a Treatment 4 b 3 a 2 a 1 a 5 a 10 b 9 a 8 a 7 a 11 a Figure 2. 2015 grain sorghum yield under diverse crop production practices at the Topeka unit of the Kansas River Valley Experiment Field. See Table 1 for treat ment details. Different letter shows statistical significance (P < 0.05). 6 a 180 170 160 150 140 130 120 110 100 90 80 70 60 50 Yield, bu/a Treatment 4 b 3 a 2 a 1 a 5 a 10 b 9 a 8 a 7 a 11 a Treatment Figure 2. 2015 grain sorghum yield under diverse crop production practices at the Topeka unit of the Kansas River Valley Experiment Field. See Table 1 for treat ment details. Different letter shows statistical significance (P < 0.05). Kansas State University Agricultural Experiment Station and Cooperative Extension Service 5 Kansas Field Research 2016 6 a 180 170 160 150 140 130 120 110 100 90 80 70 60 50 Yield, bu/a Treatment 4 c 3 a 2 b 1 a 5 a 10 c 9 c 8 a 7 a 11 a Figure 3. 2015 grain sorghum yield under diverse crop production practices at the Ottawa site of East Central Kansas. See Table 1 for treatment details. Different letter shows statis tical significance (P < 0.05). 6 a 180 170 160 150 140 130 120 110 100 90 80 70 60 50 Yield, bu/a Treatment 4 c 3 a 2 b 1 a 5 a 10 c 9 c 8 a 7 a 11 a Figure 3. 2015 grain sorghum yield under diverse crop production practices at the Ottawa site of East Central Kansas. See Table 1 for treatment details. Different letter shows statis tical significance (P < 0.05). Kansas State University Agricultural Experiment Station and Cooperative Extension Service Kansas Field Research 2016 2,000 100 80 60 40 20 0 Yield per plant, grams Grain number per head 1,000 Y = 0.023X r2 = 0.89 n = 64 0 3,000 4,000 Figure 4. Grain number per head vs. yield per plant relationship for both sites for the 2015 growing season, regardless of treatment. 2,000 100 80 60 40 20 0 Yield per plant, grams Grain number per head 1,000 Y = 0.023X r2 = 0.89 n = 64 0 3,000 4,000 Figure 4. Grain number per head vs. yield per plant relationship for both sites for the 2015 growing season, regardless of treatment. Kansas State University Agricultural Experiment Station and Cooperative Extension Service Kansas State University Agricultural Experiment Station and Cooperative Extension Service 6
https://openalex.org/W3163726975	https://link.springer.com/content/pdf/10.1007/s12094-021-02632-7.pdf	English	null	Sexual, bladder and bowel function following different minimally invasive techniques of radical hysterectomy in patients with early-stage cervical cancer	Clinical & translational oncology	2,021	cc-by	6,573	Abstract Purpose Despite the establishment of radical surgery for therapy of cervical cancer, data on quality of life and patient- reported outcomes are scarce. The aim of this retrospective cohort study was to evaluate bladder, bowel and sexual function in women who underwent minimally invasive surgery for early-stage cervical cancer. Purpose Despite the establishment of radical surgery for therapy of cervical cancer, data on quality of life and patient- reported outcomes are scarce. The aim of this retrospective cohort study was to evaluate bladder, bowel and sexual function in women who underwent minimally invasive surgery for early-stage cervical cancer. Methods From 2007–2013, 261 women underwent laparoscopically assisted radical vaginal hysterectomy (LARVH = 45), vaginally assisted laparoscopic or robotic radical hysterectomy (VALRRH = 61) or laparoscopic total mesometrial resec- Purpose Despite the establishment of radical surgery for therapy of cervical cancer, data on quality of life and patient- reported outcomes are scarce. The aim of this retrospective cohort study was to evaluate bladder, bowel and sexual function in women who underwent minimally invasive surgery for early-stage cervical cancer. Methods From 2007–2013, 261 women underwent laparoscopically assisted radical vaginal hysterectomy (LARVH = 45), vaginally assisted laparoscopic or robotic radical hysterectomy (VALRRH = 61) or laparoscopic total mesometrial resec- tion (TMMR = 25) and 131 of them completed the validated German version of the Australian Pelvic Floor Questionnaire (PFQ). Results were compared with controls recruited from gynecological clinics (n = 24) and with urogynecological patients (n = 63).i Methods From 2007–2013, 261 women underwent laparoscopically assisted radical vaginal hysterectomy (LARVH = 45), vaginally assisted laparoscopic or robotic radical hysterectomy (VALRRH = 61) or laparoscopic total mesometrial resec- tion (TMMR = 25) and 131 of them completed the validated German version of the Australian Pelvic Floor Questionnaire (PFQ). Results were compared with controls recruited from gynecological clinics (n = 24) and with urogynecological patients (n = 63).i Results Groups were similar regarding age, BMI and parity. The TMMR group had significantly shorter median follow-up (16 months versus 70 and 36 months). Postoperatively, deterioration of bladder function was reported by 70%, 57% and 44% in the LARVH, VARRVH and TMMR groups, respectively (p = 0.734). Bowel function was significantly worse after TMMR with a higher deterioration rate in 72 versus 43% (LARVH) and 47% (VARRVH) with a correspondingly higher bowel dysfunction score of 2.9 versus 1.5 and 1.8, respectively and 1.8 in urogynaecological patients. Clinical and Translational Oncology (2021) 23:2335–2343 https://doi.org/10.1007/s12094-021-02632-7 Clinical and Translational Oncology (2021) 23:2335–2343 https://doi.org/10.1007/s12094-021-02632-7 RESEARCH ARTICLE Abstract Sexual dysfunction was com- mon in all surgical groups. 38% considered their vagina too short which was significantly associated with deep dyspareunia. Compared with controls, surgical groups had significantly increased PFQ scores.l p g g p gi y Conclusion Pelvic floor dysfunction commonly deteriorates and negatively impacts on quality of life after minimally invasive radical hysterectomy, especially bowel function after TMMR. Pelvic floor symptoms should routinely be addressed pre- and postoperatively. ds Pelvic floor function · Cervical cancer · Quality of life · Minimally invasive surgery · Urinary in Keywords Pelvic floor function · Cervical cancer · Quality of life · Minimally invasive surgery · Urinary incontinence · Sexual function Sexual, bladder and bowel function following different minimally invasive techniques of radical hysterectomy in patients with early‑stage cervical cancer K. Baessler1,2 · S. Windemut3 · V. Chiantera4 · C. Köhler5 · J. Sehouli1 Received: 11 March 2021 / Accepted: 26 April 2021 / Published online: 18 May 2021 © The Author(s) 2021 * K. Baessler Kaven.baessler@franziskus-berlin.de 1 Department of Gynecology with Center for Oncological Surgery, Charité-Universitätsmedizin Berlin, Berlin, Germany Introduction * K. Baessler Kaven.baessler@franziskus-berlin.de Cervical cancer is one of the most frequent and challenging diseases worldwide [1]. Besides oncological aspects, quality of life like pelvic floor function including sexuality is rel- evant but underreported in scientific reports [2, 3]. Surgery remains the cornerstone in the treatment of early cervical cancer. Abdominal radical hysterectomy has dominated sur- gical techniques for decades with complications like persist- ing voiding problems in up to 41% [4], bowel symptoms in up to 58% [5], sexual dysfunction in up to 60% [4, 6] and lymphedema in up to 19% [7, 8] of cases. Some stud- ies comparing open and laparoscopic radical hysterectomy demonstrated similar oncological outcome [2, 9] whereas 1 Department of Gynecology with Center for Oncological Surgery, Charité-Universitätsmedizin Berlin, Berlin, Germany 2 Pelvic Floor Centre Franziskus and St Joseph Hospital Berlin, Budapester Str. 15‑19, 10787 Berlin, Germany 3 Department of Gynecology, Vivantes Hospital Am Urban, Berlin, Germany 4 Department of Gynecologic Oncology, University of Palermo, Palermo, Sicilia, Italy 5 Department of Gynecology, Medical Faculty, University of Cologne, Cologne, Germany 2 Pelvic Floor Centre Franziskus and St Joseph Hospital Berlin, Budapester Str. 15‑19, 10787 Berlin, Germany 3 Department of Gynecology, Vivantes Hospital Am Urban, Berlin, Germany 4 Department of Gynecologic Oncology, University of Palermo, Palermo, Sicilia, Italy 5 Department of Gynecology, Medical Faculty, University of Cologne, Cologne, Germany (0121 3456789) 3 2336 Clinical and Translational Oncology (2021) 23:2335–2343 The German version of the Australian Pelvic Floor Questionnaire (PFQ) [20] assesses bladder, bowel, prolapse and sexual symptoms [18] and includes a validated post- treatment module evaluating the impression of improve- ment or deterioration in each pelvic floor domain [19]. We added particularly interesting questions: do you think your vagina is too short? (yes–no); has your ability to achieve orgasm changed after the operation? (unchanged, improved, worsened); have you been diagnosed with lymphedema? (yes–no).f a recent randomized controlled trial (LACC trial) did not confirm this. i Several different minimally invasive techniques incorpo- rating varying degrees of nerve-sparing have been described. During laparoscopically assisted radical vaginal hysterec- tomy (LARVH), the vaginal parametrial resection is pre- ceded by laparoscopic staging and lymph node resection [10, 11]. Introduction Due to a long learning curve [11] and increased rates of urological complications during the vaginal part of the operation [12], an alternative technique was developed including creation of a vaginal cuff enclosing the cervix, laparoscopic radical hysterectomy with enbloc removal of uterus and parametria vaginally (vaginally assisted laparo- scopic radical hysterectomy = VALRH) [13]. The laparo- scopic part can also be performed Roboter-assisted (vagi- nally assisted robotic radical hysterectomy = VARRH) and achieved similar oncologic results [14]. Given the reported minimal important difference and effect sizes of the Australian PFQ and its German version [19, 21], we considered differences in domain scores of ≥1 as clinically important. The German validation paper [18] reported a global dysfunction score of 2.6 in healthy con- trols. To demonstrate a difference of 1.5 in the global score, 80% power and alpha = 0.05, 24 women had to be included in each group. However, we chose to invite all consecutive women to allow for multiple comparisons. To be able to judge the magnitude of pelvic floor dysfunction after sur- gery, we compared questionnaire scores with the control group recruited from gynecological clinics with non-malig- nant diseases (e.g., fibroids, n = 24) and women seeking care in our urogynecology unit (n = 66). None of these women were on anticholinergics or pessary treatment at the time of recruitment and none had undergone pelvic surgery includ- ing hysterectomy. The concept of abdominal total mesometrial resection (TMMR) is based on ontogenetic anatomy: The Müllerian compartment is completely removed during surgery apart from the vagina to maintain sexual function [15]. TMMR can also safely be performed laparoscopically [16]. Besides oncological safety, quality of life and pelvic floor function is important, especially in younger women. A recent meta-analysis described favorable bladder function after laparoscopic nerve-sparing radical hysterectomy [2]. Unfortunately, although eight studies reported bladder and bowel function, only two studies looked at sexual function. Statistical analysis was performed using IBM SPSS Sta- tistics. For normally distributed variables, ANOVA was used with post hoc Bonferoni testing. For dichotomous variables, the Chi-square test or Fisher’s exact tests as appropriate were performed. For ordinal variables, the non-parametric Kruskal–Wallis test was employed. The aim of this study was to evaluate bladder, bowel and sexual function in women who underwent LARVH, VALRH, VARRH or laparoscopic TMMR for early-stage cervical cancer. This is an ancillary report to Lucidi et al. Introduction [17] considering all women who answered a validated pel- vic floor questionnaire at one center and comparing them to urogynecological patients and gynecologic controls. In accordance with the journal’s guidelines, we will pro- vide our data for the reproducibility of this study in other centers if such is requested. Results 456a BMI (mean ± SD) 24.1 ± 4.3 25.2 ± 6.0 24.1 ± 3.6 24.3 ± 3.7 0.884a Operating time (mean min ± SD) 318.2 ± 73.0 278.3 ± 82.1 336.7 ± 66.2 228.0 ± 54.1 < 0.001a Number of lymph nodes (mean ± SD) 28.5 ± 14.0 28.4 ± 16.8 44.1 ± 15.0 15.6 ± 11.8 < 0.001a PIVER II 60 (61%) 64 (62%) 20 (83%) 20 (57%) 0.153b III 38 (39%) 40 (38%) 4 (17%) 15 (43%) TNM IA1L1 7 (7%) 11 (11%) 0 2 (6%) 0.430b IA2 8 (8%) 4 (4%) 1 (4%) 3 (9%) IB1 74 (75%) 73 (70%) 23 (96%) 27 (77%) IB2 2 (2%) 9 (9%) 0 1 (3%) IIA1 1 (1%) 0 0 0 IIA2 1 (1%) 0 0 0 IIB 5 (5%) 7 (7%) 0 2 (6%) Hospital stay (mean ± SD) 12 ± 3 10 ± 3 10 ± 3 8 ± 2 < 0.001a VALRRH and TMMR groups, respectively, reported sub- jective deterioration of bladder function (p = 0.734). Stress urinary incontinence was present postoperatively in 81/130 women (62%) and overactive bladder symptoms in 50/131 (38%). Prevalence did not differ between surgical groups. Age, operating technique and Piver-Rutledge class did not impact on bladder function scores although overactive bladder including nocturia correlated with age (r = 0.23; p = 0.009). (r = 0.28; p = 0.002). The time of first postoperative bowel motion was not different between groups (median 4, range 1–8; p = 0.265), but more women needed laxatives more than once in the TMMR group (19/35; 54%) compared to LARVH (36/98; 37%), VALRH (34/104; 34%) and VARRH groups (6/24; 25%; p < 0.001). (r = 0.28; p = 0.002). The time of first postoperative bowel motion was not different between groups (median 4, range 1–8; p = 0.265), but more women needed laxatives more than once in the TMMR group (19/35; 54%) compared to LARVH (36/98; 37%), VALRH (34/104; 34%) and VARRH groups (6/24; 25%; p < 0.001). Of the 261 women, 131 women completed the ques- tionnaire (53%; 45 LARVH, 51 VALRH, 10 VARRH, 25 TMMR). Groups did not differ regarding age, BMI and par- ity (Table 3). The analysis of women who did and those who did not reply did not show a difference in age, BMI, TNM, Piver-Rutledge classification and length of follow- up (data not shown). Results This retrospective cohort study was approved by the Char- ité Institutional Ethical Review Board. Written informed consent was obtained. All 261 women who underwent minimally invasive surgery for early-stage cervical cancer (≤ FIGO stage II) from 2005–2013, older than 18 years of age were asked to complete a validated pelvic floor question- naire [18, 19]. From 2005–2007, LARVH was performed (n = 98), thereafter VALRH (n = 104) and VARRH (n = 24). Laparoscopic TMMR was performed from 2011 (n = 35). Perioperative characteristics of all 261 women are given in Table 1. Operating time correlated negatively with age (r = − 0.144; p = 0.02) and positively with BMI (r = 0.186; p < 0.001). In the TMMR group the number of removed lymph nodes was significantly smaller compared to all other groups (p < 0.001). Radicality and perioperative complica- tions including urinary tract infections, thrombosis and infection did not differ between operating techniques. f At the time of hospital discharge, only 4/35 women (12%) in the TMMR group versus 65/226 (29%) in the other groups had a postvoid residual of more than 50 ml (p < 0.001; Chi- square test). Length of hospital stay correlated with length of catheter use (r = 0.66, p < 0.001) and operating time Demographic and perioperative data were obtained from hospital charts. Postoperative voiding dysfunction was defined as postvoid residual of more than 50 ml when discharged. We noted first postoperative defecation and required doses of laxatives. 1 3 1 3 Clinical and Translational Oncology (2021) 23:2335–2343 2337 scopically assisted radical vaginal hysterectomy, VALRH-vaginally assisted laparoscopic radical hysterectomy, TMMR- laparoscopic total mesometrial resection) Table 1 Demographic and perioperative data, PIVER classification, TNM-stage and histopathological tumor type in all 261 women who underwent surgery for early-stage cervical cancer (LARVH-laparo- Percentages may not sum to 100 due to rounding a ANOVA b Chi-square test LARVH N = 98 VALRH N = 104 Robotic VALRH N = 24 TMMR N = 35 p Age (mean ± SD) 44.6 ± 11.5 46.3 ± 11.4 45.4 ± 9.1 47.8 ± 10.8 0. Results Worsened Analysis of the subjective impression of changes (Table 2) showed that 70%, 57% and 44% in the LARVH, 1 3 Clinical and Translational Oncology (2021) 23:2335–2343 2338 Table 2 Displayed are follow-up time, radiation therapy, pelvic floor questionnaire domain scores and improvement scales as well as postoperative impression of a short vagina in the surgical groups (LARVH- laparoscopically assisted radical vaginal hysterectomy, VALRRH-vaginally assisted laparoscopic or robotic radical hysterectomy, TMMR- laparoscopic total mesometrial resection) Percentages do not necessarily sum up to 100 due to rounding a Kruskal-Wallis test b Chi-square test LARVH N = 45 VALRRH N = 61 TMMR N = 25 p Follow-up time (months; median, range) 70 (5–103) 36 (5–76) 16 (4–26) < 0.001a Radiation therapy 6 (13%) 13 (21%) 6 (24%) 0.460b Subjective impression Bladder function 0.734a No change Worsened Improved 13 (30%) 31 (70%) 22 (37%) 34 (57%) 4 (7%) 9 (36%) 11 (44%) 5 (20%) Subjective impression Bowel function 0.024a No change Worsened Improved 25 (57%) 19 (43%) 30 (50%) 28 (47%) 2 (3%) 6 (24%) 18 (72%) 1 (4%) Bladder function score (median, range) 2 (0–5.8) 1.8 (0–6.9) 1.1 (0–6.4) 0.362a Bowel function score (median, range) 1.5 (0.3–5.3) 1.8 (0–7.1) 2.9 (0.9–5.9) 0.001a Prolapse domain score (median, range) 0 (0–4) 0 (0–6.7) 0 (0–0.8) 0.278a Sexual function score (median, range) 1.4 (0–10) 2.4 (0–10) 3.3 (0–6.7) 0.178a Global Pelvic Floor dysfunction score (median, range) 5.4 (0.3–18.9) 5.9 (0.8–19.8) 8 (2–15.4) 0.316a Impression short vagina 11 (32%) 19 (41%) 6 (24%) 0.716b Table 2 Displayed are follow-up time, radiation therapy, pelvic floor questionnaire domain scores and improvement scales as well as postoperative impression of a short vagina in the surgical groups (LARVH- laparoscopically assisted radical vaginal hysterectomy, VALRRH-vaginally assisted laparoscopic or robotic radical hysterectomy, TMMR- laparoscopic total mesometrial resection) VALRH-vaginally assisted laparoscopic radical hysterectomy, TMMR-laparoscopic total mesometrial resection) VALRH-vaginally assisted laparoscopic radical hysterectomy, TMMR-laparoscopic total mesometrial resection) Table 3 Comparison of women after laparoscopic/robotic radical hysterectomy or TMMR, controls and urogynaecological patients (LARVH-laparoscopically assisted radical vaginal hysterectomy, Table 3 Comparison of women after laparoscopic/robotic radical hysterectomy or TMMR, controls and urogynaecological patients (LARVH-laparoscopically assisted radical vaginal hysterectomy, K k l W lli t t LARVH N = 45 VALRRH N = 61 TMMR N = 25 Controls N = 24 Urogyn N = 66 p Age (years; median, range) 43 (25–73) 45 (27–76) 51 (30–68) 46 (25–67) 511 (30–81) 0.015a Parity (median, range) 1 (0–3) 1 (0–4) 1 (0–4) 1 (0–4) 2 (0–5) 0.654 Bladder function score (median, range) 2 (0–5.8) 1.8 (0–6.9) 1.1b (0–6.4) 0.7b (0–2) 3.2c (0–6.7) < 0.001b, c Bowel function score (median, range) 1.5 (0.3–5.3) 1.8 (0–7.1) 2.9d (0.9–5.9) 0.9e (0–3.2) 1.8 (0–5.6) < 0.001d, e Prolapse domain score (median, range) 0 (0–4) 0 (0–6.7) 0 (0–0.8) 0 (0–4) 0.1f (0–7.3) < 0.001f Sexual function score (median, range) 1.4 (0–10) 2.4 (0–10) 3.3 (0–6.7) 0g (0–3.7) 1.9 (0–5.2) 0.001g Global PF dysfunction score (median, range) 5.4 (0.3–18.9) 5.9 (0.8–19.8) 8 (2–15.4) 2.6 (0–11.4) 9.3 (2.1–15.9) < 0.001h * Kruskal–Wallis tests a post hoc comparison shows urogynaecological patients were significantly older compared to LARVH only b Controls had a significantly lower (better) bladder domain score compared with all groups apart from TMMR c Urogynaecological patients had a significantly higher (worse) bladder domain scores compared with all other groups d TMMR patients had a significantly higher (worse) bowel function score compared with all groups e Controls had a significantly lower (better) bowel domain score f The prolapse symptom score was significantly higher (worse) in the urogynaecological patients compared with all groups g Sexual function scores were significantly lower (better) in the control group h Global PF scores in the RH/TMMR groups were significantly higher (worse) compared to the control group and lower (better) compared with urogynaecological patients n shows urogynaecological patients were significantly older compared to LARVH only introitus and deep pain 10/94 (11%) women without dif- ferences between groups. Results Given the low number in the robotic group, we combined the laparoscopic and robotic cases as operating steps were similar and analysis of complications and outcomes did not reveal statistically relevant differences apart from operating time (vaginally assisted laparoscopic or robotic radical hysterectomy = VALRRH, n = 61). Median follow-up time was significantly shorter in the TMMR group at 16 months (p < 0.001; Table 2). One woman completed the questionnaire at 4 months and five women between 6 and 7 months, the others between 12 and 26 months. In the LARVH group, 96% completed the questionnaire more than 24 months after surgery and 69% of the women in the VAL- RRH group (p > 0.05; Table 2). Bowel function worsened in 72% after TMMR which is significantly more often compared with LARVH (43%) and VALRRH (47%; p=0.024; Table 2). Women after TMMR also had worse bowel dysfunction scores and reported more frequently constipation, straining at defecation and incom- plete bowel emptying, (60 versus 39% and 44%, respectively; p=0.004). They also described bowel dysfunction as more bothersome than women in the other groups (p=0.001). Age and postoperative radiation therapy were associated with fecal urgency and fecal incontinence for loose stool (p<0.021). The sexual function domain of the questionnaire was completed by 122 women. Of those, 87 (71%) were sexu- ally active at follow-up without differences between surgical groups. Reasons for sexual abstinence were lack of partner (47%), partner impotent (15%), dyspareunia (12%), vaginal dryness (6%) and low desire (6%). Our additional questions were answered by a variable number of women. Results Thirty-eight percent of women (36/96) considered their vagina too short after the operation and 28% (27/96) too narrow or too tight without differences between groups (Table 3). These symptoms were associated introitus and deep pain 10/94 (11%) women without dif- ferences between groups. Thirty-eight percent of women (36/96) considered their vagina too short after the operation and 28% (27/96) too narrow or too tight without differences between groups (Table 3). These symptoms were associated ability to climax during sexual activity was reported by 33/99 (33%) women (10/33 after LARVH; 18/49 after VALRRH; 5/17 after TMMR; p=0.249). Reduced desire described 39/105 (37%) women, superficial dyspareunia 21/94 (22%), deep dyspareunia 32/94 (34%) and both, 1 3 Clinical and Translational Oncology (2021) 23:2335–2343 2339 impaired bladder, bowel and sexual function. Pelvic floor dysfunction scores were clinically and statistically signifi- cantly worse compared with controls. with each other (p=0.006) and also with postoperative radi- ation (p<0.034). Deep dyspareunia was associated with a short vagina (p<0.001). Women with postoperative radiation regardless of surgical technique had a significantly higher (worse) sexual function score (3.3 versus 1.4; p<0.001). Coital incontinence was present in 21/93 women (23%). Sexual symptoms were considered moderately and greatly bothersome by 40% which was associated with postoperative radiation (p=0.017). Laparoscopic TMMR resulted in early recovery of blad- der emptying but led to aggravated bowel dysfunction including constipation and incomplete bowel emptying. The difference in bowel domain scores between TMMR, other surgical groups, urogynecological patients and con- trols was ≥ 1, which is the estimated minimal important difference of the Australian Pelvic Floor Questionnaire [21]. Interestingly, subjective bothersomeness of bowel dysfunction was greater after TMMR even compared with urogynecological patients. Although bladder dysfunction after TMMR was similar to controls, 44% of women after TMMR reported worsened bladder function. Apart from sig- nificantly worse bowel function after TMMR the differences of pelvic floor dysfunction between surgical techniques were small. Surgical radicality and nerve sparing aspects can be considered similar between LARVH and VALRRH, only the TMMR technique differs with Müllerian compartment resec- tion. Although the TMMR technique claims the preservation of nerve structures, the presacral space is dissected which might lead to constipation comparable to complications after rectopexy [22]. Compared to controls, women after surgery showed sig- nificantly higher bladder, bowel and sexual dysfunction scores (p < 0.001, Table 3, Fig. 1). Results Symptoms of pelvic organ prolapse were not more common than in controls (p = 0.720). When LARVH, VALRRH and TMMR groups were sepa- rately compared with controls, bladder function was similar to controls only in the TMMR group. Bowel dysfunction was significantly more frequently reported as bothersome after TMMR compared to other surgical groups as well as to urogynecological patients and controls (p ≤ 0.004). Uro- gynecological patients were significantly older than women in the LARVH group, but there were no differences to all other groups (Table 3). After LARVH, 28/45 women (62%) had clinically rele- vant self-reported lymphedema, after VALRRH 39/61 (64%) and after TMMR 9/25 (36%; p = 0.045; Chi-square test). Recurrent disease at follow-up was present in two (4%), three (5%) and one (4%), respectively (p = 0.981). We found a high rate of dyspareunia with 67% of all women complaining of introitus and/or deep pain during intercourse. A shortened vagina was reported frequently after surgery and was associated with deep dyspareunia. Radiation therapy aggravated sexual function.i Discussion In contrast to our findings, published urinary function data for abdominal TMMR show higher rates of overactive bladder and stress urinary incontinence but less constipation [23]. Whether this is an effect of the laparoscopic technique remains open. A small prospective case series demonstrated a high rate of early postoperative urodynamic detrusor overactivity which persisted long term in 6/9 women after This study focused on patient-centered outcomes and uti- lized a validated pelvic floor questionnaire to evaluate the impact of different minimally invasive radical hysterec- tomy techniques or total mesometrial resection on pelvic floor function. All evaluated surgical techniques resulted in 1 3 Fig. 1 Stacked column plot of median pelvic floor domain scores in the different surgical groups compared to controls and urogynaecological patients. The global pelvic floor ques- tionnaire scores in the surgical groups were significantly higher (worse) compared to the control group and lower (better) compared with urogynaeco- logical patients (LARVH- laparoscopically assisted radical vaginal hysterectomy, VALRH- vaginally assisted laparoscopic radical hysterectomy, TMMR- laparoscopic total mesometrial resection) 1 1 3 3 2340 Clinical and Translational Oncology (2021) 23:2335–2343 The rate of dyspareunia was high at 67%. A similar rate was reported in women after robotic radical hysterectomy [34]. Given the creation of a vaginal cuff enclosing the cervix prior to radical hysterectomy, the VALRRH groups would be most prone to a shortened vagina. More women in this group (41 versus 32% and 24%) reported this without a statistically significant difference. Although the symptom of a short vagina has been reported before [34–36], our study demonstrated that a subjectively short and narrow vagina interferes with sexual intercourse. Höckel et al. reported only few problems after abdominal TMMR but there was no systematic analysis of sexual aspects [23]. Sowa et al. com- pared TMMR with the classic Wertheim-Meigs operation and did not find differences regarding sexuality [37]. Some studies imply that sexual problems increase with length of follow-up [27]. Long-term evaluation should be considered in all patients after radical hysterectomy due to the fact that survival rates are high and morbidity data after 5 years are scarce. nerve-sparing radical hysterectomy [24]. Given our study design, we might have missed this effect. Discussion Compared with our control group as well as published community cohorts, the prevalence of stress and urge urinary incontinence appeared similar [25].i Our data for first postoperative defecation on day 3 or 4 are similar to other studies [8, 26]. Postoperative con- stipation after radical hysterectomy has been reported in 19%[27]-35% [28], decreasing to 9% after 5 years [29]. This corresponds to our results with exception of the TMMR group, which had significantly more often defecation prob- lems. Given the significantly shorter follow-up time after TMMR, it remains open whether these symptoms recover after 2 years. A postvoid residual of more than 50 ml was considered pathological in our institution [16]. This cut-off might not be standard in other centers and we might overestimate voiding dysfunction. Hospital stay was rather long between 8–12 days, but is consistent with national practice. It can also be explained with the longer catheter use. Postoperative lymphedema is common [7] and our rates are similar to reports after robotic radical hysterectomy [34]. The rate of postoperative lymphedema was lowest after TMMR which also corresponds to the fact that less lymph nodes were dissected (Table 1). We did not assess how much lymphedema interferes with quality of life but others described high levels of distress [7, 34] When interpreting our results, it has to be taken into account that pelvic floor dysfunction is very common in women. Urinary incontinence has a high prevalence of more than 50% in postmenopausal women [25]. Data evaluated in this study do not seem particularly high compared with urogynecological patients but well above rates established in non-urogynecological controls. Symptoms were also con- sidered more bothersome than in controls. Our results are in contrast to Novackova et al. [30]. They found less stress uri- nary incontinence and postoperative urodynamic evaluation did not differ significantly 12 months after nerve-sparing radical hysterectomy. The differences might be attributed to different nerve-sparing techniques but also to their lack of validated questionnaires and short follow-up time [30]. Strengths and weaknesses Limitations of this study include lack of preoperative data including pelvic floor dysfunction and different lengths of follow-up. This study was not prospectively planned, not randomized and institutional surgical techniques had been transformed according to complication profiles. Further- more, the Australian Pelvic Floor Questionnaire and its German version has been validated in community-dwelling women [38], patients attending gynecological and urogynae- cologic clinics [20] as well as in pregnant and postpartum women [39] but not specifically in gynecological cancer patients. Nevertheless, it is a strength of this study that a validated self-administered instrument including postop- erative scales of impression of improvement was used and the minimal important difference is known. Also, the com- parison to healthy controls and urogynecological patients provide information on the magnitude of the reported pelvic floor symptoms. In our view, it helps to interpret the severity of pelvic floor symptoms. Apart from bowel function after TMMR, PFQ domain scores were lower compared with urogynecological patients. As women in the control groups had not undergone hyster- ectomy, comparison of pelvic floor symptoms might be con- sidered inappropriate. However, a systematic review of uro- dynamic outcomes after hysterectomy for benign diseases showed that urinary function is not adversely affected and might improve after hysterectomy [31]. We had purposely chosen urogynecological patients without any pelvic surgery to reduce possible influences as historically many hysterec- tomies had been performed for pelvic organ prolapse [32]. Regardless of the surgical technique, adjuvant radiation therapy lead to more pelvic floor dysfunction including con- stipation, excessive straining and incomplete bowel empty- ing as well as sexual problems. This is in accordance with others who also reported lower general health-related quality of life after radiotherapy [33]. Especially sexual dysfunc- tion was worse after radiotherapy which has been described before [27, 33]. l Further strengths include that the calculated sample size was reached and the study is powered to evaluate subjective pelvic floor function as a patient-centered outcome. Due to the methodological limitations, effect of postoperative radio- therapy cannot be analyzed in detail. Furthermore, stand- ardization of nerve-sparing techniques are ongoing [40]. References 1. Sant M, Chirlaque Lopez MD, Agresti R, Sanchez Perez MJ, Holleczek B, Bielska-Lasota M, Dimitrova N, Innos K, Katalinic A, Langseth H, Larranaga N, Rossi S, Siesling S, Minicozzi P. Survival of women with cancers of breast and genital organs in Europe 1999–2007: results of the EUROCARE-5 study. Eur J Cancer. 2015;51(15):2191–205. https://doi.org/10.1016/j.ejca. 2015.07.022. 2. Xue Z, Zhu X, Teng Y. Comparison of nerve-sparing radical hysterectomy and radical hysterectomy: a systematic review and meta-analysis. Cell Physiol Biochem. 2016;38(5):1841–50. https://doi.org/10.1159/000443122. Raising awareness of possible deterioration of pelvic floor function would enable health care providers to offer better support and early therapy. Future studies on cervical cancer surgery should include validated outcome meas- ures to assess pelvic floor function and especially sexual function. Also, possible prevention strategies should be addressed prospectively. p g 3. Laterza RM, Sievert KD, de Ridder D, Vierhout ME, Haab F, Cardozo L, van Kerrebroeck P, Cruz F, Kelleher C, Chapple C, Espuna-Pons M, Koelbl H. Bladder function after radical hyster- ectomy for cervical cancer. Neurourol Urodyn. 2015;34(4):309– 15. https://doi.org/10.1002/nau.22570. 4. Kenter GG, Ansink AC, Heintz AP, Aartsen EJ, Delemarre JF, Hart AA. Carcinoma of the uterine cervix stage I and IIA: results of surgical treatment: complications, recurrence and survival. Eur J Surg Oncol. 1989;15(1):55–60.ff 5. Griffenberg L, Morris M, Atkinson N, Levenback C. The effect of dietary fiber on bowel function following radical hysterectomy: a randomized trial. Gynecol Oncol. 1997;66(3):417–24. https://doi. org/10.1006/gyno.1997.4797. Acknowledgements We thank Prof. Achim Schneider for initializing this research when he was head of the Department of Gynecology at Charité Campus Benjamin Franklin and Mitte. 6. Schover LR, Fife M, Gershenson DM. Sexual dysfunc- tion and treatment for early stage cervical cancer. Cancer. 1989;63(1):204–12. Author’s contributions BK conceptualization, methodology, validation, formal analysis, writing—review and editing, project administration, supervision. WS investigation, formal analysis, writing—original draft. CV investigation, writing—review and editing, project administration. KC investigation, writing—review and editing, project administration. SJ resources, writing—review and editing, supervision. 7. Bergmark K, Avall-Lundqvist E, Dickman PW, Henningsohn L, Steineck G. Lymphedema and bladder-emptying difficulties after radical hysterectomy for early cervical cancer and among popula- tion controls. Int J Gynecolog Cancer. 2006;16(3):1130–9. https:// doi.org/10.1111/j.1525-1438.2006.00601.x. Funding Open Access funding enabled and organized by Projekt DEAL. Funding Open Access funding enabled and organized by Projekt DEAL. 8. Pieterse QD, Maas CP, ter Kuile MM, Lowik M, van Eijkeren MA, Trimbos JB, Kenter GG. Conclusions Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Despite the limitations of this retrospective study, we believe we provided relevant information on patient- centered outcomes of pelvic floor function after different techniques of minimally invasive radical hysterectomy and TMMR to improve counseling of women with cervical cancer. All evaluated minimally invasive techniques for patients with early-stage cervical cancer had a negative impact on pelvic floor function exceeding symptom scores in controls. Laparoscopic TMMR was initially better for bladder function but not long term and resulted in a per- sisting and significantly higher rate of bowel dysfunction. When choosing an operation for cervical cancer, pelvic floor symptoms should be taken into account and should be part of preoperative counseling, provided that onco- logic safety can be assured. For women with existing con- stipation, e.g., TMMR with a higher rate of postoperative bowel dysfunction might be less appropriate. Preopera- tive and postoperative assessment of pelvic floor function including sexuality should be mandatory in patients under- going surgery for cervical cancer. 3 3 Clinical and Translational Oncology (2021) 23:2335–2343 2341 References An observational longitudinal study to evaluate miction, defecation, and sexual function after radical hysterectomy with pelvic lymphadenectomy for early-stage cervi- cal cancer. Int J Gynecolog Cancer. 2006;16(3):1119–29. https:// doi.org/10.1111/j.1525-1438.2006.00461.x. Data availability Data will be made available on reasonable request. Declarations No sur- vival difference between robotic and open radical hysterectomy for women with early-stage cervical cancer: results from a nationwide population-based cohort study. Eur J Cancer. 2019;116:169–77. https://doi.org/10.1016/j.ejca.2019.05.016. 29. Bergmark K, Avall-Lundqvist E, Dickman PW, Henningsohn L, Steineck G. Patient-rating of distressful symptoms after treat- ment for early cervical cancer. Acta Obstet Gynecol Scand. 2002;81(5):443–50. 15. Hockel M, Horn LC, Manthey N, Braumann UD, Wolf U, Teich- mann G, Frauenschlager K, Dornhofer N, Einenkel J. Resection of the embryologically defined uterovaginal (Mullerian) compart- ment and pelvic control in patients with cervical cancer: a pro- spective analysis. Lancet Oncol. 2009;10(7):683–92. https://doi. org/10.1016/S1470-2045(09)70100-7. 30. Novackova M, Pastor Z, Chmel R Jr, Brtnicky T, Chmel R. Uri- nary tract morbidity after nerve-sparing radical hysterectomy in women with cervical cancer. Int Urogynecol J. 2020;31(5):981–7. https://doi.org/10.1007/s00192-019-04083-9. 31. Duru C, Jha S, Lashen H. Urodynamic outcomes after hysterec- tomy for benign conditions: a systematic review and meta-anal- ysis. Obstet Gynecol Surv. 2012;67(1):45–54. https://doi.org/10. 1097/OGX.0b013e318240aa28. 16. Chiantera V, Vizzielli G, Lucidi A, Gallotta V, Petrillo M, Legge F, Fagotti A, Sehouli J, Scambia G, Muallem MZ. Laparoscopic radical hysterectomy in cervical cancer as total mesometrial resection (L-TMMR): a multicentric experience. Gynecol Oncol. 2015;139(1):47–51. https://doi.org/10.1016/j.ygyno.2015.07.010. 32. Ross WT, Meister MR, Shepherd JP, Olsen MA, Lowder JL. Uti- lization of apical vaginal support procedures at time of inpatient hysterectomy performed for benign conditions: a national esti- mate. Am J Obstet Gynecol. 2017;217(4):436–436. https://doi. org/10.1016/j.ajog.2017.07.010. 17. Lucidi A, Windemut S, Petrillo M, Dessole M, Sozzi G, Vercel- lino GF, Baessler K, Vizzielli G, Sehouli J, Scambia G, Chiantera V. Self-reported long-term autonomic function after laparoscopic total mesometrial resection for early-stage cervical cancer: a multicentric study. Int J Gynecolog Cancer. 2017;27(7):1501–7. https://doi.org/10.1097/IGC.0000000000001045.f 33. Frumovitz M, Sun CC, Schover LR, Munsell MF, Jhingran A, Wharton JT, Eifel P, Bevers TB, Levenback CF, Gershenson DM, Bodurka DC. Quality of life and sexual functioning in cervical cancer survivors. J Clin Oncol. 2005;23(30):7428–36. https://doi. org/10.1200/JCO.2004.00.3996. p g 18. Baessler K, Kempkensteffen C. Validation of a comprehensive pelvic floor questionnaire for the hospital, private practice and research. Gynakol Geburtshilfliche Rundsch. 2009;49(4):299–307.l 34. Wallin E, Falconer H, Radestad AF. Sexual, bladder, bowel and ovarian function 1 year after robot-assisted radical hysterectomy for early-stage cervical cancer. Acta Obstet Gynecol Scand. 2019;98(11):1404–12. https://doi.org/10.1111/aogs.13680. l 19. Baessler K, Junginger B. Declarations 9. Bogani G, Rossetti DO, Ditto A, Signorelli M, Martinelli F, Mosca L, Scaffa C, Leone Roberti Maggiore U, Chiappa V, Sabatucci I, Lorusso D, Raspagliesi F. Nerve-sparing approach improves out- comes of patients undergoing minimally invasive radical hyster- ectomy: a systematic review and meta-analysis. J Minim Invasive Gynecol. 2018;25(3):402–10. https://doi.org/10.1016/j.jmig.2017. 11.014. Conflict of interest None of the authors have any conflicts of interest to declare. Ethical approval The local Ethics committee of Charité Universitäts- medizin approved this study (EA4/073/13). Ethical approval The local Ethics committee of Charité Universitäts- medizin approved this study (EA4/073/13). 10. Schneider A, Possover M, Kamprath S, Endisch U, Krause N, Noschel H. Laparoscopy-assisted radical vaginal hysterec- tomy modified according to Schauta-Stoeckel. Obstet Gynecol. Consent to participate and for publication All patients signed an informed consent form that included consent for publication. Consent to participate and for publication All patients signed an informed consent form that included consent for publication. 1 3 2342 Clinical and Translational Oncology (2021) 23:2335–2343 1996;88(6):1057–60. https://doi.org/10.1016/S0029-7844(96) 00307-9. 26. Long Y, Yao DS, Pan XW, Ou TY. Clinical efficacy and safety of nerve-sparing radical hysterectomy for cervical cancer: a sys- tematic review and meta-analysis. PLoS ONE. 2014;9(4):e94116. https://doi.org/10.1371/journal.pone.0094116. 11. Roy M, Plante M, Renaud MC. Laparoscopically assisted vagi- nal radical hysterectomy. Best Pract Res Clin Obstet Gynaecol. 2005;19(3):377–86. https://doi.org/10.1016/j.bpobgyn.2004.12. 001. 27. Pieterse QD, Kenter GG, Maas CP, de Kroon CD, Creutzberg CL, Trimbos JB, Ter Kuile MM. Self-reported sexual, bowel and bladder function in cervical cancer patients following different treatment modalities: longitudinal prospective cohort study. Int J Gynecolog Cancer. 2013;23(9):1717–25. https://doi.org/10.1097/ IGC.0b013e3182a80a65. 12. Hertel H, Kohler C, Michels W, Possover M, Tozzi R, Schneider A. Laparoscopic-assisted radical vaginal hysterectomy (LARVH): prospective evaluation of 200 patients with cervical cancer. Gynecol Oncol. 2003;90(3):505–11. y 13. Leblanc E. How I do a vaginal vault reconstruction after radical surgery. Gynecol Obstet Fertil. 2010;38(2):147–8. https://doi.org/ 10.1016/j.gyobfe.2009.12.009. 28. Plotti F, Terranova C, Capriglione S, Crispino S, Li Pomi A, de Cicco NC, Montera R, Panici PB, Angioli R, Scaletta G. Assess- ment of quality of life and urinary and sexual function after radi- cal hysterectomy in long-term cervical cancer survivors. Int J Gynecolog Cancer. 2018;28(4):818–23. https://doi.org/10.1097/ IGC.0000000000001239. 14. Alfonzo E, Wallin E, Ekdahl L, Staf C, Radestad AF, Reynisson P, Stalberg K, Falconer H, Persson J, Dahm-Kahler P. 40. Muallem MZ, Diab Y, Sehouli J, Fujii S. Nerve-sparing radi- cal hysterectomy: steps to standardize surgical technique. Int J Gynecolog Cancer. 2019;29(7):1203–8. https://doi.org/10.1136/ ijgc-2019-000410. Clinical and Translational Oncology (2021) 23:2335–2343 Declarations Validation of a pelvic floor question- naire with improvement and satisfaction scales to assess symptom severity, bothersomeness and quality of life before and after pelvic floor therapy. Aktuelle Urol. 2011;42(5):316–22. https://doi.org/ 10.1055/s-0031-1271544. 35. Bergmark K, Avall-Lundqvist E, Dickman PW, Henningsohn L, Steineck G. Vaginal changes and sexuality in women with a history of cervical cancer. N Engl J Med. 1999;340(18):1383–9. https://doi.org/10.1056/NEJM199905063401802. 20. Baessler K, O’Neill SM, Maher CF, Battistutta D. A validated self- administered female pelvic floor questionnaire. Int Urogynecol J. 2010;21(2):163–72. https://doi.org/10.1007/s00192-009-0997-4.f 36. Wang X, Chen C, Liu P, Li W, Wang L, Liu Y. The morbidity of sexual dysfunction of 125 Chinese women following different types of radical hysterectomy for gynaecological malignancies. Arch Gynecol Obstet. 2018;297(2):459–66. https://doi.org/10. 1007/s00404-017-4625-0. p g 21. Baessler K, Mowat A, Maher CF. The minimal important differ- ence of the Australian Pelvic floor questionnaire. Int Urogynecol J. 2019;30(1):115–22. https://doi.org/10.1007/s00192-018-3724-1. 22. Auguste T, Dubreuil A, Bost R, Bonaz B, Faucheron JL. Tech- nical and functional results after laparoscopic rectopexy to the promontory for complete rectal prolapse. Prospective study in 54 consecutive patients. Gastroenterol Clin Biol. 2006;30(5):659–63. 37. Sowa E, Kuhnt S, Hinz A, Schroder C, Deutsch T, Geue K. Postoperative health-related quality of life of cervical cancer patients—a comparison between the Wertheim-Meigs operation and Total Mesometrial Resection (TMMR). Geburtshilfe Frauen- heilkd. 2014;74(7):670–6. https://doi.org/10.1055/s-0034-13686 00. 23. Hockel M, Horn LC, Hentschel B, Hockel S, Naumann G. Total mesometrial resection: high resolution nerve-sparing radical hys- terectomy based on developmentally defined surgical anatomy. Int J Gynecolog Cancer. 2003;13(6):791–803. 38. Baessler K, O’Neill SM, Maher CF, Battistutta D (2008) An inter- viewer-administered validated female pelvic floor questionnaire for community-based research. Menopause. 2008;15(5):973–7. https://doi.org/10.1097/gme.0b013e3181671b89 24. Kruppa J, Kavvadias T, Amann S, Baessler K, Schuessler B. Short and long-term urodynamic and quality of life assessment after nerve sparing radical hysterectomy: a prospective pilot study. Eur J Obstet Gynecol Reprod Biol. 2016;201:131–4. https://doi.org/ 10.1016/j.ejogrb.2016.03.026. 39. Metz M, Junginger B, Henrich W, Baessler K. Development and validation of a questionnaire for the assessment of pelvic floor disorders and their risk factors during pregnancy and post partum. Geburtshilfe Frauenheilkd. 2017;77(4):358–65. https://doi.org/10. 1055/s-0043-102693. 25. Hunskaar S, Lose G, Sykes D, Voss S. The prevalence of uri- nary incontinence in women in four European countries. BJU Int. 2004;93(3):324–30. 1 3 2343 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3 1 3 3
https://openalex.org/W2888015317	https://www.frontiersin.org/articles/10.3389/fdata.2018.00003/pdf	English	null	An Application of Data Mining Techniques to Explore Congressional Lobbying Records for Patterns in Pediatric Special Interest Expenditures Prior to the Affordable Care Act	Frontiers in big data	2,018	cc-by	2,944	1 Data Science Institute, University of Virginia, Charlottesville, VA, United States, 2 School of Medicine, University of Virginia, Charlottesville, VA, United States, 3 School of Nursing, University of Virginia, Charlottesville, VA, United States, 4 Department of Computer Science, University of Virginia, Charlottesville, VA, United States Edited by: Frank Emmert-Streib, Tampere University of Technology, Finland Keywords: lobbying, data mining, data science, Affordable Care Act, pediatrics Keywords: lobbying, data mining, data science, Affordable Care Act, pediatrics Reviewed by: Soumya Sarkar, Indian Institute of Technology Kharagpur, India Kristina Hettne, Leiden University Medical Center, Netherlands Ruoyu Li, Facebook, United States Reviewed by: Soumya Sarkar, Indian Institute of Technology Kharagpur, India Kristina Hettne, Leiden University Medical Center, Netherlands Ruoyu Li, Facebook, United States An Application of Data Mining Techniques to Explore Congressional Lobbying Records for Patterns in Pediatric Special Interest Expenditures Prior to the Affordable Care Act Elizabeth Harrison 1,2, Caitlin Dreisbach 1,3, Nada Basit 1,4 and Jessica Keim-Malpass 3 1 Data Science Institute, University of Virginia, Charlottesville, VA, United States, 2 School of Medicine, University of Virginia, Charlottesville, VA, United States, 3 School of Nursing, University of Virginia, Charlottesville, VA, United States, 4 Department of Computer Science, University of Virginia, Charlottesville, VA, United States 1. BACKGROUND Since 2006, 1.7 billion dollars has been spent lobbying Congress and federal agencies in the area of health (Steinbrook, 2009). Despite this, there has been a dearth of empirical studies focusing on the relationship between ﬁnancial lobby inﬂuence and associated health legislation (Steinbrook, 2009). Most of the existing empirical examples assessing the impact of lobbying on legislation focus on the relationship between either lobbying and tobacco or pharmaceutical and medical device regulations (Steinbrook, 2009; Jorgensen, 2013; Savell et al., 2014). Associated concerns over transparency and public access to lobbying ﬁnancial disclosures have only recently begun to be addressed (Steinbrook, 2009). Correspondence: Caitlin Dreisbach cnd2y@virginia.edu In the United States, there has been a long history of advocacy on the behalf of children (Denne and Hay, 2013). The year 1921 marked the ﬁrst federal legislative eﬀort (the Sheppard-Towner Act) to designate federal funds for prenatal programs targeting poor women and children (Denne and Hay, 2013). Since then, federal programs focused on child health have increased in scope and quantity, including numerous federal programs that expanded with the 2010 passage of the Patient Protection and Aﬀordable Care Act (ACA) (Farrell et al., 2011; Keim-Malpass et al., 2013, 2015). This period, in particular, gave an opportunity for researchers and clinicians to advocate for their pediatric speciﬁc interests (Steinbrook, 2009). Specialty section: This article was submitted to Medicine and Public Health, a section of the journal Frontiers in Big Data Received: 24 April 2018 Accepted: 02 August 2018 Published: 20 August 2018 The use of data science applications provides a scientiﬁc framework to describe the ﬁnancial impact of legislation and lobbying for pediatric issues. Emerging trends in the use of data science methods within a social science domain have focused on social network analyses, surveying sentiment through text, and understanding patterns in publicly available datasets (Bellazzi et al., 2011; Asch et al., 2015). A recent study in the European Union on quantitative textual analysis highlighted the importance of rich, public data sources that have the potential to highlight incongruities between lobbying positions and legislative success (Bunea and Ibenskas, 2015). In the United States, the Lobbying Disclosure Act (LDA), signed into law by President Bill Clinton, requires the Secretary of the Senate and the Clerk of the House of Representatives to make publicly in Big Data in Big Data DATA REPORT published: 20 August 2018 doi: 10.3389/fdata.2018.00003 420.5. The ElementTree XML API (2018). Available online at: https://docs.python. org/3/library/xml.etree.elementtree.html. 1104th Congress. Lobbying Disclosure Act of 1995. Available online at: https:// lobbyingdisclosure.house.gov/lda.html. 2US Senate Oﬃce of Public Records US. Lobbying Disclosure Act (LDA) Downloadable Lobbying Databases (2017). Available online at: https://www. senate.gov/legislative/Public_Disclosure/database_download.htm. 320.4. XML Processing Modules - Python 3.6.4 documentation (2018). Available online at: https://docs.python.org/3/library/xml.html. 2.3. Data Preprocessing To begin, all necessary packages are loaded, including ElementTree for parsing, Pandas for the creation of multiple data frames, and defaultdict to organize the central dictionary for the soon-to-be parsed data. Next, the LDA site is accessed to download the folder with lobbying data from the fourth quarter of 2009. An os.listdir function is applied to extract all individual ﬁles from this folder. Finally, an algorithm is used to parse through the XML ﬁles and convert the congressional lobbying data into a more usable form. See Figure 2 for this algorithm in pseudo-code. 2. DATA AND METHODOLOGY Data science techniques such as parsing, cleaning, and transforming raw data to identify patterns and trends can be used to focus on important lobbying issues published through the LDA. Coding languages –s in this case Python – can be critical tools to extract meaningful and actionable items within troves of data (Wiedeman, 2017). As a dynamically-typed and general- use programming language, Python provides a new technical toolbox for political researchers, clinicians, and data scientists. By leveraging data science for the investigation of lobbying for pediatric research and clinical trials, this data report aims to illustrate how one might investigate deeper trends regarding the inclusion of pediatric health concerns in the political environment. After extracting the data, but prior to performing any analysis, a master dictionary is used to create eight individual data frames, one for each of several keys in the master dictionary: “Filing,” “Issue,” “Entity,” “Org,” “Lobbyist,” “Registrant,” “Client,” and “Government Entity.” Subsequently, another data frame is created by merging “Filing,” which includes amounts of money reported per ﬁling, and “Issue,” which includes umbrella codes and more speciﬁc issues of interest. Finally, a Pandas series is formed from the new data frame by grouping amounts by code. 2.1. Data Source This data report focuses on data pertaining to lobbying records submitted in the fourth quarter of 2009, a period of intense healthcare lobbying prior to the passage of the Aﬀordable Care Act. Currently, the public LDA database can be accessed online. The website provides both a search engine for speciﬁc database queries and downloadable copies of the raw XML data from 1999 to the current year2. The data from each quarter comprises about 25 separate congressional lobbying XML ﬁles with approximately 1,000 lines of code each. Thus, the ﬁnal dataset used for the analysis in this report includes over 25,000 lines of XML code. Citation: Harrison E, Dreisbach C, Basit N and Keim-Malpass J (2018) An Application of Data Mining Techniques to Explore Congressional Lobbying Records for Patterns in Pediatric Special Interest Expenditures Prior to the Affordable Care Act. Front. Big Data 1:3. doi: 10.3389/fdata.2018.00003 August 2018 \| Volume 1 \| Article 3 Frontiers in Big Data \| www.frontiersin.org Data Mining Healthcare Lobbying Records Harrison et al. available all registrations and reports related to lobbying activity1. The intended purpose of this act was to increase the accountability of federal lobbying practice. However, the relatively unexplored nature of the LDA database suggests the need for advanced methods to provide clarity to the power of lobbying. both humans and machines4. ET runs through the naturally hierarchical format of XML ﬁles to parse out root and child attributes, which can then be used to sort the information into other data structures. For reference, the organizational structure of the data is outlined in Figure 1, including the component extracted for this data report. Analyses of congressional lobbying activities focused on child health prior to the ACA oﬀer a unique perspective on the various factors that contribute to child health advocacy and the eventual passage of relevant legislation. This data report illustrates several data parsing principles that can be used to mine lobbying records and can serve as the basis for future inquiry and analysis. 2.4. Queries and Searching To set up the code structure for more speciﬁc queries, a search function is developed to identify output based on user input. This function creates an empty list and appends each dictionary item that contains the search term in its “Speciﬁc Issues” section, which is a subcategory of each individual “Issue” section. Because the search is based on user input, any term can be searched within the list of dictionaries. The ﬁnal appended list can then be easily converted into a data frame. 2.2. ElementTree and XML Overview The authors’ extraction method uses a popular web-parsing package in Python called ElementTree. The ElementTree (ET) module utilizes a simple and eﬃcient Application Programming Interface (API) for parsing, sorting, and organizing XML data3. Extensible Markup Language (XML) was designed for widespread use across the internet, and is readable by 1104th Congress. Lobbying Disclosure Act of 1995. Available online at: https:// lobbyingdisclosure.house.gov/lda.html. 2 3. ANALYSIS A sorted amount-by-code Pandas series shows that the largest sum of lobbying dollars spent and ﬁled in the fourth quarter of 2009 has been ﬁled under “Taxation/Internal Revenue Code,” with an associated value of $527,147,333. Next is “Health Issues,” with $436,651,121. Analysis of the pediatric search results shows that these lobbying disclosures have been ﬁled under a multitude of umbrella codes, including “Health Issues,” “Budget Appropriations,” “Science/Technology,” “Education,” “Disaster Planning & Emergencies,” and “Medicare/Medicaid.” Although this data can be merged with the “Filing”/“Issue” data frame, it remains impossible to make meaningful deductions about amounts spent on pediatric lobbying activities speciﬁcally because multiple issues are grouped together with the pediatric- related items, such as one ﬁling that cites the following speciﬁc issues in one group: Another unexpected but important ﬁnding of this data parsing method relates to the actual dollar amounts that can be extracted from the XML ﬁles. First, and most importantly, the XML ﬁles do not diﬀerentiate lobbying expenditures from income; these amounts are indistinguishable and are not linked to any identiﬁers that would allow a researcher to parse them out separately. Second, although the amounts appear to be speciﬁc to the single dollar on the results from the queries, the forms used to ﬁle lobbying reports allow a box to be checked for all lobbying expenses under $5,000, without any space to indicate the actual amount. Finally, in regards to income from clients, the forms used to gather lobbying data allow amounts over $5,000 to be rounded to the nearest $10,000: “Support legislation to amend title XVIII of the Social Security Act to provide for coverage of federally recommended vaccines under Medicare, Part B. Support legislation to amend title XIX of the Social Security Act to include all public clinics for the distribution of pediatric vaccines under the Medicaid program. Support legislation to amend the Social Security Act, the Federal Food, Drug and Cosmetic Act, and the Public Health Service Act to ensure a suﬃcient supply of vaccines and for other purposes. Lobbied to ensure that the Internet would not be used to import counterfeit and defective medical products. Support a rigorous and workable Aﬀordable Biologics for Consumers Act. Support enactment and follow-on Biologics legislation with amendments that strengthen FDA pathway and provide data exclusivity. General matters related to vaccine industry stability, pandemic preparedness, avian inﬂuenza”4. 2.2. ElementTree and XML Overview Consideration of a wide variety of related terms is essential in the creation of an eﬀective initial query. For the purposes of this data report, the term “pediatric” is chosen because it is both sensitive and speciﬁc for medical data in the under 18-year-old population. Using this single term, 35 results are pulled from the entire fourth quarter data frame. Then, key pieces of action and legislation are identiﬁed, such as the Improving Access to Clinical Trials Act of 2009, programs August 2018 \| Volume 1 \| Article 3 Frontiers in Big Data \| www.frontiersin.org 2 Data Mining Healthcare Lobbying Records Harrison et al. FIGURE 1 \| Data organization tree. FIGURE 1 \| Data organization tree. to expand medical device testing in children, and budget appropriations for generic pediatric drug trials. Finally, the search results are compared to the previously saved data regarding codes and lobbying dollar amounts spent per issue. FIGURE 2 \| XML parsing algorithm pseudocode. FIGURE 2 \| XML parsing algorithm pseudocode. Frontiers in Big Data \| www.frontiersin.org REFERENCES Keim-Malpass, J., Letzkus, L., and Kennedy, C. (2015). Health literacy and the Aﬀordable Care Act: a policy analysis for children with special health care needs in the USA. Risk Manage. Healthcare Policy 8, 31–36. doi: 10.2147/RMHP.S80699 Asch, D. A., Rader, D. J., and Merchant, R. M. (2015). Mining the social mediome. Trends Mol. Med. 21, 528–529. doi: 10.1016/j.molmed.2015.06.004 Bellazzi, R., Diomidous, M., Sarkar, I., Takabayashi, K., Ziegler, A., McCray, A. (2011). Data analysis and data mining: current issues in biomedical informatics. Methods Inform. Med. 50, 536–544. doi: 10.3414/ME11- 06-0002 Savell E, Gilmore, A. B., and Fooks, G. (2014). How does the tobacco industry attempt to inﬂuence marketing regulations? A systematic review. PLoS ONE 9:e87389. doi: 10.1371/journal.pone.0087389 Savell E, Gilmore, A. B., and Fooks, G. (2014). How does the tobacco industry attempt to inﬂuence marketing regulations? A systematic review. PLoS ONE 9:e87389. doi: 10.1371/journal.pone.0087389 Steinbrook, R. (2009). Lobbying, campaign contributions, and health care reform. N. Engl. J. Med. 361:e52. doi: 10.1056/NEJMp0910879 Steinbrook, R. (2009). Lobbying, campaign contributions, and health care reform. N. Engl. J. Med. 361:e52. doi: 10.1056/NEJMp0910879 Bunea, A., and Ibenskas, R.(2015). Quantitative text analysis and the study of EU lobbying and interest groups. Eur. Union Polit. 16, 429–455. doi: 10.1177/1465116515577821 Wiedeman, G. (2017) Python for Archivists: Breaking Down Barriers Between Systems. University Libraries Faculty Scholarship. Available online at: https:// scholarsarchive.library.albany.edu/ulib_fac_scholar/93. Wiedeman, G. (2017) Python for Archivists: Breaking Down Barriers Between Systems. University Libraries Faculty Scholarship. Available online at: https:// scholarsarchive.library.albany.edu/ulib_fac_scholar/93. Denne, S. C., and Hay, W. W. (2013). Advocacy for research that beneﬁts children. JAMA Pediatr. 167:792. doi: 10.1136/archdischild-2014-306259 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Farrell, K., Hess, C., and Justice, D. (2011). The Aﬀordable Care Act and Children with Special Health Care Needs: An Analysis and Steps for State Policymakers Available online at: http://cahpp.org/resources/the-aﬀordable-care-act-and- children-with-special-health-care-needs-an-analysis-and-steps-for-state- policymakers/. Copyright © 2018 Harrison, Dreisbach, Basit and Keim-Malpass. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 3. ANALYSIS “Provide a good faith estimate, rounded to the nearest $10,000, of all lobbying related income from the client (including all August 2018 \| Volume 1 \| Article 3 3 Data Mining Healthcare Lobbying Records Harrison et al. payments to the registrant by any other entity for lobbying activities on behalf of the client)”5. More importantly, deeper analytical processes – including greater statistical manipulation of the currency values that are associated with each lobbying record – could be assessed. As mentioned in our results, the limitation in greater statistical analyses is the innate inability to identify the exact amount of money spent on any particular lobbying activity. Only when the forms and procedures used to collect lobbying data are adjusted will data mining techniques be helpful in shedding light on federal lobbying practices, both in and out of the healthcare policy realm. Due to the clear ineﬃciency of the original reporting forms, this report identiﬁes concerns with the process of lobby reporting at its source. Although the current public LDA database is searchable and the raw XML data is available to download, the system continues to lack transparency. AUTHOR CONTRIBUTIONS Future research could leverage other data science techniques to assess the value of the underlying text in lobbying records. Potentially helpful methods include natural language processing (NLP), topic modeling, and generalized text analysis algorithms. Reﬁning methods to the text level may illuminate greater detail about inconsistencies in the way lobbying records are written. However, while a greater understanding of lobbying language might be beneﬁcial, this kind of analysis would largely ignore the immense ﬁnancial representation that lobbying groups have in the United States Congress. EH and CD contributed to conception, design, analysis, and interpretation. EH parsed the data, led all coding, and developed the extraction algorithm. CD wrote the ﬁrst draft of the report. JK-M and NB contributed to concept development and review. All authors contributed to manuscript revision and read and approved the submitted version. DATA AVAILABILITY STATEMENT Pediatric research is a topic for which critical analysis of lobbying records may be clinically meaningful, particularly with regards to records originating at the time of the ACA’s passage. Further, supporting the development of major healthcare bills that inﬂuence patients across the age continuum is essential to countering current governmental eﬀorts to decrease healthcare appropriations. The datasets analyzed for this report can be found on the United States Senate website: https://www.senate.gov/legislative/ Public_Disclosure/database_download.htm. All pertinent code is accessible via Github: https://github.com/eih2nn/ UnitedStatesLobbyingDisclosureData. FUNDING This research was funded in part by the University of Virginia Data Science Institute and, in part, by the University of Virginia School of Nursing. This research was funded in part by the University of Virginia Data Science Institute and, in part, by the University of Virginia School of Nursing. 5Lobbying Registration (LD-1DS) Sample Form (2017). Available from: https://lobbyingdisclosure.house.gov/help/default.htm?turl=WordDocuments %2Flobbyingregistrationld1dssampleform.htm. REFERENCES Jorgensen, P. D. (2013). Pharmaceuticals, political money, and public policy: a theoretical and empirical agenda. J. Law. Med. Ethics 41, 561–570. doi: 10.1111/jlme.12065 Keim-Malpass, J., Hart, T. G., and Miller, J. R. (2013). Coverage of palliative and hospice care for pediatric patients With a life-limiting illness: a policy brief. J. Pediatr. Health Care 27, 511–516. doi: 10.1016/j.pedhc.2013.07.011 August 2018 \| Volume 1 \| Article 3 Frontiers in Big Data \| www.frontiersin.org
https://openalex.org/W1935656259	https://europepmc.org/articles/pmc4577823?pdf=render	English	null	Estimating rates of de novo infection and mitotic replication in HTLV-1 persistence: de novo infection continues after early infection	Retrovirology	2,015	cc-by	708	Laydon et al. Retrovirology 2015, 12(Suppl 1):P46 http://www.retrovirology.com/content/12/S1/P46 POSTER PRESENTATION Open Access Estimating rates of de novo infection and mitotic replication in HTLV-1 persistence: de novo infection continues after early infection Daniel J Laydon, Vikram Sunkara, Charles RM Bangham, Becca Asquith From 17th International Conference on Human Retroviruses: HTLV and Related Viruses Trois Ilets, Martinique. 18-21 June 2015 From 17th International Conference on Human Retroviruses: HTLV and Related Viruses Trois Ilets, Martinique. 18-21 June 2015 Human T-lymphotropic virus type-1 (HTLV-1) persists within hosts via de novo infection (infectious spread) and infected cell proliferation (mitotic spread), creating a population structure of multiple clones (infected cell populations with identical genomic proviral integration sites). However, the relative contributions of these mechanisms are unknown, and will determine the effi- cacy of antiretroviral therapy. The prevailing view is that infectious spread does not contribute to HTLV-1 pro- viral load after early infection. However, this notion is based on low-throughput and imprecise data on clonal integration site abundance and diversity. We recently used our high-throughput protocol that maps and quan- tifies the abundance of HTLV-1 integration sites to esti- mate a diversity of between 10^4 and 10^5 HTLV-1 T cell clones in the body of an asymptomatic carrier or patient with HAM/TSP. Since infectious spread estab- lishes new clones, high clonal diversity implies substan- tial infectious spread. HTLV-1 clonal abundance varies within individual hosts by several orders of magnitude. Therefore quantifying within-host HTLV-1 dynamics requires mathematical modelling at multiple scales, and stochastic processes are necessary to describe the beha- viour of small clones. Here we apply a hybrid model of deterministic and stochastic processes to time-series patient data, to estimate the relative contributions of infectious spread and mitotic spread. We find, contrary to previous belief that infectious spread persists during chronic infection, even after the proviral load has reached its set point. The risk of HTLV-1-associated malignant and inflammatory disease is strongly corre- lated with the proviral load: the load in turn is corre- lated with the total number of HTLV-1-infected clones, but not with the degree of oligoclonal proliferation. Our results therefore suggest that attempts to suppress de novo infection may reduce the risk of malignant transformation. Published: 28 August 2015 doi:10.1186/1742-4690-12-S1-P46 Cite this article as: Laydon et al.: Estimating rates of de novo infection and mitotic replication in HTLV-1 persistence: de novo infection continues after early infection. Retrovirology 2015 12(Suppl 1):P46. Published: 28 August 2015 doi:10.1186/1742-4690-12-S1-P46 Cite this article as: Laydon et al.: Estimating rates of de novo infection and mitotic replication in HTLV-1 persistence: de novo infection continues after early infection. Retrovirology 2015 12(Suppl 1):P46. © 2015 Laydon et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Estimating rates of de novo infection and mitotic replication in HTLV-1 persistence: de novo infection continues after early infection Daniel J Laydon, Vikram Sunkara, Charles RM Bangham, Becca Asquith From 17th International Conference on Human Retroviruses: HTLV and Related Viruses Trois Ilets, Martinique. 18-21 June 2015 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit le distributed under the terms of the Creative Commons Attribution License (http:// rmits unrestricted use, distribution, and reproduction in any medium, provided the mons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ this article, unless otherwise stated. Submit your next manuscript to BioMed Central and take full advantage of: Department of Medicine, Imperial College London, UK © 2015 Laydon et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated.
https://openalex.org/W4362549578	https://vtechworks.lib.vt.edu/bitstream/10919/115498/1/RiggsTurning.pdf	English	null	Turning lakes into river gauges using the LakeFlow algorithm	Authorea (Authorea)	2,023	cc-by	8,962	© 2023. The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Key Points: Ryan M. Riggs1 , George H. Allen2 , Craig B. Brinkerhoff3 , Md. Safat Sikder4, and Jida Wang4 • LakeFlow is a new algorithm that uses Surface Water and Ocean Topography (SWOT) satellite data to estimate river inflow and outflow at lakes via mass conservation 1Department of Geography, Texas A&M University, College Station, TX, USA, 2Department of Geosciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA, 3Department of Civil and Environmental Engineering, University of Massachusetts Amherst, Amherst, MA, USA, 4Department of Geography and Geospatial Sciences, Kansas State University, Manhattan, KS, USA • Applying LakeFlow to three sample lake systems shows promising performance for estimating lake inflows and outflows (median Nash-Sutcliffe Efficiency = 0.88) Abstract Rivers and lakes are intrinsically connected waterbodies yet they are rarely used to hydrologically constrain one another with remote sensing. Here we begin to bridge the gap between river and lake hydrology with the introduction of the LakeFlow algorithm. LakeFlow uses river-lake mass conservation and observations from the Surface Water and Ocean Topography (SWOT) satellite to provide river discharge estimates of lake and reservoir inflows and outflows. We test LakeFlow performance at three lakes using a synthetic SWOT data set assuming the maximum measurement errors defined by the mission science requirements, and we include modeled lateral inflow and lake evaporation data to further constrain the mass balance. We find that LakeFlow produces promising discharge estimates (median Nash-Sutcliffe efficiency = 0.88, relative bias = 14%). LakeFlow can inform water resources management by providing global lake inflow and outflow estimates, highlighting a path for recognizing rivers and lakes as an interconnected system. • Including modeled estimates of non SWOT-observed evaporation and tributary inflows can further improve LakeFlow discharge estimates Supporting Information: Supporting Information may be found in the online version of this article. Supporting Information: Supporting Information may be found in the online version of this article. Correspondence to: R. M. Riggs, rriggs@tamu.edu Correspondence to: R. M. Riggs, rriggs@tamu.edu Citation: Riggs, R. M., Allen, G. H., Brinkerhoff, C. B., Sikder, M. S., & Wang, J. (2023). Turning lakes into river gauges using the LakeFlow algorithm. Geophysical Research Letters, 50, e2023GL103924. https://doi.org/10.1029/2023GL103924 Plain Language Summary Effective water resource management depends on our ability to monitor and understand lake and reservoir inflows and outflows. Citation: Received 30 MAR 2023 Accepted 3 MAY 2023 Turning Lakes Into River Gauges Using the LakeFlow Algorithm Turning Lakes Into River Gauges Using the LakeFlow 10.1029/2023GL103924 RESEARCH LETTER Key Points: Correspondence to: R. M. Riggs, rriggs@tamu.edu Citation: Riggs, R. M., Allen, G. H., Brinkerhoff, C. B., Sikder, M. S., & Wang, J. (2023). Turning lakes into river gauges using the LakeFlow algorithm. Geophysical Research Letters, 50, e2023GL103924. https://doi.org/10.1029/2023GL103924 Turning Lakes Into River Gauges Using the LakeFlow Algorithm ER Key Points: Satellite remote sensing of lakes and rivers has become increasingly important for water management but little work has been done to estimate streamflow at river- lake interfaces. Here we present the LakeFlow algorithm that leverages satellite observations of lakes and rivers to estimate streamflow at lake inflows and outflows. We test LakeFlow using synthetic data at three U.S. lakes in Georgia, Arizona and Kansas, and find that it yields promising estimates of streamflow at river-lake boundaries. LakeFlow provides valuable insights into river-lake streamflow dynamics, which can inform water management decisions and is a step forward in the integration of river and lake studies. 1. Introduction Rivers and lakes serve as vital sources of freshwater for ecosystems and civilizations worldwide (Everard & Powell, 2002; Macklin & Lewin, 2015; Yevjevich, 1992). While rivers and lakes are often treated as separate systems in large-scale remote sensing studies, their hydrologies are intimately related such that hydrologic changes in one water body type can be used to constrain the hydrology of an adjacent water body of a differ- ent type (Vörösmarty et al., 2000). For example, the relationship between inflow and outflow of a natural lake or human-made reservoir (hereinafter collectively referred to as a “lake” unless otherwise stated) can control the lake's volumetric water storage and water surface elevation. Natural lakes located along river networks can attenuate local discharge downstream and actively managed reservoirs can significantly affect downstream flow regime by altering the natural timing and quantity of river discharge (Doll et al., 2009; Wang et al., 2017; Yang et al., 2022). Reservoir inflow and outflow dynamics are key for modeling reservoir operations, which can be difficult to simulate from water mass balance alone, especially at the continental to global scale (Cohen et al., 2014; Harrigan et al., 2020). At these large scales, understanding of the hydrologic interplay between rivers and lakes has largely been devel- oped through the analysis of streamflow gauges located on lake inflows and outflows (i.e., the rivers flowing into and out of a lake), as well as lake-level gauges (Batalla et al., 2004; Shiklomanov & Lammers, 2009; Yang et al., 2008). Unfortunately, most lakes do not have publicly available gauge data and those that do are primar- ily located on large lakes or in a few geographically isolated regions (Brazil National Water Agency, 2022; Do et al., 2018; Gudmundsson et al., 2018; U.S. Geological Survey, 2022). This lack of observational data limits our 1 of 11 1 of 11 RIGGS ET AL. RIGGS ET AL. Geophysical Research Letters 10.1029/2023GL103924 understanding of how impoundments impact surface water flows and has motivated the development of alterna- tive techniques for supplementing river and lake gauge observations. understanding of how impoundments impact surface water flows and has motivated the development of alterna- tive techniques for supplementing river and lake gauge observations. Satellite remote sensing is uniquely capable of providing observation-based discharge estimates in near-real time and at the global scale (Smith, 1997). 1. Introduction Although remote sensing of discharge (RSQ) has been performed using a variety of satellite data and techniques (Gleason & Durand, 2020), much of the recent focus has been in preparation for the recently launched Surface Water and Ocean Topography (SWOT) mission (Biancamaria et al., 2016). Though SWOT cannot directly observe river discharge, it can potentially provide unprecedented cotemporal measurements of river area, elevation, width, and slope for all rivers within the SWOT River Data- base (SWORD) (Altenau et al., 2021). The SWOT mission will also produce discharge estimates calculated by combining cotemporal SWOT observations with flow laws (e.g., hydraulic geometry, Manning's equation), mass conservation principles, and a priori estimates of non-SWOT-observable flow-law parameters (FLP) such as frictional resistance (Manning's n) and bathymetry (Brinkerhoff et al., 2020; Durand et al., 2014). These SWOT discharge estimates will be practically produced using the Confluence program which houses several different RSQ algorithms (Durand et al., 2023). SWOT RSQ algorithms are sensitive to FLP estimates (Durand et al., 2016) which are provided by the SWORD of Science (SoS) database for all SWOT observable rivers (Brinkerhoff et al., 2020). SoS priors of Manning's n and bathymetry are developed using in situ measurements that are then paired with river attributes such as mean width, allowing for mean width alone to provide prior estimates of these FLPs. Although SWOT discharge is expected to improve our understanding of global river discharge (Pavelsky et al., 2014), existing SWOT RSQ algorithms do not leverage SWOT observations of lakes into their workflow, which could improve performance. In lakes, SWOT can observe lake surface area and elevation, which together can be combined to estimate volu- metric storage change (Busker et al., 2019; Crétaux et al., 2011; Gao, 2015; Zhao & Gao, 2019). Storage change estimates are valuable for understanding seasonal and long-term trends in water availability and usage (Cooley et al., 2021; Keys & Scott, 2018; Ryan et al., 2020). Storage change fluctuations also influence downstream river discharge (Nickles & Beighley, 2021; Wang et al., 2013) but very few remote sensing applications consider lakes and rivers as an interconnected system (Gardner et al., 2019). The few remote sensing studies that do assess lakes and rivers together rely on modeled discharge and use satellite estimates of lake storage change to revise the modeled outflow discharge (Bonnema & Hossain, 2019; Yoon & Beighley, 2015; Yoon et al., 2016). 1. Introduction This calibration only improves the difference between the inflow and outflow discharge, leaving the original bias in the modeled inflow (or outflow) discharge uncorrected (Bonnema, Sikder, Miao, et al., 2016). However, the accura- cies for both inflow and outflow discharge are important because together they provide key insights into human water management and the impact lakes have on river flow regime. Currently, SWOT RSQ algorithms have neither been assessed nor are specifically designed to run at river-lake boundaries (Bonnema, Sikder, Hossain, et al., 2016; Durand et al., 2016; Frasson et al., 2021). To address these gaps in our ability to monitor the river-lake continuum, we develop LakeFlow, an algorithm which applies river-lake mass conservation to estimate both lake inflow and outflow discharge. Like other SWOT RSQ algorithms, LakeFlow relies on Manning's equation and mass conservation (Feng et al., 2021; Hagemann et al., 2017) but also leverages additional SWOT observations of lake storage change to further constrain river discharge. In addition to discharge, LakeFlow estimates Manning's n and bathymetry of lake inflow and outflow channels, which can be used to inform or improve other SWOT RSQ algorithms. LakeFlow could potentially be applied to the nearly 17 thousand SWOT observable lakes that are located along the SWORD network and have at least one inflow and one outflow reach that are observable from SWOT (Figure 1). In total, LakeFlow could possibly provide valuable insights into discharge dynamics at 19,380 inflow and 16,959 outflow reaches that are connected to SWOT observable lakes. Ultimately, LakeFlow bridges the gap between lake storage and river discharge to improve SWOT discharge coverage and accuracy. e governed by the applicable Creative Commons License 2. Methods 2. Methods 2.1. LakeFlow Algorithmic Design The LakeFlow algorithm uses SWOT observed river and lake variables to estimate discharge. LakeFlow uses the modified version of Manning's equation from Durand et al. (2014) to describe discharge dynamics for the inflow and outflow reaches, 2 of 11 RIGGS ET AL. Geophysical Research Letters 10.1029/2023GL103924 Figure 1. Global distribution of lakes suitable for LakeFlow implementation (N = 16,610) with three sample lakes highlighted. Each of these lakes is observable by SWOT (Sheng et al., 2016) and contains at least one SWOT observable inflow and one SWOT observable outflow (Allen & Pavelsky, 2018; Altenau et al., 2021). Note the Lake Allatoona inflow gauge is located on the inflow mainstem (dashed orange line) but is located 7 km upstream of the SWORD reach (orange line). Figure 1. Global distribution of lakes suitable for LakeFlow implementation (N = 16,610) with three sample lakes highlighted. Each of these lakes is observable by SWOT (Sheng et al., 2016) and contains at least one SWOT observable inflow and one SWOT observable outflow (Allen & Pavelsky, 2018; Altenau et al., 2021). Note the Lake Allatoona inflow gauge is located on the inflow mainstem (dashed orange line) but is located 7 km upstream of the SWORD reach (orange line). 𝑄𝑄= 𝑛𝑛−1(𝐴𝐴0 + 𝛿𝛿𝛿𝛿)5∕3𝑊𝑊−2∕3𝑆𝑆1∕2, (1) 𝑄𝑄= 𝑛𝑛−1(𝐴𝐴0 + 𝛿𝛿𝛿𝛿)5∕3𝑊𝑊−2∕3𝑆𝑆1∕2, (1) where Q is discharge and n is the frictional resistance of the river channel, referred to as Manning's n. A0 repre- sents the unobservable cross-sectional area that extends beyond the minimum observed water level, hereinafter referred to as bathymetry, δA is the SWOT observable change in cross-sectional area, W is river width, and S is slope. LakeFlow leverages SWOT estimated lake storage change (δV) during a time period such as between two consecutive SWOT overpasses (p) to constrain inflow and outflow discharge based on mass conservation, 𝛿𝛿𝛿𝛿𝑝𝑝= ∫ 𝑡𝑡=0 ( 𝑛𝑛𝑖𝑖−1(𝐴𝐴0𝑖𝑖+ 𝛿𝛿𝛿𝛿𝑖𝑖)5∕3𝑊𝑊𝑖𝑖 −2∕3𝑆𝑆𝑖𝑖 1∕2 −𝑛𝑛𝑜𝑜−1(𝐴𝐴0𝑜𝑜+ 𝛿𝛿𝛿𝛿𝑜𝑜)5∕3𝑊𝑊−2∕3 𝑜𝑜 𝑆𝑆𝑜𝑜 1∕2 + 𝑄𝑄𝑙𝑙−𝐸𝐸 ) 𝑡𝑡𝑑𝑑𝑑𝑑𝑑 (2) (2) here t represents any time during period p, Ql is lateral inflows from channels too small to be observed by SWOT, E is lake evaporation, and all other variables are the same as Equation 1 with i and o denoting the variables of the SWOT observable inflow and outflow reaches, respectively (Figure 2). Simply put, LakeFlow assumes that lake storage change is equal to inflow minus outflow discharge while accounting for lateral inflows and evaporation. 2. Methods Bayesian infer- ence aims to approximate the posterior distribution by assuming proportion- ality (p(Θ\|x) ∝ f(x\|Θ)p(Θ)) and using Monte Carlo sampling. To implement the Bayesian inference, we log transform and scale Manning's equation to have integer coefficients, 6 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙= −6 log 𝑛𝑛+ 10 log(𝐴𝐴0 + 𝛿𝛿𝛿𝛿) −4 log 𝑊𝑊+ 3 log 𝑆𝑆𝑆 (4) (4) To provide a likelihood equation, we rearrange Equation 4 to isolate the measured variables for both the inflow and outflow reaches, 4 log 𝑊𝑊−3 log 𝑆𝑆= −6 log 𝑛𝑛−6 log 𝑄𝑄+ 10 log(𝐴𝐴0 + 𝛿𝛿𝛿𝛿), (5) (5) and the likelihood equation for river-lake mass conservation is, likelihood equation for river-lake mass conservation is, 𝛿𝛿𝛿𝛿−𝑄𝑄𝑙𝑙+ 𝐸𝐸= exp(log 𝑄𝑄𝑖𝑖) −exp(log 𝑄𝑄𝑜𝑜). (6) (6) The Bayesian approach requires prior estimates of all unknown parameters in Equation 2 which are taken from the SWOT SoS. In addition to esti- mates of Manning's n and bathymetry, the SoS provides gauge-constrained and unconstrained modeled estimates of mean flow and LakeFlow uses the gauge-constrained estimate, taken from the Global Reach-Level A Priori Discharge Estimates for SWOT (GRADES) model product (Lin et al., 2019). The Bayesian inference uses the Stan probabilistic programming language (Stan Development Team, 2023) to approximate the posterior distribution and provide estimates of all unknowns in Equation 2. Figure 2. Conceptual diagram of the LakeFlow algorithm which uses repeat SWOT observations of lakes and rivers to estimate the inflows and outflows of lakes in cubic meters per second. See Equations 1 and 2 for variable definitions. Shown are two snapshots of a lake system corresponding to two SWOT overpasses (t = 0 and t = p). Note that time 0 corresponds to the minimum observed flow on record and that only SWOT observable variables are shown for t = p. 2. Methods While SWOT provides estimates of lake storage change (δV), change in river cross-sectional area (δA), slope (S), and width (W), it does not observe Manning's n (n) or bathymetry (A0) for the inflow and outflow reaches, leaving four unknown variables in Equation 2. Note that for simplicity, we only include one inflow reach and one outflow reach for Equation 2 but LakeFlow has the capability to be applied on lakes with multiple inflow and outflow reaches. Like many other SWOT RSQ algorithms, LakeFlow struggles from parameter equifinality; there are roughly equal numbers of known and unknown parameters in Equation 2. Following the approach of Hagemann et al. (2017) and Brinkerhoff et al. (2022), we use Bayesian inference to constrain the uncertainty in LakeFlow's unknown 3 of 11 3 of 11 RIGGS ET AL. Geophysical Research Letters 10.1029/2023GL103924 Figure 2. Conceptual diagram of the LakeFlow algorithm which uses repeat SWOT observations of lakes and rivers to estimate the inflows and outflows of lakes in cubic meters per second. See Equations 1 and 2 for variable definitions. Shown are two snapshots of a lake system corresponding to two SWOT overpasses (t = 0 and t = p). Note that time 0 corresponds to the minimum observed flow on record and that only SWOT observable variables are shown for t = p. parameters (ni, A0i, no, A0o) given repeated SWOT observations. Bayesian approaches start from Bayes rule, parameters (ni, A0i, no, A0o) given repeated SWOT observations. Bayesian approaches start from Bayes rule, 𝑝𝑝(Θ\|𝑥𝑥) = 𝑓𝑓(𝑥𝑥\|Θ)𝑝𝑝(Θ) 𝑝𝑝(𝑥𝑥) , (3) (3) (3) where Θ is a set of unobserved SWOT parameters, x is the SWOT observed data, f(x\|Θ) is the sampling model where data are conditional on the param- eters, and p(Θ) is the joint prior distribution of the parameters. Thus, we are interested in approximating p(Θ\|x), the posterior distribution. Bayesian infer- ence aims to approximate the posterior distribution by assuming proportion- ality (p(Θ\|x) ∝ f(x\|Θ)p(Θ)) and using Monte Carlo sampling. To implement the Bayesian inference, we log transform and scale Manning's equation to have integer coefficients, where Θ is a set of unobserved SWOT parameters, x is the SWOT observed data, f(x\|Θ) is the sampling model where data are conditional on the param- eters, and p(Θ) is the joint prior distribution of the parameters. Thus, we are interested in approximating p(Θ\|x), the posterior distribution. 2.2. Data Sets We investigate the performance of LakeFlow in three sample lakes spanning a range of climate regions as seen in Figure 1: Lake Allatoona (humid); Lake Mohave (arid); and Tuttle Creek Reservoir (semi-arid). Lake Allatoona (area: 36 km 2) is a flood control reservoir along the Etowah River in northwestern Georgia. Lake Mohave (area: 99 km 2) is a hydropower reservoir on the Colorado River spanning the border of Arizona and Nevada. Tuttle Creek Reservoir (area: 43 km 2) is located in northeastern Kansas and is built to control floods on the Little Blue and Big Blue Rivers. These lakes each have a U.S. Geological Survey (USGS) gauge station on or near their SWOT observable inflow and outflow reaches as well as on the lakes themselves. Lake Allatoona and Lake Mohave each contain one inflow and one outflow reach and Tuttle Creek Reservoir has two inflow reaches. Because SWOT data are not yet available, we generate a synthetic data set of SWOT observable variables by utilizing gauge records from the USGS (U.S. Geological Survey, 2022), a Landsat-based water occurrence map (Pekel et al., 2016), and a priori channel attributes provided in SWORD. We then corrupt these data to produce SWOT-like observations by using the measurement errors defined by the mission science requirements and limit the number of observations to one observation per week corresponding to the approximate average overpass rate of SWOT over these lakes (Biancamaria et al., 2016). The synthetic data set is developed using hydraulic princi- ples and contains values of non-SWOT observed Manning's n and bathymetry, with ranges of 0.030–0.035 s/m 1/3 and 12–59 m 2, respectively (see Text S1 in Supporting Information S1 for details of the synthetic data set). The historical timespan of the synthetic data set is determined by the availability of USGS gauge records, such that the measurements of the lake and its inflow and outflow must all overlap in time. As a result, the timespan for 4 of 11 RIGGS ET AL. Geophysical Research Letters 10.1029/2023GL103924 the three lakes is 10/01/2009 to 09/30/2014 for Lake Allatoona, 10/01/2008 to 09/30/2013 for Lake Mohave, and 10/01/2006 to 09/30/2011 for Tuttle Creek Reservoir. 3. Results The results of the analysis, generated from synthetic SWOT data at the three test sites, indicate that the LakeFlow algorithm will be able to successfully estimate lake inflows and outflows from SWOT observations. In general, we find that LakeFlow estimated discharge skillfully resembles the gauge hydrograph for all of the inflow and outflow reaches (Figure 3). However, there is clear bias on some reaches, namely the Allatoona Lake Inflow and Tuttle Creek Reservoir Inflow 1. Even where there are biases present, LakeFlow captures flow variability for each of the reaches analyzed here as evidenced by a positive NSE for all reaches and a median NSE and NRMSE of 0.88% and 29.0%, respectively. While two reaches have relatively large rBias values, all of the other reaches have an absolute rBias less than 15% with a median rBias of 13.5%, indicating that on average, LakeFlow provides near-zero bias discharge estimates at river-lake interfaces. LakeFlow accurately estimates discharge dynamics across all seven study reaches (Figure 4a). Overall, LakeFlow discharge performance tends to modestly improve with the addition of the lateral inflow and evaporation ancil- lary datasets but does not tend to improve with the addition of only a single one of these datasets (Figures S1 and S2 in Supporting Information S1). This discrepancy is likely due to the inherent bias introduced when only including one of these ancillary terms. LakeFlow discharge mean absolute error (MAE) improves by 1.6% when both ancillary datasets are included compared to including neither. However, the bias marginally increases when both ancillary data are used but remains near-zero (Figure 4a). With and without the ancillary data, LakeFlow discharge for each study location correlates well with same-day gauge discharge observations with marginal over- estimations and underestimations in low and high flows, respectively (Pearson correlation coefficient, R ranges from 0.95 to 0.99). Across all reaches, we find that discharge performance modestly improves with the addition of the ancillary data (Figure 4b). For example, the mean NSE and NRMSE improve by 4.8% and 6.7%, respectively, when including ancillary data. Conversely, there is a positive bias present in most reaches and the mean rBias is unaffected by the inclusion of the ancillary data. Nearly all of the metrics have a negatively skewed distribution, indicating that LakeFlow performs well on average but occasionally exhibits poor performance. 2.2. Data Sets Where the LakeFlow algorithm can run (i.e., SWOT observable lakes with SWOT observable inflow and outflow reaches), lake storage change is predominantly governed by large-river inflows and outflows that are observable by SWOT, but lake storage change can also be influenced by other factors including inflow from groundwater runoff, small lateral streams (pink lines in Figure 1), and evaporation loss (Tayfur et al., 2007; Tian et al., 2022; Zhao et al., 2022). To study the impact of including these factors on LakeFlow's performance, we run two scenarios of LakeFlow: one that only includes SWOT-based observations and a second that includes SWOT observations and also ancillary datasets of lateral inflow and evaporation, represented by Ql and E in Equa- tion 2, respectively. We estimate lateral inflow using high-resolution simulated discharge from GRADES (Lin et al., 2019) and we estimate evaporation losses using modeled data from the Global Lake Evaporation Volume (GLEV) data set (Zhao et al., 2022) (see Text S2 in Supporting Information S1 for details of these ancillary datasets). We then assess LakeFlow's performance related to the ancillary datasets for each of the three study sites by comparing same-day LakeFlow estimated discharge with gauge discharge from the USGS and calculate Nash-Sutcliffe Efficiency (NSE), relative bias (rBias), normalized root-mean-square error (NRMSE), and mean absolute error (MAE) (Table S1 in Supporting Information S1). In addition to assessing discharge accuracy, we compare LakeFlow FLP estimates with the synthetic data set's values of Manning's n and bathymetry. We further compare LakeFlow FLP estimates with the SoS prior estimates to assess LakeFlow's capabilities for informing other SWOT-based RSQ algorithms. The SoS FLPs are chosen for comparison as these are the default prior FLP estimates for SWOT RSQ algorithms (Durand et al., 2023) (see Section 1 for more information). 3. Results LakeFlow estimated discharge for all lake inflows and outflows compared to gauge records. These LakeFlow discharge estimates are produced using ancillary data (“SWOT + EQl”). Figure 3. LakeFlow estimated discharge for all lake inflows and outflows compared to gauge records. These LakeFlow discharge estimates are produced using ancillary data (“SWOT + EQl”). 3. Results In addition to estimating discharge, LakeFlow can estimate unobserved bathymetry (A0) and Manning's n, with MAE values of 44 m 2 and 0.37 s/m 1/3 (log), respectively. Across all reaches and scenarios, LakeFlow MAE for bathymetry is on average 80% lower than the SoS MAE (Figure 4c) while LakeFlow estimated Manning's n values are marginally worse than the SoS (Figure 4d). LakeFlow tends to overestimate Manning's n values in the three test lakes, which may be related to bathymetry estimates having a positive bias. Bathymetric accuracy declines by 2.8% and Manning's n accuracy remains stable with the inclusion of the ancillary data. 5 of 11 RIGGS ET AL. Geophysical Research Letters 10.1029/2023GL103924 k Fl ti t d di h f ll l k i fl d tfl d t d Th L k Fl di h ti t d d LakeFlow estimated discharge for all lake inflows and outflows compared to gauge records. These LakeFlow discharge estimates are produced usi RIGGS ET AL. 6 of 11 4. Discussion The LakeFlow algorithm can provide useful discharge estimates at river-lake interfaces and will enhance the SWOT mission's capabilities for monitoring surface water dynamics. We do not test LakeFlow in locations where other SWOT RSQ algorithms have been assessed, but our findings indicate that LakeFlow's discharge accuracy is comparable or better than other SWOT RSQ algorithms (Frasson et al., 2021), thus providing the capability to extend the SWOT discharge product to river-lake boundaries with no expected decline in accuracy. These Figure 3. LakeFlow estimated discharge for all lake inflows and outflows compared to gauge records. These LakeFlow discharge estimates are produced using ancillary data (“SWOT + EQl”). RIGGS ET AL. 6 of 11 4. Discussion The LakeFlow algorithm can provide useful discharge estimates at river-lake interfaces and will enhance the SWOT mission's capabilities for monitoring surface water dynamics. We do not test LakeFlow in locations where other SWOT RSQ algorithms have been assessed, but our findings indicate that LakeFlow's discharge accuracy is comparable or better than other SWOT RSQ algorithms (Frasson et al., 2021), thus providing the capability to extend the SWOT discharge product to river-lake boundaries with no expected decline in accuracy. These Figure 3. LakeFlow estimated discharge for all lake inflows and outflows compared to gauge records. These LakeFlow discharge estimates are produced using ancillary data (“SWOT + EQl”). Figure 3. 4. Discussion The LakeFlow algorithm can provide useful discharge estimates at river-lake interfaces and will enhance the SWOT mission's capabilities for monitoring surface water dynamics. We do not test LakeFlow in locations where other SWOT RSQ algorithms have been assessed, but our findings indicate that LakeFlow's discharge accuracy is comparable or better than other SWOT RSQ algorithms (Frasson et al., 2021), thus providing the capability to extend the SWOT discharge product to river-lake boundaries with no expected decline in accuracy. These 6 of 11 RIGGS ET AL. RIGGS ET AL. Geophysical Research Letters 10.1029/2023GL103924 Figure 4. LakeFlow performance without (“SWOT only”) and with (“SWOT + EQl”) ancillary data. (a) Scatterplots of same-day gauge discharge versus LakeFlow estimated discharge across all reaches. (b) Boxplots and half violin plots of LakeFlow discharge performance metrics across all reaches: NSE (scaled by 100), rBias (%), and NRMSE (%). (c) Scatterplots of synthetic bathymetry versus LakeFlow estimated bathymetry across all reaches. (d) Scatterplots of log synthetic Manning's n versus log LakeFlow estimated Manning's n across all reaches. Figure 4. LakeFlow performance without (“SWOT only”) and with (“SWOT + EQl”) ancillary data. (a) Scatterplots of same-day gauge discharge versus LakeFlow estimated discharge across all reaches. (b) Boxplots and half violin plots of LakeFlow discharge performance metrics across all reaches: NSE (scaled by 100), rBias (%), and NRMSE (%). (c) Scatterplots of synthetic bathymetry versus LakeFlow estimated bathymetry across all reaches. (d) Scatterplots of log synthetic Manning's n versus log LakeFlow estimated Manning's n across all reaches. Figure 4. LakeFlow performance without (“SWOT only”) and with (“SWOT + EQl”) ancillary data. (a) Scatterplots of same-day gauge discharge versus LakeFlow estimated discharge across all reaches. (b) Boxplots and half violin plots of LakeFlow discharge performance metrics across all reaches: NSE (scaled by 100), rBias (%), and NRMSE (%). (c) Scatterplots of synthetic bathymetry versus LakeFlow estimated bathymetry across all reaches. (d) Scatterplots of log synthetic Manning's n versus log LakeFlow estimated Manning's n across all reaches. discharge data can inform hydroelectric and water management decisions and improve our understanding of how reservoir dynamics affect the surrounding environment (Barnett & Pierce, 2008; Chadwick et al., 2021; Huang et al., 2019; Wang et al., 2018). Reservoir operations are particularly important in transboundary water basins where water management in upstream portions of the basin can lead to actual or perceived inequities in downstream water distribution (UNEP, 2016). 4. Discussion However, LakeFlow inflow and outflow discharge estimates can potentially increase the transparency of reservoir management practices with implications for water management decisions within transboundary basins (Gleason & Hamdan, 2017). In addition to discharge, LakeFlow's ability to estimate Manning's n and bathymetry values could provide useful geomorphic insights near river-lake interfaces. Compared to the SoS, LakeFlow provides marginally worse Manning's n estimates but significantly more accurate bathymetric estimates (assuming the synthetic FLPs are a reasonably valid benchmark). However, Manning's n values are inherently limited to a small range of 0.02–0.07 s/m 1/3 (Arcement & Schneider, 1989) whereas bathymetry varies widely globally. Since RSQ algo- rithms are sensitive to prior FLP estimates (Bonnema, Sikder, Hossain, et al., 2016; Durand et al., 2016; Tuozzolo et al., 2019), the more accurate LakeFlow bathymetries could improve the performance and efficiency of other RSQ algorithms near river-lake interfaces. Thus, there is potential to implement LakeFlow into the SWOT Confluence program (Durand et al., 2023) to inform other SWOT RSQ algorithms while also extending the SWOT discharge product to river-lake boundaries. While LakeFlow is shown to perform well at the three study sites presented here, further work should be done to fully assess LakeFlow's performance. First, expanding the analysis to contain many more lakes spanning a variety of conditions would help to determine which factors (e.g., lake size, climate) are the dominant control on LakeFlow performance. To determine LakeFlow's benefits beyond discharge information, studies should quantify the effect of using LakeFlow estimates of Manning's n and bathymetry as a priori information in other SWOT RSQ algorithms. Further work is also needed to better characterize the importance of including ancillary data in LakeFlow as these data, on average, improve LakeFlow discharge estimates while decreasing bathymetric 7 of 11 RIGGS ET AL. Geophysical Research Letters 10.1029/2023GL103924 accuracy. Future work should also investigate whether additional ancillary data (e.g., water withdrawal, ground- water outflows) can improve LakeFlow's ability to estimate inflows and outflows. Errors in LakeFlow outputs may largely be driven by inaccuracies in the input data. The GRADES discharge product reports a Kling-Gupta efficiency of ≥0.2 at 62% of validation sites and the GLEV evaporation data set has an estimated uncertainty of 10%. While these errors are relatively low for global-scale products, their influence on LakeFlow's performance should be evaluated once SWOT data are available for use. 4. Discussion Finally, running LakeFlow with real SWOT data will allow for a more accurate assessment of LakeFlow performance. Running LakeFlow at the global scale using SWOT observations requires a harmonized lake and river data set, which is currently in development and will enable the further understanding of river-lake interactions worldwide. Overall, this study presents a first step in bridging river and lake hydrology with satellite remote sensing, illumi- nating a path forward for monitoring river-lake dynamics globally. Potential applications of LakeFlow include informing reservoir operations for flood control or optimizing the distribution of freshwater resources to humans and ecosystems (Boulange et al., 2021; Grimaldi et al., 2016; Munier et al., 2015). LakeFlow could also be used to provide estimates of water residence time in lakes which could offer insights into the variability of lake greenhouse gas emissions (Maavara et al., 2019, 2020), sediment supply of rivers and lakes (Kondolf et al., 2014; Lewis et al., 2013; Wisser et al., 2013), and lotic-lentic ecosystem connectivity (Harvey & Schmadel, 2021). Applied at the global scale, LakeFlow could potentially enhance our ability to monitor and understand the impact of reservoir operations on the global water cycle. 5. Conclusion The LakeFlow algorithm applies observations from SWOT to a river-lake mass conservation framework to esti- mate river discharge at lake inflows and outflows. We applied LakeFlow on three sample lakes spanning a vari- ety of physiographic conditions using a synthetic data set of SWOT-like measurements. Our findings suggest that LakeFlow can provide promising discharge estimates of river-lake boundaries using data from the SWOT satellite. Specifically, LakeFlow captures the flow dynamics at all of the SWOT-observable inflow and outflow reaches in this study with NSE values ranging from 0.46 to 0.95, similar or better to other SWOT RSQ algo- rithm performance (Frasson et al., 2021). Incorporating lateral inflow and lake evaporation ancillary datasets into LakeFlow typically improves performance, although the impact of ancillary datasets on algorithm efficacy will be clearer once SWOT data becomes available in sufficient quantities. LakeFlow can improve upon prior esti- mates of bathymetry, which may prove beneficial for other SWOT RSQ algorithms, with relevance to the SWOT Confluence program. Estimating discharge at reservoir inflow and outflow reaches will improve our understand- ing of reservoir regulations' effect on river discharge. LakeFlow is a step toward integrating remote sensing of lake storage variability and river discharge to provide a more comprehensive view of surface water dynamics. Acknowledgments Acknowledgments g This work was supported by the NASA SWOT Science Team (Grant 80NSSC20K1143), the Texas A&M Presidential Excellence Fund, and the Texas Space Grant Consortium. Craig Brinkerhoff was funded by a NASA Future Investigators in Earth and Space Science Fellowship (80NSSC21K1591). The authors would like to acknowledge Aote Xin and Sarah Sorel at Kansas State University for their assistance in synthetic data generation. The authors acknowledge members of the SWOT discharge algo- rithm working group for their feedback in the early stages of algorithm develop- ment. The authors would like to also acknowledge Valeriy Ivanov, Cassandra Nickles and an anonymous reviewer for their feedback which helped to improve the manuscript quality. Allen, G. H., & Pavelsky, T. M. (2018). Global extent of rivers and streams. Science, 361(6402), 585–588. https://doi.org/10.1126/science.aat0636 Altenau, E. H., Pavelsky, T. M., Durand, M. T., Yang, X., Frasson, R. P. D. M., & Bendezu, L. (2021). The surface water and ocean topog- raphy (SWOT) mission river database (SWORD): A global river network for satellite data products. Water Resources Research, 57(7), e2021WR030054. https://doi.org/10.1029/2021WR030054 Allen, G. H., & Pavelsky, T. M. (2018). Global extent of rivers and streams. Science, 361(6402), 585–588. https://doi.org/10.1126/science.aat0636 Altenau, E. H., Pavelsky, T. M., Durand, M. T., Yang, X., Frasson, R. P. D. M., & Bendezu, L. (2021). The surface water and ocean topog- raphy (SWOT) mission river database (SWORD): A global river network for satellite data products. Water Resources Research, 57(7), e2021WR030054. https://doi.org/10.1029/2021WR030054 Arcement, G. J., & Schneider, V. R. (1989). Guide for selecting Manning’s roughness coefficients for natural channels and flood plains (No. 2339). U.S. Geological Survey. https://doi.org/10.3133/wsp2339 Barnett, T. P., & Pierce, D. W. (2008). When will Lake Mead go dry? Water Resources Research, 44(3), W03201. https://doi. org/10.1029/2007WR006704 Batalla R J Gómez C M & Kondolf G M (2004) Reservoir-induced hydrological changes in the Ebro River basin (NE Spain) Journal of g Batalla, R. J., Gómez, C. M., & Kondolf, G. M. (2004). Reservoir-induced hydrological changes in the Ebro River basin (NE Spain). Journal of Hydrology, 290(1–2), 117–136. https://doi.org/10.1016/j.jhydrol.2003.12.002 Data Availability Statement The LakeFlow outputs and synthetic SWOT data are openly available on Zenodo (https://doi.org/10.5281/ zenodo.7781510) and the code used in this analysis can be found on GitHub (https://github.com/Ryan-Riggs/ Lakeflow). All data used to develop the synthetic datasets are publicly available: Landsat data (https://global-sur- face-water.appspot.com/download), U.S. Geological Survey gauge data (https://waterdata.usgs.gov/nwis/rt), evaporation data (https://doi.org/10.5281/zenodo.4646621), and GRADES hydrological model outputs (https:// www.reachhydro.org/home/records/grades). Acknowledgments Geophysical Research Letters https://doi.org/10.5194/hess-23-669 Busker, T., de Roo, A., Gelati, E., Schwatke, C., Adamovic, M., Bisselink, B., et al. (2019). A global lake and reservoir volume analysis using a surface water dataset and satellite altimetry. Hydrology and Earth System Sciences, 23(2), 669–690. https://doi.org/10.5194/hess-23-669-2019 Ch d i k C Gi á J B í P Vi ñ S & M F (2021) A i i f d li t h Wh i it i t Chadwick, C., Gironás, J., Barría, P., Vicuña, S., & Meza, F. (2021). Assessing reservoir performance under climate chan be too late if current water management is not changed? Water, 13(1), 64. https://doi.org/10.3390/w13010064 Cohen, S., Kettner, A. J., & Syvitski, J. P. M. (2014). Global suspended sediment and water discharge dynamics between 1960 and 2010: C nental trends and intra-basin sensitivity. Global and Planetary Change, 115, 44–58. https://doi.org/10.1016/j.gloplacha.2014.01.011 nental trends and intra-basin sensitivity. Global and Planetary Change, 115, 44–58. https://doi.org/10.1016/j.gloplacha.2014.01.011 Cooley, S. W., Ryan, J. C., & Smith, L. C. (2021). Human alteration of global surface water storage variability. Nature, 591(7848), 78–81. https:// doi.org/10.1038/s41586-021-03262-3 oley, S. W., Ryan, J. C., & Smith, L. C. (2021). Human alteration of global surface water storage variability. Nature, 591(7848), 78–81. https:// doi.org/10.1038/s41586-021-03262-3 Crétaux, J.-F., Arsen, A., Calmant, S., Kouraev, A., Vuglinski, V., Bergé-Nguyen, M., et al. (2011). SOLS: A lake database to monitor in the near real time water level and storage variations from remote sensing data. Advances in Space Research, 47(9), 1497–1507. https://doi. org/10.1016/j.asr.2011.01.004 g j Do, H. X., Gudmundsson, L., Leonard, M., & Westra, S. (2018). The global streamflow indices and metadata archive (GSIM) – Part 1: The production of a daily streamflow archive and metadata. Earth System Science Data, 10(2), 765–785. https://doi.org/10.5194/essd-10-765-2018 ll, P., Fiedler, K., & Zhang, J. (2009). Global-scale analysis of river ﬂow alterations due to water withdrawals and reservoirs. Hydrology and Earth System Sciences, 20. Durand, M., Gleason, C. J., Garambois, P. A., Bjerklie, D., Smith, L. C., Roux, H., et al. (2016). An intercomparison of remote sensing river discharge estimation algorithms from measurements of river height, width, and slope. Water Resources Research, 52(6), 4527–4549. https:// doi.org/10.1002/2015WR018434 g Durand, M., Gleason, C. J., Pavelsky, T. M., Frasson, R. P. D. M., Turmon, M. J., David, C. H., et al. (2023). A framework for estimating global river discharge from the Surface Water and Ocean Topography satellite mission. https://doi.org/10.1002/essoar.10508946.1 Durand, M., Neal, J., Rodríguez, E., Andreadis, K. M., Smith, L. Geophysical Research Letters C., & Yoon, Y. (2014). Estimating reach-averaged discharge for the River Severn from measurements of river water surface elevation and slope. Journal of Hydrology, 511, 92–104. https://doi.org/10.1016/j. jhydrol.2013.12.050 j y Everard, M., & Powell, A. (2002). Rivers as living systems. Aquatic Conservation: Marine and Freshwater Ecosystems, 12(4), 329–337. https:// doi.org/10.1002/aqc.533 ng, D., Gleason, C. J., Lin, P., Yang, X., Pan, M., & Ishitsuka, Y. (2021). Recent changes to Arctic river discharge. Nature Communications, 12(1), 6917. https://doi.org/10.1038/s41467-021-27228-1 Frasson, R. P. D. M., Durand, M. T., Larnier, K., Gleason, C., Andreadis, K. M., Hagemann, M., et al. (2021). Exploring the factors controlling the error characteristics of the surface water and ocean topography mission discharge estimates. Water Resources Research, 57(6), e2020WR028519. https://doi.org/10.1029/2020WR028519 Gao, H. (2015). Satellite remote sensing of large lakes and reservoirs: From elevation and area to storage. WIREs Water, 2, 147–157. https://doi. org/10.1002/wat2.1065 Gardner, J. R., Pavelsky, T. M., & Doyle, M. W. (2019). The abundance, size, and spacing of lakes and reservoirs connected to river netwo Geophysical Research Letters, 46(5), 2592–2601. https://doi.org/10.1029/2018GL080841 eason, C. J., & Durand, M. (2020). Remote sensing of river discharge: A review and a framing for the discipline. Remote Sensing, 12(7), 1107. https://doi.org/10.3390/rs12071107 p g eason, C. J., & Hamdan, A. N. (2017). Crossing the (watershed) divide: Satellite data and the changing politics of international river basins. Geographical Journal, 183(1), 2–15. https://doi.org/10.1111/geoj.12155 Grimaldi, S., Li, Y., Pauwels, V. R. N., & Walker, J. P. (2016). Remote sensing-derived water extent and level to constrain hydraulic flood casting models: Opportunities and challenges. Surveys in Geophysics, 37(5), 977–1034. https://doi.org/10.1007/s10712-016-9378-y Gudmundsson, L., Do, H. X., Leonard, M., & Westra, S. (2018). The global streamflow indices and metadata archive (GSIM) – Part 2: Quality control, time-series indices and homogeneity assessment. Earth System Science Data, 10(2), 787–804. https://doi.org/10.5194/ essd-10-787-2018 Hagemann, M. W., Gleason, C. J., & Durand, M. (2017). BAM: Bayesian AMHG-manning inference of discharge using remotely sensed stream width, slope, and height. Water Resources Research, 53(11), 9692–9707. https://doi.org/10.1002/2017WR021626 Harrigan, S., Zsoter, E., Alfieri, L., Prudhomme, C., Salamon, P., Wetterhall, F., et al. (2020). GloFAS-ERA5 operational global river reanalysis 1979 present Earth System Science Data 12(3) 2043 2060 https://doi org/10 5194/essd 12 2043 2020 Harrigan, S., Zsoter, E., Alfieri, L., Prudhomme, C., Salamon, P., Wetterhall, F., et al. (2020). GloFAS-ERA5 operational global river disch reanalysis 1979–present. Earth System Science Data, 12(3), 2043–2060. https://doi.org/10.5194/essd-12-2043-2020 Harvey, J. W., & Schmadel, N. M. (2021). Geophysical Research Letters Geophysical Research Letters 10.1029/2023GL103924 Biancamaria, S., Lettenmaier, D. P., & Pavelsky, T. M. (2016). The SWOT mission and its capabilities for land hydrology. Surveys in Geophysics, 37(2), 307–337. https://doi.org/10.1007/s10712-015-9346-y Biancamaria, S., Lettenmaier, D. P., & Pavelsky, T. M. (2016). The SWOT mission and its capabilities for land hydrology. Surveys in Geophysics, 37(2), 307–337. https://doi.org/10.1007/s10712-015-9346-y Bonnema, M. G., & Hossain, F. (2019). Assessing the potential of the surface water and ocean topography mission for reservoir mo M k Ri B i W R R h 55(1) 444 461 h //d i /10 1029/2018WR023743 nnema, M. G., & Hossain, F. (2019). Assessing the potential of the surface water and ocean topography mission for reservoir monitoring in the Mekong River Basin. Water Resources Research, 55(1), 444–461. https://doi.org/10.1029/2018WR023743 Bonnema, M. G., & Hossain, F. (2019). Assessing the potential of Bonnema, M. G., & Hossain, F. (2019). Assessing the potential of the surface water and ocean topography mission for reservoir monitoring in Mekong River Basin. Water Resources Research, 55(1), 444–461. https://doi.org/10.1029/2018WR023743 Basin. Water Resources Research, 55(1), 444–461. https://doi.org/10.10 Bonnema, M. G., Sikder, S., Hossain, F., Durand, M., Gleason, C. J., & Bjerklie, D. M. (2016). Benchmarking wide swath altimetry-based river discharge estimation algorithms for the Ganges river system. Water Resources Research, 52(4), 2439–2461. https://doi. org/10.1002/2015WR017296 nnema, M. G., Sikder, S., Miao, Y., Chen, X., Hossain, F., Ara Pervin, I., et al. (2016). Understanding satellite-based monthly-to-seasonal reservoir outflow estimation as a function of hydrologic controls. Water Resources Research, 52(5), 4095–4115. https://doi.org/10.1002/2015WR017830 Boulange, J., Hanasaki, N., Yamazaki, D., & Pokhrel, Y. (2021). Role of dams in reducing global flood exposure under climate change. Na Communications, 12(1), 417. https://doi.org/10.1038/s41467-020-20704-0 Brazil National Water Agency. (2022). National water and sanitation agency (ANA) [WWW Document]. Agência Nac. Águas E Saneam. Bá ANA. Retrieved from https://www.gov.br/ana/en/national_water_agency Brinkerhoff, C. B., Gleason, C. J., Feng, D., & Lin, P. (2020). Constraining remote river discharge estimation using reach-scale geomorphol Water Resources Research, 56(11), e2020WR027949. https://doi.org/10.1029/2020WR027949 Brinkerhoff, C. B., Gleason, C. J., Zappa, C. J., Raymond, P. A., & Harlan, M. E. (2022). Remotely sensing river greenhouse gas exchange v ity using the SWOT satellite. Global Biogeochemical Cycles, 36(10), e2022GB007419. https://doi.org/10.1029/2022GB007419 Busker, T., de Roo, A., Gelati, E., Schwatke, C., Adamovic, M., Bisselink, B., et al. (2019). A global lake and reservoir volume analysis u surface water dataset and satellite altimetry. Hydrology and Earth System Sciences, 23(2), 669–690. References Batalla, R. J., Gómez, C. M., & Kondolf, G. M. (2004). Reservoir-induced hydrological changes in the Ebro River basin (NE Spain). Journal of Hydrology, 290(1–2), 117–136. https://doi.org/10.1016/j.jhydrol.2003.12.002 8 of 11 RIGGS ET AL. RIGGS ET AL. Geophysical Research Letters Geophysical Research Letters 10.1029/2023GL103924 Kondolf, G. M., Gao, Y., Annandale, G. W., Morris, G. L., Jiang, E., Zhang, J., et al. (2014). Sustainable sediment management in reservoirs and regulated rivers: Experiences from five continents. Earth's Future, 2(5), 256–280. https://doi.org/10.1002/2013EF000184 Lewis, S. E., Bainbridge, Z. T., Kuhnert, P. M., Sherman, B. S., Henderson, B., Dougall, C., et al. (2013). Calculating sediment trapping efficien- cies for reservoirs in tropical settings: A case study from the Burdekin Falls Dam, NE Australia. Water Resources Research, 49(2), 1017–1029. https://doi.org/10.1002/wrcr.20117 n, P., Pan, M., Beck, H. E., Yang, Y., Yamazaki, D., Frasson, R., et al. (2019). Global reconstruction of naturalized river flows at 2.94 million reaches. Water Resources Research, 55(8), 6499–6516. https://doi.org/10.1029/2019WR025287 aavara, T., Chen, Q., Van Meter, K., Brown, L. E., Zhang, J., Ni, J., & Zarfl, C. (2020). River dam impacts on biogeochemical cycling. Nature Reviews Earth & Environment, 1(2), 103–116. https://doi.org/10.1038/s43017-019-0019-0 Maavara, T., Lauerwald, R., Laruelle, G. G., Akbarzadeh, Z., Bouskill, N. J., Van Cappellen, P., & Regnier, P. (2019). Nitrous oxide emissions from inland waters: Are IPCC estimates too high? Global Change Biology, 25(2), 473–488. https://doi.org/10.1111/gcb.14504 Macklin, M. G., & Lewin, J. (2015). The rivers of civilization. Quaternary Science Reviews, 114, 228–244. https://doi.org/10.10 quascirev.2015.02.004 unier, S., Polebistki, A., Brown, C., Belaud, G., & Lettenmaier, D. P. (2015). SWOT data assimilation for operational reservoir management on the upper Niger River Basin. Water Resources Research, 51(1), 554–575. https://doi.org/10.1002/2014WR016157 ckles, C., & Beighley, E. (2021). Leveraging river network topology and regionalization to expand SWOT-derived river discharge time series in the Mississippi River Basin. Remote Sensing, 13(8), 1590. https://doi.org/10.3390/rs13081590 Pavelsky, T. M., Durand, M. T., Andreadis, K. M., Beighley, R. E., Paiva, R. C. D., Allen, G. H., & Miller, Z. F. (2014). Assessing the potential global extent of SWOT river discharge observations. Journal of Hydrology, 519, 1516–1525. https://doi.org/10.1016/j.jhydrol.2014.08.044 kel, J.-F., Cottam, A., Gorelick, N., & Belward, A. S. (2016). High-resolution mapping of global surface water and its long-term changes. Nature, 540(7633), 418–422. https://doi.org/10.1038/nature20584 an, J. C., Smith, L. C., Cooley, S. W., Pitcher, L. H., & Pavelsky, T. M. (2020). Global characterization of inland water reservoirs using ICESat-2 altimetry and climate reanalysis. Geophysical Research Letters, 47(17), e2020GL088543. https://doi.org/10.1029/2020GL088543 Sheng, Y., Song, C., Wang, J., Lyons, E. A., Knox, B. R., Cox, J. S., & Gao, F. (2016). Representative lake water extent mapping at contine Sheng, Y., Song, C., Wang, J., Lyons, E. A., Knox, B. R., Cox, J. Geophysical Research Letters S., & Gao, F. (2016). Representative lake water extent mapping at continental scales using multi-temporal Landsat-8 imagery. Remote Sens. Environ., Landsat 8 Science Results, 185, 129–141. https://doi.org/10.1016/j. rse.2015.12.041 g g g y p pp g scales using multi-temporal Landsat-8 imagery. Remote Sens. Environ., Landsat 8 Science Results, 185, 129–141. https://doi.org/10.10 rse.2015.12.041 Shiklomanov, A. I., & Lammers, R. B. (2009). Record Russian river discharge in 2007 and the limits of analysis. Environmental Research Letters, 4, 045015. https://doi.org/10.1088/1748-9326/4/4/045015 Smith, L. C. (1997). Satellite remote sensing of river inundation area, stage, and discharge: A review. Hydrological Processes, 11(10), 1427– 1439. https://doi.org/10.1002/(SICI)1099-1085(199708)11:10<1427::AID-HYP473>3.0.CO;2-S Stan Development Team. (2023). Stan modeling language users guide and reference manual. Tayfur, G., Moramarco, T., & Singh, V. P. (2007). Predicting and forecasting flow discharge at sites receiving significant lateral inflow. Hydro- logical Processes, 21(14), 1848–1859. https://doi.org/10.1002/hyp.6320 ogical Processes, 21(14), 1848–1859. https://doi.org/10.1002/hyp.6320 Tian, W., Liu, X., Wang, K., Bai, P., Liu, C., & Liang, X. (2022). Estimation of global reservoir evaporation losses. Journal of Hydrology, 127524. https://doi.org/10.1016/j.jhydrol.2022.127524 Tuozzolo, S., Lind, G., Overstreet, B., Mangano, J., Fonstad, M., Hagemann, M., et al. (2019). Estimating river discharge with swath altimetry: A proof of concept using AirSWOT observations. Geophysical Research Letters, 46(3), 1459–1466. https://doi.org/10.1029/2018GL080771 UNEP (2016) Transboundary river basins status and trends summary for policy makers United Nations Environment Programme p p g p y , ( ), p g UNEP. (2016). Transboundary river basins status and trends, summary for policy makers. United Nations Environment Programme. U.S. Geological Survey. (2022). USGS current water data for the nation [WWW Document]. United States Geological Survey. Retrieved from h // d / i / UNEP. (2016). Transboundary river basins status and trends, summary for policy makers. United Nations Environment Programme. U S G l i l S (2022) USGS d f h i [WWW D ] U it d St t G l i l S R t i U.S. Geological Survey. (2022). USGS current water data for the nation [WWW Document]. United States Geological Survey. Retrieved from https://waterdata usgs gov/nwis/rt U.S. Geological Survey. (2022). USGS current water data for the nation [WWW Document]. United States Geological Survey. Retrieved https://waterdata.usgs.gov/nwis/rt Vörösmarty, C. J., Green, P., Salisbury, J., & Lammers, R. B. (2000). Global water resources: Vulnerability from climate change and population growth. Science, 289(5477), 284–288. https://doi.org/10.1126/science.289.5477.284 ang, F., Maberly, S. C., Wang, B., & Liang, X. (2018). Effects of dams on riverine biogeochemical cycling and ecology. Geophysical Research Letters The river corridor’s evolving connectivity of lotic and lentic waters. Frontiers in Water, 2. https://doi. org/10.3389/frwa.2020.580727 g Huang, L., Li, X., Fang, H., Yin, D., Si, Y., Wei, J., et al. (2019). Balancing social, economic and ecological benefits of reservoir operation during the flood season: A case study of the Three Gorges Project, China. Journal of Hydrology, 572, 422–434. https://doi.org/10.1016/j. jhydrol.2019.03.009 Keys, T. A., & Scott, D. T. (2018). Monitoring volumetric fluctuations in tropical lakes and reservoirs using satellite remote sensing. Lake and Reservoir Management, 34(2), 154–166. https://doi.org/10.1080/10402381.2017.1402226 9 of 11 RIGGS ET AL. Geophysical Research Letters Inland Waters, 8(2), 130–140. https://doi.org/10.1080/20442041.2018.1469335 p g Wang, J., Sheng, Y., Gleason, C. J., & Wada, Y. (2013). Downstream Yangtze River levels impacted by Three Gorges Dam. Environmental Research Letters, 8(4), 044012. https://doi.org/10.1088/1748-9326/8/4/044012 Wang, J., Sheng, Y., & Wada, Y. (2017). Little impact of the Three Gorges Dam on recent decadal lake decline across China’s Yangtze P Water Resources Research, 53(5), 3854–3877. https://doi.org/10.1002/2016WR019817 Wisser, D., Frolking, S., Hagen, S., & Bierkens, M. F. P. (2013). Beyond peak reservoir storage? A global estimate of declining water s capacity in large reservoirs. Water Resources Research, 49(9), 5732–5739. https://doi.org/10.1002/wrcr.20452 p y g ( ) p g Yang, T., Zhang, Q., Chen, Y. D., Tao, X., Xu, C., & Chen, X. (2008). A spatial assessment of hydrologic alteration caused by dam construction in the middle and lower Yellow River, China. Hydrological Processes, 22(18), 3829–3843. https://doi.org/10.1002/hyp.6993 Yang, T., Zhang, Q., Chen, Y. D., Tao, X., Xu, C., & Chen, X. (2008). A spatial assessment of hydrologic alteration caused by dam constru Yang, X., Pavelsky, T. M., Ross, M. R. V., Januchowski-Hartley, S. R., Dolan, W., Altenau, E. H., et al. (2022). Mapping flow-obstr tures on global rivers. Water Resources Research, 58(1), e2021WR030386. https://doi.org/10.1029/2021WR030386 Yoon, Y., & Beighley, E. (2015). Simulating streamflow on regulated rivers using characteristic reservoir storage patterns derived fr remote sensing data. Hydrological Processes, 29(8), 2014–2026. https://doi.org/10.1002/hyp.10342 Yoon, Y., Beighley, E., Lee, H., Pavelsky, T., & Allen, G. (2016). Estimating flood discharges in reservoir-regulated river basins by integrat- ing synthetic SWOT satellite observations and hydrologic modeling. Journal of Hydrologic Engineering, 21(4), 05015030. https://doi. org/10.1061/(ASCE)HE.1943-5584.0001320 Yoon, Y., Beighley, E., Lee, H., Pavelsky, T., & Allen, G. (2016). Estimating flood discharges in reservoir-regulated river basins by integrat- ing synthetic SWOT satellite observations and hydrologic modeling. Journal of Hydrologic Engineering, 21(4), 05015030. https://doi. org/10.1061/(ASCE)HE.1943-5584.0001320 Zhao, G., & Gao, H. (2019). Estimating reservoir evaporation losses for the United States: Fusing remote sensing and modeling approaches. Remote Sensing of Environment, 226, 109–124. https://doi.org/10.1016/j.rse.2019.03.015 Zhao, G., & Gao, H. (2019). Estimating reservoir evaporation losses for the United States: Fusing remote sensing and modeling approaches. Remote Sensing of Environment, 226, 109–124. https://doi.org/10.1016/j.rse.2019.03.015 Zhao, G., Li, Y., Zhou, L., & Gao, H. (2022). Evaporative water loss of 1.42 million global lakes. Nature Communications, 13(1), 3686. htt doi.org/10.1038/s41467-022-31125-6 ao, G., Li, Y., Zhou, L., & Gao, H. (2022). Evaporative water loss of 1.42 million global lakes. Nature Communications, 13(1), 3686. Geophysical Research Letters https:// doi.org/10.1038/s41467-022-31125-6 Zhao, G., Li, Y., Zhou, L., & Gao, H. (2022). Evaporative water loss of 1.42 million global lak doi.org/10.1038/s41467-022-31125-6 10 of 11 RIGGS ET AL. Geophysical Research Letters 10.1029/2023GL103924 References From the Supporting Information Abatzoglou, J. T., Dobrowski, S. Z., Parks, S. A., & Hegewisch, K. C. (2018). TerraClimate, a high-resolution global dataset of monthly climate and climatic water balance from 1958–2015. Scientific Data, 5(1), 170191. https://doi.org/10.1038/sdata.2017.191 ck, H. E., Wood, E. F., Pan, M., Fisher, C. K., Miralles, D. G., van Dijk, A. I. J. M., et al. (2019). MSWEP V2 Global 3-hourly 0.1° precipi- Beck, H. E., Wood, E. F., Pan, M., Fisher, C. K., Miralles, D. G., van Dijk, A. I. J. M., et al. (2019). MSWEP V2 Global 3-hourly 0.1° precipi- tation: Methodology and quantitative assessment. Bulletin of the American Meteorological Society, 100(3), 473–500. https://doi.org/10.1175/ BAMS-D-17-0138.1 Chow, V. T. (1959). Open-channel hydraulics. McGraw-Hill. ( ) p y David, C. H., Maidment, D. R., Niu, G.-Y., Yang, Z.-L., Habets, F., & Eijkhout, V. (2011). River network routing on the NHDPlus dataset. Journal of Hydrometeorology, 12(5), 913–934. https://doi.org/10.1175/2011JHM1345.1 Desrochers, N. M., Trudel, M., Biancamaria, S., Siles, G., Desroches, D., Carbonne, D., & Leconte, R. (2021). Effects of aquatic and emergent riparian vegetation on SWOT mission capability in detecting Surface water extent. Ieee Journal of Selected Topics in Applied Earth Observa- tions and Remote Sensing, 14, 12467–12478. https://doi.org/10.1109/JSTARS.2021.3128133 Leopold, L. B., & Maddock, T. (1953). The hydraulic geometry of stream channels and some physiographic implications. U.S. Governm Printing Office. Liang, X., Lettenmaier, D. P., Wood, E. F., & Burges, S. J. (1994). A simple hydrologically based model of land surface water and energy fluxes for general circulation models. Journal of Geophysical Research, 99(D7), 14415–14428. https://doi.org/10.1029/94JD00483 Penman, H. L. (1948). Natural evaporation from open water, bare soil and grass. Proceedings of the Royal Society A: Mathematical, Physical Sciences, 193, 120–145. https://doi.org/10.1098/rspa.1948.0037 dell, M., Houser, P. R., Jambor, U., Gottschalck, J., Mitchell, K., Meng, C.-J., et al. (2004). The global land data assimilation system. Bulletin of the American Meteorological Society, 85(3), 381–394. https://doi.org/10.1175/BAMS-85-3-381 Xia, X., Zhang, S., Li, S., Zhang, L., Wang, G., Zhang, L., et al. (2018). The cycle of nitrogen in river systems: Sources, transformation, and Xia, X., Zhang, S., Li, S., Zhang, L., Wang, G., Zhang, L., et al. (2018). The cycle of nitrogen in river syste Environmental Science: Processes & Impacts, 20(6), 863–891. https://doi.org/10.1039/C8EM00042E Xia, X., Zhang, S., Li, S., Zhang, L., Wang, G., Zhang, L., et al. (2018). The cycle of nitrogen in river systems: Sources, transformation, and flux. Environmental Science: Processes & Impacts, 20(6), 863–891. References From the Supporting Information https://doi.org/10.1039/C8EM00042E Zhao, G., & Gao, H. (2018). Automatic correction of contaminated images for assessment of reservoir surface area dynamics. Geophysical Research Letters, 45(12), 6092–6099. https://doi.org/10.1029/2018GL078343 Environmental Science: Processes & Impacts, 20(6), 863–891. https://doi.org/10.1039/C8EM00042E Zhao, G., & Gao, H. (2018). Automatic correction of contaminated images for assessment of reservoir surface area dynamics. Geophysical Research Letters 45(12) 6092 6099 https://doi org/10 1029/2018GL078343 p , ( ), p g Zhao, G., & Gao, H. (2018). Automatic correction of contaminated images for assessment of reservoir surface area dynamics. Geophy Zhao, G., & Gao, H. (2018). Automatic correction of contaminated images for assessment of reservoir surface area dynamics. Geophysical Research Letters, 45(12), 6092–6099. https://doi.org/10.1029/2018GL078343 Zhao, G., & Gao, H. (2018). Automatic correction of contaminated images for Research Letters, 45(12), 6092–6099. https://doi.org/10.1029/2018GL078343 , , , ( ) g Research Letters, 45(12), 6092–6099. https://doi.org/10.1029/2018GL078343 , , , ( ) g y p y Research Letters, 45(12), 6092–6099. https://doi.org/10.1029/2018GL078343 earch Letters, 45(12), 6092–6099. https://doi.org/10.1029/2018GL0783 Research Letters, 45(12), 6092–6099. https://doi.org/10.1029/2018GL078343 11 of 11 RIGGS ET AL.
https://openalex.org/W2944710832	https://www.mdpi.com/2073-4360/11/5/873/pdf?version=1559038133	English	null	Twisting of Fibers Balancing the Gel–Sol Transition in Cellulose Aqueous Suspensions	Polymers	2,019	cc-by	6,636	Received: 13 March 2019; Accepted: 28 April 2019; Published: 13 May 2019 Abstract: Cellulose hydrogels and ﬁlms are advantageous materials that are applied in modern industry and medicine. Cellulose hydrogels have a stable scaffold and never form ﬁlms upon drying, while viscous cellulose hydrosols are liquids that could be used for ﬁlm production. So, stabilizing either a gel or sol state in cellulose suspensions is a worthwhile challenge, signiﬁcant for the practical applications. However, there is no theory describing the cellulose ﬁbers’ behavior and processes underlying cellulose-gel-scaffold stabilizing. In this work, we provide a phenomenological mechanism explaining the transition between the stable-gel and shapeless-sol states in a cellulose suspension. We suppose that cellulose macromolecules and nanoﬁbrils under strong dispersing treatment (such as sonication) partially untwist and dissociate, and then reassemble in a 3D scaffold having the individual elements twisted in the nodes. The latter leads to an exponential increase in friction forces between the ﬁbers and to the corresponding fastening of the scaffold. We conﬁrm our theory by the data on the circular dichroism of the cellulose suspensions, as well as by the direct scanning electron microscope (SEM) observations and theoretical assessments. Keywords: cellulose hydrogel; cellulose ﬁlms; twisting; dispersing polymers polymers Twisting of Fibers Balancing the Gel–Sol Transition in Cellulose Aqueous Suspensions Aleksey A. Skoblin 2, Valery P. Melnikov 2, Maria G. Mikhaleva 2 and Sergey V. Stovbun 2 1 Faculty of Biology, M.V. Lomonosov Moscow State University, Lenin Hills 1/12, 119192 Moscow, Russia 2 N.N. Semenov Institute of Chemical Physics, RAS. Kosygina 4, 119991 Moscow, Russia; nikolskij56@mail.ru (S.N.N.); a.s.vedenkin@gmail.com (A.S.V.); g_politenkova@mail.ru (G.G.P.); ab1954@yandex.ru (A.A.S.); melnikov@chph.ras.ru (V.P.M.); wawe@bk.ru (M.G.M.); s.stovbun@yandex.ru (S.V.S.) Faculty of Biology, M.V. Lomonosov Moscow State University, Lenin Hills 1/12, 119192 Moscow, Russia 2 N.N. Semenov Institute of Chemical Physics, RAS. Kosygina 4, 119991 Moscow, Russia; nikolskij56@mail.ru (S.N.N.); a.s.vedenkin@gmail.com (A.S.V.); g_politenkova@mail.ru (G.G.P.); ab1954@yandex.ru (A.A.S.); melnikov@chph.ras.ru (V.P.M.); wawe@bk.ru (M.G.M.); s.stovbun@yandex.ru (S.V.S.) y gy, y, / , , 2 N.N. Semenov Institute of Chemical Physics, RAS. Kosygina 4, 119991 Moscow, Russia; nikolskij56@mail.ru (S.N.N.); a.s.vedenkin@gmail.com (A.S.V.); g_politenkova@mail.ru (G.G.P.); ab1954@yandex.ru (A.A.S.); melnikov@chph.ras.ru (V.P.M.); wawe@bk.ru (M.G.M.); s.stovbun@yandex.ru (S.V.S.) * Correspondence: dvzlenko@gmail.com; Tel.: +7-926-683-3300 * Correspondence: dvzlenko@gmail.com; Tel.: +7-926-683-3300 Polymers 2019, 11, 873; doi:10.3390/polym11050873 Polymers 2019, 11, 873 2 of 11 Polymers 2019, 11, 873 Polymers 2019, 11, 873 Once the concentrated aqueous solutions of nanocellulose are dried, dense ﬁlms may form [2,19,20]. Nanocellulose ﬁlms often demonstrate unique optical properties due to the chiral nematic phase formation [2,18,20,21]. Water evaporation decreases the cellulose nanoﬁbers’ helix pitch, which, in its turn, results in coloration [2,19,22]. The latter is related to the supramolecular packing of nanoﬁbrils within the ﬁlm, and strongly depends on the heterogeneity of the ﬁbrils. Thus, the coloration effect is tied to the protocol of the raw-material treatment [19]. Although many works focused on adjusting nanocellulose properties, the structure–property relationship between nanocellulose ﬁbers and their chiral phases and gels is still relatively unstudied [2,8]. For example, increasing cellulose nanoﬁbers’ surface-bound charge can increase the pitch of the chiral phase in solution, and, thus, promote the liquid crystalline phase formation [23]. Meanwhile, the density of the cellulose ﬁlms is also related with the charge density on the ﬁbers’ surface, while the mechanical properties of the ﬁlms correlate mostly with the crystallinity index [17]. Cellulose, chitin, and chitosan hydrogels are themselves advantageous materials, applied in many ﬁelds of medicine and agriculture [24–26]. For example, cellulose-based hydrogels can be used as water/nutrient reservoirs [24], for stomach bulking, for drug delivery [27], or for wound dressing [27,28]. However, cellulose ﬁlms obtained by the hydrogels’ drying are also of particular interest, for example, as biodegradable and safe material for food packing [29]. The properties of nanocellulose suspensions critically depend on the preparation conditions and cellulose’s origin [2,8,16,17,30]. In some cases, the hydrogel properties can signiﬁcantly restrict the formation of the dense ﬁlms. This phenomenon was described as the “nonequilibrium kinetic arrest” of the gel-like state in the cellulose suspension preventing the formation of dense ﬁlms upon water evaporation [23,31]. Yet, in case the gel-like state was not “arrested”, suspension become unsuitable for stable hydrogel production. So, the problem to stabilize one of these states in nanocellulose suspensions is of crucial importance for modern high-end industrial applications. Nevertheless, there is no universal theoretical description of the processes stabilizing the 3D scaffold of nanocellulose gels. The cellulose hydrogel formation and stabilization are usually explained through van der Waals interactions and hydrogen bonding between nanocellulose ﬁbers [25,30,32,33]. However, in natural cellulose, hydrogen bonds predominantly form between the macromolecules rather than between the nanoﬁbrils [24,34,35]. Polymers 2019, 11, 873 The spatial complementarity of cellulose chains is essential for short-range hydrogen-bond formation, and this condition seems to not be satisﬁed in case of an irregular gel lattice. Still, nanocellulose forms stable hydrogels [23,24]. In this work, we demonstrate that the extent to which cellulose is ground determines the stability of the resulting hydrogel. Mild grinding and dispersing results in less stable hydogels that could be used to produce dense and transparent ﬁlms via simple drying. On the other hand, intensive dispersing (such as sonication) leads to the gel-like hydrogels that form white, opaque, and shapeless dense pieces upon drying. Such hydrogels could not be used to produce ﬁlms, but are stable enough to be utilized as hydrogels themselves [24]. Based on our previous works, we provide a phenomenological description of the mechanism explaining the behavior of the nanocellulose suspensions and its dependence on the degree of the milling. The main idea is that, in case of intense dispersing, cellulose was able to reassemble into the morphological structures resembling natural supramolecular packing. This leads to stabilizing the hydrogel scaffold in 3D form and preventing ﬁlm formation. 1. Introduction Cellulose is a common biological polymer, widely used in industry due to its excellent mechanical properties, biodegradability, and comparatively low cost. Natural cellulose (and chitin) consists of macroscopic ﬁbers composed of smaller and mechanically stronger helical elements [1–3]. The smallest building block of the natural cellulose is an elementary ﬁbril or nanoﬁbril, which is a twisted bundle of parallel cellulose chains [2,4,5]. The lateral size of the nanoﬁbril depends on the origin of the cellulose, and is in the range of 3–4 nm in softwood [6–9], to 9 nm in cotton [10], and up to 20 nm in cellulose from tunicates [5]. Nanoﬁbrils twist into microﬁbrils forming helical bundles [11–13]. The hierarchical supramolecular organization of cellulose provides for its mechanical properties in nature [1,14]. The enzymatic hydrolysis of cellulose, followed by ultraﬁne mechanical milling in water, leads to forming hydrogels that contain 1%–3% cellulose by mass [15]. The same result could be achieved through aggressive acid hydrolysis followed by sonication [16]. The lattice of cellulose hydrogels is made up of nanoﬁbers (nanocellulose) having a rather high aspect ratio, enriched with a crystalline fraction [17] and demonstrating a prominent helical structure [8,18]. Increasing hydrolysis time or intensifying the dispersion process leads to a decrease in both the length of the nanoﬁbers and their polydispersion [2,18]. Polymers 2019, 11, 873; doi:10.3390/polym11050873 www.mdpi.com/journal/polymers www.mdpi.com/journal/polymers 2. Materials and Methods The hardwood bleached craft cellulose (GOST 28172-89, Figure 1A) was obtained from Arkhangelsk pulp-and-paper mill (Russia). The cellulose suspensions were prepared in 3 steps: 1. oxidative hydrolysis; 2. preliminary grinding; 3. ﬁnal dispersing. Acid hydrolysis or oxidative acid hydrolysis is a standard initial step for cellulose hydrogel preparation [2,23,36,37]. The subsequent treatment could be mechanical grinding [8,15] or sonication [2,16,23]. Here, we used both approaches. Polymers 2019, 11, 873 3 of 11 10 m B 30 m A 30 m 10 m A B 5 m C 5 m D E F Figure 1. (A–D) Typical scanning electron microscope (SEM) images of cellulose pulp in the course of milling. (A) Initial raw; (B) pulp after oxidative hydrolysis; (C) pulp treated with a colloidal mill; (D) cellulose after ﬁnal dispersing with a high-pressure homogenizer (hydrosol). White blocks correspond to salt crystals. Pulp after high-pressure homogenization (hydrosol) was a viscous liquid (E) that became a jellylike substance (hydrogel) after sonication (F). B 5 m D 5 m C E F Figure 1. (A–D) Typical scanning electron microscope (SEM) images of cellulose pulp in the course of milling. (A) Initial raw; (B) pulp after oxidative hydrolysis; (C) pulp treated with a colloidal mill; (D) cellulose after ﬁnal dispersing with a high-pressure homogenizer (hydrosol). White blocks correspond to salt crystals. Pulp after high-pressure homogenization (hydrosol) was a viscous liquid (E) that became a jellylike substance (hydrogel) after sonication (F). Cellulose hydrolysis (2 h) was held in the mixed aqueous solution of sulfuric acid (10%) and hydrogen peroxide (3%) at a temperature of 95–97 ◦C and a solvent-to-pulp ratio of 30 (per mass). After cooling the mixture, the reaction was stopped by ﬂushing with cold distilled water on a glass ﬁlter. Then, the pulp was drained with cold distilled water until pH reached 7.0. Then, the suspension of cellulose (2.5%–2.8% per mass) was ground by a colloidal mill MK 2000 (IKA, Germany) at 4 of 11 Polymers 2019, 11, 873 the room temperature. The rotor–stator clearance was smaller than 10 µm, and milling time was 30 min (Figure 1C). The obtained cellulose pulp (2.5%–2.8%) was diluted 10 times with distilled water and dispersed using high-pressure homogenizer HPH 2000/4-DH5 (IKA, Germany), at pressure of 1.8–1.9 kBar. The procedure was repeated 10 times. The obtained cellulose suspensions (∼2.5 mg/mL, Figure 1D) were white viscous liquids (Figure 1E), so we refer to them as “hydrosols”. 2. Materials and Methods Hydrosols were diluted twofold with distilled water and sonicated using immersible source UZG 8 −0.4/22 (22 kHz, 0.4 kW, VNIITVCH, Sent-Petersburg, Russia) for 5 min. The resulting suspensions (∼1 mg/mL) were jellylike nonﬂowing white substances (Figure 1F) as was previously shown [29]. So, here and hereafter this type of the cellulose suspension is referred to as “hydgels”. The grinding procedures were independently repeated 5 times. The morphology of the cellulose in suspensions was examined microscopically in the xerogel and aerogel samples. Xerogel samples were prepared as follows: the samples of the cellulose hydrosol were diluted 100 times (down to ∼25 ng/mL) with the distilled water and incubated under stirring for 1 hour. The aliquots of the obtained material were dripped on the glass slide and dried at room temperature for 3 h. Prior to the experiments, the glass surface was sonicated and then washed with 100% ethanol. The aerogel samples [25,33] were prepared as follows: the cellulose hydrogel droplets (2–4 mm in diameter) were frozen in liquid nitrogen and lyophilized at −25 ◦C. The samples of the cellulose suspensions in the course of preliminary treatment and milling (Figure 1A–D) were investigated using a Phenom XL (Phenom World) scanning electron microscope. After each stage of treatment, the samples of the cellulose pulp were placed on the glass slide, dried at 70 ◦C for 12 h, and examined. The xerogel samples were examined using a Solver UHV (NT-MDT, Russia) atomic force microscope (AFM) operating in a semicontact mode. Semicontact mode is preferable for the soft and easily destroyable materials, and it allows for achieving a resolution of 1 nm. Aerogel samples were investigated by a JSM-7500F (Jeol, Japan) scanning electron microscope (SEM) operating at low accelerating voltage (0.5–1.5 kV). Aerogel samples were not covered by metal. The twisting/untwisting of the cellulose ﬁbers was monitored by circular-dichroism (CD) spectra. CD spectra were measured using an SKD-2 CD spectrometer [38]. Cellulose ﬁlms were prepared from liquid mass by drying at the glass slide at room temperature. The chemicals were obtained from ChimMed (Moscow, Russia). 3.1. Hydrosol Sedimentation Stability and Structure The problem to ﬁnd the right equilibrium between stabilizing and destabilizing the cellulose gel structure is important for practical applications [8,16,23,30,31], as cellulose can be used in the form of the hydrogel itself [25,39] or in the form of the ﬁlm [2,24]. At the same time, even the simple sedimental stability of the cellulose gels was not previously estimated, so we believe this task is noteworthy. To verify the cellulose suspensions’ long-term stability, 12 independent samples of hydrosol (1.1%, 1.4%, 1.8%, and 2.7%, by mass, three repeats per each concentration) were incubated at room temperature for two years. During this period, we did not observe any precipitates or optical inhomogeneities, irrespective of gel concentration. So, in the hydrosol, the characteristic time of cellulose precipitation or aggregation lies in a yearly time-scale. The AFM investigation of dried hydrosols revealed chaos of the short (<1 µm on average) and rather thick (∼100 nm on average) ﬁbers. Let us estimate the characteristic time of precipitation of 5 of 11 Polymers 2019, 11, 873 such ﬁbers. The velocity (v) of the sedimentation of spherical particles having a diameter d could be estimated according to the expression: such ﬁbers. The velocity (v) of the sedimentation of spherical particles having a diameter d could be estimated according to the expression: v = g(ρ −ρ0)d2 18η v = g(ρ −ρ0)d2 18η where ρ and ρ0 are the density of the particles and surrounding medium, correspondingly; g, gravity acceleration; and η, viscosity of the medium. The same estimate can be considered as an upper-bound assessment of the sedimentation rate of the cylinder-shaped particles of the same diameter (d). So, sedimentation of the micron-sized cellulose hydrosol elements should become noticeable in a time scale of days, while hydrosols were stable for at least two years. where ρ and ρ0 are the density of the particles and surrounding medium, correspondingly; g, gravity acceleration; and η, viscosity of the medium. The same estimate can be considered as an upper-bound assessment of the sedimentation rate of the cylinder-shaped particles of the same diameter (d). So, sedimentation of the micron-sized cellulose hydrosol elements should become noticeable in a time scale of days, while hydrosols were stable for at least two years. The AFM investigation of the hydrosols also revealed elements of a smaller diameter of about 100 nm (Figure 2B). These particles should also precipitate in the yearly time-scale. 3.1. Hydrosol Sedimentation Stability and Structure However, sedimentation was not observed, which implies that cellulose ﬁbers were interacting with each other, forming a continuous lattice, which accounts for the sedimental stability of the hydrosol. 0.6 0 0.2 0.8 1.0 1.2 distance, m distance, m 0 20 40 60 80 height, nm 0 40 80 120 160 distance, m distance, m height, m A 0 0.2 0.4 0.6 0.8 1.0 1.2 0.4 0 100 200 300 400 500 10 20 30 40 50 60 0.4 0.6 1.2 1.6 2.0 0 0 0.4 0.8 1.2 1.6 2.0 B distance, m height, m Figure 2. Typical atomic force microscope (AFM) images of the dried cellulose hydrosol. Cellulose was passed through the high-pressure homogenizer but was not sonicated. Some regions were composed of the more-or-less parallel elements (A), while in others, the cellulose elements crossed (B). The inset in (A) shows the horizontal relief proﬁle. 0.6 0 0.2 0.8 1.0 1.2 distance, m distance, m 0 20 40 60 80 height, nm A 0 0.2 0.4 0.6 0.8 1.0 1.2 0.4 0 100 200 300 400 500 10 20 30 40 50 60 distance, m height, m 0 40 80 120 160 distance, m distance, m height, m 0.4 0.6 1.2 1.6 2.0 0 0 0.4 0.8 1.2 1.6 2.0 B distance, m Figure 2. Typical atomic force microscope (AFM) images of the dried cellulose hydrosol. Cellulose was passed through the high-pressure homogenizer but was not sonicated. Some regions were composed of the more-or-less parallel elements (A), while in others, the cellulose elements crossed (B). The inset in (A) shows the horizontal relief proﬁle. Figure 2. Typical atomic force microscope (AFM) images of the dried cellulose hydrosol. Cellulose was passed through the high-pressure homogenizer but was not sonicated. Some regions were composed of the more-or-less parallel elements (A), while in others, the cellulose elements crossed (B). The inset in (A) shows the horizontal relief proﬁle. Let us estimate the energy of interaction between cellulose ﬁbers in hydrosol. The energy (W) of interaction between two parallel cellulose ﬁbers having radius R ∼100 nm and length L ∼1 µm could be estimated as [40]: √ Let us estimate the energy of interaction between cellulose ﬁbers in hydrosol. 3.1. Hydrosol Sedimentation Stability and Structure The energy (W) of interaction between two parallel cellulose ﬁbers having radius R ∼100 nm and length L ∼1 µm could be estimated as [40]: √ W = AL √ R 24 √ h3 , W = AL √ R 24 √ h3 , where h is the distance between the ﬁbers, and A = 10−20 J – Hamaker constant [40]. The Coulomb friction force (Ff ) is related to downforce Fd and a friction coefﬁcient (k f ): where h is the distance between the ﬁbers, and A = 10−20 J – Hamaker constant [40]. The Coulomb friction force (Ff ) is related to downforce Fd and a friction coefﬁcient (k f ): Ff = k f Fd Ff = k f Fd The downforce could be calculated as a partial derivative of the interaction energy between the ﬁbrils per the distance between them: √ Fd = −∂W ∂h = AL √ R 16 √ h5 Fd = −∂W ∂h = AL √ R 16 √ h5 Assuming k f ∼1, and the distance between the ﬁbers ∼nm (Figure 2), force Ff would be about 10−9 N. For the crossed ﬁbrils, the friction force would be smaller in proportion to the ratio of the ﬁber length and diameter, i.e., approximately ∼10−10 N. Polymers 2019, 11, 873 6 of 11 Polymers 2019, 11, 873 The gravity force acting on the cellulose ﬁber and responsible for sedimentation could be assessed as follows (regardless the Archimedes buoyant force): The gravity force acting on the cellulose ﬁber and responsible for sedimentation could be assessed as follows (regardless the Archimedes buoyant force): FG = mg = Vρg = πR2Lρg ∼10−16 N FG = mg = Vρg = πR2Lρg ∼10−16 N where the density on cellulose ρ was assumed to be ∼1.5 g/mL. Therefore, the gravity force is many orders of magnitude smaller than the friction force between the crossed and tightly clamped-down cellulose ﬁbers. So, hydrosol’s stability could be explained by van der Waals interaction between the ﬁbrils. 3.3. Cellulose Films Fine cellulose suspensions are known to form dense ﬁlms upon drying, but the properties of the obtained suspensions and ﬁlms strongly depend on the treatment protocol [2,18,31,43]. For example, the ﬁlms’ coloration depends on the size distribution of the cellulose nanocrystals [19], while some treatment conditions (acid hydrolysis time [44]), as well as the system composition [23] may lock the suspension in a gel-like state. We attempted to prepare the ﬁlms using a cellulose suspension taken at different stages of treatment (Figure 1). Upon drying, the cellulose only pretreated with a colloidal mill (Figure 1C) formed white, opaque, and spongy ﬁlms (Figure 4, top). On the other hand, after the high-pressure treatment, the obtained hydrosols formed dense, transparent, and colorless ﬁlms (Figure 4, bottom). The latter ﬁlms were signiﬁcantly thinner. Figure 4. Cellulose ﬁlms obtained from the pulp only treated with a colloidal mill (top, Figure 1C), and additionally treated with the high-pressure homogenizer (bottom, Figure 1D). Hydrogels obtained after sonication of the cellulose suspension did not form ﬁlms upon drying. Figure 4. Cellulose ﬁlms obtained from the pulp only treated with a colloidal mill (top, Figure 1C), and additionally treated with the high-pressure homogenizer (bottom, Figure 1D). Hydrogels obtained after sonication of the cellulose suspension did not form ﬁlms upon drying. The difference in behavior of the milled and additionally homogenized cellulose could be explained by the crucial difference in the dimension of the elements present in the mixture (Figure 1C,D). The suspension treated with a colloidal mill still contained the intact fragments of plant cells that are absent in the hydrosol. Too-large and hollow fragments form a cavernulous and highly scattering ﬁlm (Figure 4, top). On the other hand, the hydrosol was composed of much smaller elements that were able to agglomerate in a dense and uniform ﬁlm. It would be reasonable to propose that the sonicated cellulose suspension (hydrogel) would form denser ﬁlms than the hydrosol, as the additional dispersing treatment would decrease the average size of the cellulose fragments and increase their ability to agglutinate. However, upon drying, the pieces of hydrogel formed irregular-shaped opaque blocks instead of ﬁlms. It seems that there is a clear analogy between the observed effect and the earlier-reported “kinetic arrest” of the gel-like state described for concentrated cellulose suspensions [23]. 3.2. Hydrogel Structure The morphology of the cellulose hydrogels was examined in the aerogel samples. The lyophilization of gel droplets frozen at 77 K preserves the original structure of the gel lattice [25,33] that can then be visualized using SEM (Figure 3). The scaffold of the cellulose hydrogel looks like a continuous irregular net of the helical cellulose ﬁbers with a diameter of about 10–20 nm, occasionally united in the thicker ﬁbers, up to 100 nm in diameter. On average, the length of the thin ﬁbers was about several hundred nanometers. Similar dimensions were earlier reported for cellulose hydrogels treated in a similar manner [15,18,31]. 100nm 100nm A B Figure 3. Typical SEM images of the lyophilized nanocellulose hydrogels (aerogels). Aerogel samples were rather heterogeneous, some areas were sparse (A), while others were dense (B). Arrows indicate helical and twisted zones of contact between ﬁbrils. 100nm B 100nm A Figure 3. Typical SEM images of the lyophilized nanocellulose hydrogels (aerogels). Aerogel samples were rather heterogeneous, some areas were sparse (A), while others were dense (B). Arrows indicate helical and twisted zones of contact between ﬁbrils. The helical structure and dimensions of the cellulose ﬁbrils in the hydrogel strongly resemble the hierarchical structure of the native cellulose [1,3]. The ﬁbers of several dozen nanometers in diameter correspond to native cellulose microﬁbrils usually having a diameter of about 15–30 nm [12,24]. Thicker ﬁbers could be considered as the bundles of the microﬁbrils that were also observed in plant cell walls [12,24,35]. The relatively small length of the ﬁbers (∼1 µm) in the hydrogel implies the breaking of the microﬁbrils in the course of the milling and homogenization (Figure 1B). The congruence of elements’ sizes in the hydrogel and natural cellulose most probably points at preserving the native cellulose structure despite of the acid hydrolysis, grinding, and sonication. The main structural difference between native cellulose and the hydrogel’s scaffold is that ﬁbrils composing the hydogel wind around each other and form a 3D net (Figure 3, arrows) instead of the regular layered structure typical of the plant cell wall [6,41,42]. Similar images were demonstrated earlier for the sonicated cellulose hydrogels [2,5,18]. This observation implies that, in the hydrogel’s scaffold, the cellulose ﬁbrils have, in part, preserved their native structure and, in part, have formed some new contacts resembling the native ones. 3.2. Hydrogel Structure The latter were randomly formed and accounted for 7 of 11 Polymers 2019, 11, 873 stabilizing the hydrogel’s scaffold. The friction force between the twisted ﬁbers increases exponentially according to the Capstan equation: 2 k stabilizing the hydrogel’s scaffold. The friction force between the twisted ﬁbers increases exponentially according to the Capstan equation: k Ff ∼e2παk f Ff ∼e2παk f where α is the number of turns. This effect is absent in a more-or-less liquid hydrosol, and makes the hydrogel a jellylike substance capable of shape-keeping. where α is the number of turns. This effect is absent in a more-or-less liquid hydrosol, and makes the hydrogel a jellylike substance capable of shape-keeping. 3.4. Optical Activity of Cellulose Hydrosol Based on SEM observations, we proposed that gel-state stabilization after sonication occurs due to the occasional mutual twisting of the cellulose ﬁbrils that leads to forming the continuous 3D lattice. Twisting cellulose ﬁbrils into novel structures should be precluded by a partial untwisting of the existed structures [3]. The latter can be monitored through measuring the optical activity (circular dichroism spectra) of the samples, as the contribution of twisting the crystalline nanoﬁbrils seems to be greater than the optical activity of individual glucose monomers [38,45]. So, variations in the CD spectra observed under nondestructive conditions clearly indicate the twisting/untwisting processes in cellulose. The cellulose hydrosols demonstrated prominent optical activity in the near-UV region (Figure 5A). The maximum of the CD spectrum shifted hypsochromically with the decrease of cellulose concentration (Figure 5A, inset). This effect can be explained by the cellulose ﬁbers untwisting caused by dilution, as untwisting leads to increasing the void volume in the samples and decreasing the density of the ﬁbers’ packing, correspondingly. The latter leads to widening the band gap and the corresponding shift of the absorption maximum, which directly affects the CD maximum position. The UV absorption spectra themselves were crucially corrupted by light scattering in the turbid cellulose hydrosol samples (data not shown). So, the observed hypsochromic shift could be considered as an indication of an at least partial untwisting of the nanoﬁbrils. 220 240 260 280 300 320 340 360 380 Wavelength (nm) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 CD signal (a.u.) 220 230 240 250 260 270 280 290 300 310 0.0 0.2 0.4 0.6 0.8 1.0 1.2 CD signal (a.u.) Wavelength (nm) 0.0 1.0 2.0 3.0 240 280 320 cellulose concentration (%) Wavelength (nm) [NaHSO3] (%) Amplitude(a.u.) 0.4 2.0 2.8 0.6 0.8 1.0 1.2 A B Figure 5. Circular-dichroism spectra of the cellulose hydrosols of different concentration (A), and in the presence on sodium hydrosulﬁte (B). (A) purple—3.0% of cellulose by weight, blue—1.5%, green—0.75%, orange—0.38%, and red—0.19%. Inset: position of the maximum as a function of cellulose concentration. (B) violet—pure cellulose hydrosol, cyan—1.0% of NaHSO3 by weight, yellow—2.0%, and red—3.0% (cellulose concentration, 0.38%). Inset: spectra maximum amplitude as a function of NaHSO3 concentration. 3.3. Cellulose Films Note that, in our experiments, the hydrogel samples were diluted twice, as compared to the hydrosol, so, the concentration cannot explain the gel stabilization. Polymers 2019, 11, 873 8 of 11 Sonication is a widely used approach for dispersing cellulose and preparing hydrogels [2,16,23]. However, our results demonstrate that if the ﬁnal goal of the work is ﬁlm production, so sonication seems to be too strong, thus promoting locking the suspension in a gel state. 3.4. Optical Activity of Cellulose Hydrosol 220 230 240 250 260 270 280 290 300 310 0.0 0.2 0.4 0.6 0.8 1.0 1.2 CD signal (a.u.) Wavelength (nm) [NaHSO3] (%) Amplitude(a.u.) 0.4 2.0 2.8 0.6 0.8 1.0 1.2 B 220 240 260 280 300 320 340 360 380 Wavelength (nm) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 CD signal (a.u.) 0.0 1.0 2.0 3.0 240 280 320 cellulose concentration (%) Wavelength (nm) A Wavelength (nm) Figure 5. Circular-dichroism spectra of the cellulose hydrosols of different concentration (A), and in Figure 5. Circular dichroism spectra of the cellulose hydrosols of different concentration (A), and in the presence on sodium hydrosulﬁte (B). (A) purple—3.0% of cellulose by weight, blue—1.5%, green—0.75%, orange—0.38%, and red—0.19%. Inset: position of the maximum as a function of cellulose concentration. (B) violet—pure cellulose hydrosol, cyan—1.0% of NaHSO3 by weight, yellow—2.0%, and red—3.0% (cellulose concentration, 0.38%). Inset: spectra maximum amplitude as a function of NaHSO3 concentration. The untwisting o cellulose ﬁbers should be intensiﬁed by the electrostatic repulsion of the nanoﬁbrils in case of charging their surface [17,23]. For example, the sulphiting of the cellulose (in the course of the sulﬁte pulping process) should result in negatively charged nanoﬁbrils. Charging the nanoﬁbrils’ surface was reported even for a much-stronger sulphuric acid [23]. Treating hydrosol samples with a sodium hydrosulphite affected the CD spectra in accordance with our assumption (Figure 5B). The amplitude of the CD signal decreased with increasing NaHSO3 concentration (Figure 5B, inset). So, the twisting–untwisting processes take place in cellulose suspensions upon some external stimuli. SEM observations and data on the optical activity, taken 9 of 11 Polymers 2019, 11, 873 together, brought us to the conclusion that stabilization of the cellulose hydrogel structure can occur due to the mutual twisting of ﬁbers detached from each other by sonication. The following abbreviations are used in this manuscript: The following abbreviations are used in this manuscript: The following abbreviations are used in this manuscript: The following abbreviations are used in this manuscript: AFM Atomic Force Microscopy CD Circular Dichroism SEM Scanning Electron Microscopy The following abbreviations are used in this manuscript: AFM Atomic Force Microscopy CD Circular Dichroism SEM Scanning Electron Microscopy AFM Atomic Force Microscopy CD Circular Dichroism SEM Scanning Electron Microscopy 4. Conclusions Cellulose nanoﬁbrils can stick together to form a 3D net that serves as the gel scaffold [25,39], or to form the dense and transparent or even colored ﬁlms [2,31]. Here, we demonstrated the crucial inﬂuence of cellulose treatment intensity on the properties of the ﬁnal cellulose suspensions and ﬁlms. In particular, sonication seems to be strong enough to cause partial cellulose untwisting, which leads to reverse twisting after the ultrasound was switched off. The twisting back can occur randomly in the bulk of the suspension that stabilizes the 3D scaffold of the hydrogel and prevents dense-ﬁlm formation. On the other hand, the mechanical strength of individual nanoﬁbrils is much greater than the strength of macroscopic cellulose ﬁbers [46]. The mutual arrangement of the cellulose nanoﬁbrils in the natural materials is optimized for their functions in the organism, but not for industrial applications. Rearrangement of the natural nanoﬁbrils’ packing is a very promising way to produce the novel materials. For example, ﬂow-assisted orientation and agglomeration of the nanocellulose ﬁbers allowed to create a ﬁber with tensile strength up to 1.5 GPa [36]. Considering our hypothesis on the role of the twisting in the hydrogels’ scaffold stabilization and its role in other processes in cellulose pulp, we anticipate that the next step in the design of cellulose-based materials would be controlled aggregation and twisting of the individual macromolecules into a thick ﬁber (“huge nanoﬁbril”) having overwhelming mechanical properties. Author Contributions: Conceptualization, D.V.Z.; Data curation, S.N.N. and A.S.V.; Formal analysis, D.V.Z., A.A.S. and S.V.S.; Funding acquisition, S.V.S.; Investigation, S.N.N., G.G.P. and V.P.M.; Methodology, S.N.N.; Visualization, D.V.Z.; Writing—original draft, M.G.M.; Writing—review & editing, D.V.Z. and S.V.S. Funding: This research was funded by FASO Russia, theme number 45.9, 0082-2014-0011, AAAA-17-117111600093-8. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Funding: This research was funded by FASO Russia, theme number 45.9, 0082-2014-0011, AAAA-17-1171 Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Author Contributions: Conceptualization, D.V.Z.; Data curation, S.N.N. and A.S.V.; Formal analysis, D.V.Z., A.A.S. and S.V.S.; Funding acquisition, S.V.S.; Investigation, S.N.N., G.G.P. and V.P.M.; Methodology, S.N.N.; Visualization, D.V.Z.; Writing—original draft, M.G.M.; Writing—review & editing, D.V.Z. and S.V.S. Funding: This research was funded by FASO Russia, theme number 45.9, 0082-2014-0011, AAAA-17-117111600093-8. Conﬂicts of Interest: The authors declare no conﬂicts of interest. References Cellulose Fibril Aggregation—An Inherent Property of Kraft Pulps. 2001, 42, 3309–3314. [CrossRef] 10. Newman, R. Estimation of the Lateral Dimensions of Cellulose Crystallites Using 13C NMR Signal Strengths. Solid State Nucl. Magn. Reson. 1999, 15, 21–29. [CrossRef] 11. Ruben, G.; Bokelman, G. Triple-Stranded, Left-Hand-Twisted Cellulose Microﬂbril. Carbohydr. Res. 1987, 160, 434–443. [CrossRef] 12. Hanley, S.J.; Giasson, J.; Revol, J.F.; Gray, D.G. Atomic Force Microscopy of Cellulose Microﬁbrils: Comparison with Transmission Electron Microscopy. Polymer 1992, 33, 4639–4642. [CrossRef] 13. Hanley, S.J.; Revol, J.F.; Godbout, L.; Gray, D.G. Atomic Force Microscopy and Transmission Electron Microscopy of Cellulose from Micrasterias Denticulata; Evid. A Chiral Helical Microﬁbril Twist. Cellulose 1997, 4, 209–220. [CrossRef] 14. Tseng, P.; Napier, B.; Zhao, S.; Mitropoulos, A.N.; Applegate, M.B.; Marelli, B.; Kaplan, D.L.; Omenetto, F.G. Directed Assembly of Bio-Inspired Hierarchical Materials with Controlled Nanoﬁbrillar Architectures. Nat. Nanotechnol. 2017, 12, 474–480. [CrossRef] 15. Pääkkö, M.; Ankerfors, M.; Kosonen, H.; Nykänen, A.; Ahola, S.; Österberg, M.; Ruokolainen, J.; Laine, J.; Larsson, P.; Ikkala, O.; Lindström, T. Enzymatic Hydrolysis Combined with Mechanical Shearing and High-Pressure Homogenization for Nanoscale Cellulose Fibrils and Strong Gels. Biomacromolecules 2007, 8, 1934–1941. [PubMed] 6. Fleming, K.; Gray, D.G.; Matthews, S. Cellulose Crystallites. Chem. Eur. J. 2001, 7, 1831–1835. [CrossRe 17. Zhao, Y.; Moser, C.; Lindström, M.E.; Henriksson, G.; Li, J. Cellulose Nanoﬁbers (CNF) from Softwood, Hardwood and Tunicate: Preparation-Structure-Film Performance Interrelation. Appl. Mater. Interfaces 2017, 9, 13508–13519. [CrossRef] [PubMed] 18. Beck-Candanedo, S.; Roman, M.; Gray, D.G. Effect of Reaction Conditions on the Properties and Behavior of Wood Cellulose Nanocrystal Suspensions. Biomacromolecules 2005, 6, 1048–1054. 19. Dumanli, A.G.; van der Kooij, H.M.; Kamita, G.; Reisner, E.; Baumberg, J.J.; Steiner, U.; Vignolini, S. Color in Cellulose Nanocrystal Films. Appl. Mater. Interfaces 2014, 6, 12302–12306. [CrossRef] 20. Fernandes, S.N.; Almeida, P.L.; Monge, N.; Aguirre, L.E.; Reis, D.; de Oliveira, C.L.; Neto, A.M.; Pieranski, P.; Godinho, M.H. Mind the Microgap in Iridescent Cellulose Nanocrystal Films. Adv. Mater. 2016, doi:10.1002/adma.201603560. [CrossRef] 21. Majoinen, J.; Kontturi, E.; Gray, O.I.D.G. SEM Imaging of Chiral Nematic Films Cast from Cellulose Nanocrystal Suspensions. Cellulose 2012, 19, 1599–1605. [CrossRef] 22. Kamita, G.; Frka-Petesic, B.; Allard, A.; Dargaud, M.; King, K.; Dumanli, A.G.; Vignolini, S. Biocompatible and Sustainable Optical Strain Sensors for Large-Area Applications. Adv. Opt. Mater. 2016, 4, 1950–1954. [CrossRef] 23. Honorato-Rios, C.; Kuhnhold, A.; Bruckner, J.R.; Dannert, R. Equilibrium Liquid Crystal Phase Diagrams and Detection of Kinetic Arrest in Cellulose Nanocrystal Suspensions. Front. Matter. References 1. Raabe, D.; Sachs, C.; Romano, P. The crustacean Exoskeleton as an Example of a Structurally and Mechanically Graded Biological Nanocomposite Material. Acta Mater. 2005, 53, 4281–4292. [CrossRef] 1. Raabe, D.; Sachs, C.; Romano, P. The crustacean Exoskeleton as an Example of a Structurally and Mechanically Graded Biological Nanocomposite Material. Acta Mater. 2005, 53, 4281–4292. [CrossRef] 2. Korolovych, V.F.; Cherpak, V.; Nepal, D.; Ng, A.; Shaikh, N.R.; Grant, A.; Xiong, R.; Bunning, T.J.; Tsukruk, V.V. Cellulose Nanocrystals with Different Morphologies and Chiral Properties. Polymer 2018, 145, 334–347. [CrossRef] 2. Korolovych, V.F.; Cherpak, V.; Nepal, D.; Ng, A.; Shaikh, N.R.; Grant, A.; Xiong, R.; Bunning, T.J.; Tsukruk, V.V. Cellulose Nanocrystals with Different Morphologies and Chiral Properties. Polymer 2018, 145, 334–347. [CrossRef] 3. Nikolsky, S.N.; Zlenko, D.V.; Melnikov, V.P.; Stovbun, S.V. The Fibrils Untwisting Limits the Rate of Cellulose Nitration Process. Cabrb. Polym. 2019, 204, 232–237. [CrossRef] 3. Nikolsky, S.N.; Zlenko, D.V.; Melnikov, V.P.; Stovbun, S.V. The Fibrils Untwisting Limits the Rate of Cellulose Nitration Process. Cabrb. Polym. 2019, 204, 232–237. [CrossRef] 4. Jarvis, M. Cellulose Stacks Up. Nature 2003, 426, 611–612. [CrossRef] 5. Zhao, Y.; Li, J. Excellent Chemical and Material Cellulose from Tunicates: Diversity in Cellulose Production Yield and Chemical and Morphological Structures from Different Tunicate Species. Cellulose 2014, 21, 3427–3441. [CrossRef] 5. Zhao, Y.; Li, J. Excellent Chemical and Material Cellulose from Tunicates: Diversity in Cellulose Production Yield and Chemical and Morphological Structures from Different Tunicate Species. Cellulose 2014, 21, 3427–3441. [CrossRef] 6. Muhlethaler, K. Ultrastructure and Formation of Plant Cell Walls. Annu. Rev. Plant. Phys. 1967, 18, 1–24. [CrossRef] 6. Muhlethaler, K. Ultrastructure and Formation of Plant Cell Walls. Annu. Rev. Plant. Phys. 1967, 18, 1–24. [CrossRef] 7. Fernandes, A.; Thomas, L.; Altaner, C.; Callow, P.; Forsyth, V.; Apperley, D.; Kennedy, C.; Jarvish, M. Nanostructure of Cellulose Microﬁbrils in Spruce Wood. Proc. Natl. Acad. Sci. USA 2011, 108, E1195–E1203. [CrossRef] [PubMed] 7. Fernandes, A.; Thomas, L.; Altaner, C.; Callow, P.; Forsyth, V.; Apperley, D.; Kennedy, C.; Jarvish, M. Nanostructure of Cellulose Microﬁbrils in Spruce Wood. Proc. Natl. Acad. Sci. USA 2011, 108, E1195–E1203. [CrossRef] [PubMed] 10 of 11 Polymers 2019, 11, 873 8. Usov, I.; Nyström, G.; Adamcik, J.; Handschin, S.; Schültz, C.; Fall, A.; Bergström, L.; Mezzenga, R. Understanding nanocellulose chirality and structure–properties relationship at the single ﬁbril level. Nat. Commun. 2015, 6, 7564. [CrossRef] 9. Hult, E.; Larsson, P.; Iversen, T. References 2016, 3, 21. [CrossRef] 24. Klemm, D.; Kramer, F.; Moritz, S.; Lindström, T.; Ankerfors, M.; Gray, D.; Dorris, A. Nanocelluloses: A New Family of Nature-Based Materials. Angew. Chem. Int. Ed. 2011, 50, 5438–5466. [CrossRef] 25. Shen, X.; Shamshina, J.L.; Berton, P.; Gurauc, G.; Rogers, R.D. Hydrogels Based on Cellulose and Chitin: Fabrication, Properties, and Applications. Green Chem. 2016, 18, 53–75. [CrossRef] 26. France, K.J.D.; Hoare, T.; Cranston, E.D. Review of Hydrogels and Aerogels Containing Nanocellulose. Chem. Matter. 2017, 29, 4609–4631. [CrossRef] 27. Ye, C.; Malak, S.T.; Hu, K.; Wu, W.; Tsukruk, V.V. Cellulose Nanocrystal Microcapsules as Tunable Cages for Nano- and Microparticles. ACS Nano 2015, 9, 10887–10895. [CrossRef] 28. Sannino, A.; Demitri, C.; Madaghiele, M. Biodegradable Cellulose-based Hydrogels: Design and Applications. Materials 2009, 2, 353–373. [CrossRef] 29. Lavoine, N.; Desloges, I.; Dufresne, A.; Bras, J. Microﬁbrillated Cellulose – Its Barrier Properties and Applications in Cellulosic Materials: A review. Carbohydr. Polym. 2012, 90, 735–764. [CrossRef] 30. Yue, Y.; Zhou, C.; French, A.D.; Xia, G.; Han, G.; Wang, Q.; Wu, Q. Comparative Properties of Cellulose Nano-Crystals from Native and Mercerized Cotton Fibers. Cellulose 2012, 19, 1173–1187. [CrossRef] 11 of 11 Polymers 2019, 11, 873 31. Lagerwall, J.P.; Schültz, C.; Salajkova, M.; Noh, J.H.; Park, J.H.; Scalia, G.; Bergström, L. Cellulose Nanocrystal-Based Materials: From Liquid Crystal Self-Assembly and Glass Formation to Multifunctional Thin Films. NPG Asia Mater. 2014, 6, e80. [CrossRef] 32. Qiu, Y.; Park, K. Environment-Sensitive Hydrogels for Drug Delivery. Adv. Drug. Deliv. Rev. 2001, 52, 3 [CrossRef] 33. Miao, C.; Hamad, W.Y. Cellulose Reinforced Polymer Composites and Nanocomposites: A Critical Review. Cellulose 2013, 20, 2221–2262. [CrossRef] 34. Sarko, A.; Marchessault, R. Supermolecular Structure of Polysaccharides. J. Polym. Sci. Part C 1969, 28, 317–331. [CrossRef] 5. O’Sullivan, A.C. Cellulose: The Structure Slowly U 35. O’Sullivan, A.C. Cellulose: The Structure Slowly Unravels. Cellulose 1997, 4, 173–207. [CrossRef] 36. Mittal, N.; Ansari, F.; Gowda, K.V.; Brouzet, C.; Chen, P.; Larsson, P.T.; Roth, S.V.; Lundell, F.; Wågberg, L.; Kotov, N.A.; et al. Multiscale Control of Nanocellulose Assembly: Transferring Remarkable Nanoscale Fibril Mechanics to Macroscale Fibers. ACS Nano 2018, 12, 6378–6388. [CrossRef] [PubMed] g g Kotov, N.A.; et al. Multiscale Control of Nanocellulose Assembly: Transferring Remarkable Nanoscale Fibril Mechanics to Macroscale Fibers. ACS Nano 2018, 12, 6378–6388. [CrossRef] [PubMed] 37. Gray, D.G. Order and Gelation of Cellulose Nanocrystal Suspensions: An Overview of Some Issues. Philos. Trans. A Math. Phys. Eng. Sci. 2018, 376, 2112. References [CrossRef] [PubMed] 37. Gray, D.G. Order and Gelation of Cellulose Nanocrystal Suspensions: A Trans. A Math. Phys. Eng. Sci. 2018, 376, 2112. [CrossRef] [PubMed] 38. Stovbun, S.V.; Skoblin, A.A.; Zlenko, D.V. Self Assembly and Gelation in Solutions of Chiral N-triﬂuoroacetylated α-aminoalcohols. Chem. Phys. 2018, 508, 34–44. [CrossRef] 39. Kalia, S.; Dufresne, A.; Cherian, B.M.; Kaith, B.; Avéros, L.; Njuguna, J.; Nassiopoulos, E. Cellulose-Based Bio- and Nanocomposites: A Review. Int. J. Polym. Sci. 2011, 2011, 837875. [CrossRef] 40. Israelachvili, J.N. Intermolecular and Surface Forces; Academic Press: Cambridge, MA, USA, 2011. 41. Preston, R. The Organisation of the Cell Wall of the Conifer Tracheid. Philos. Trans. B 1934, 61, 131–174 42. Bardage, S.; Donaldson, L.; Tokoh, C.; Daniel, G. Ultrastructure of the Cell Wall of Unbeaten Norway Spruce Pulp Fibre Surfaces. Nord. Pulp Pap. Res. J. 2004, 19, 448–452. [CrossRef] 43. Liu, D.; Wang, S.; Ma, Z.; Tian, D.; Gu, M.; Lin, F. Structure Nanocrystal Films. RSC Adv. 2014, 4, 39322–39331. [CrossRef] 43. Liu, D.; Wang, S.; Ma, Z.; Tian, D.; Gu, M.; Lin, F. Structure–Color Mechanism of Iridescent Cellulose Nanocrystal Films. RSC Adv. 2014, 4, 39322–39331. [CrossRef] 44. Li, M.; Wu, Q.; Song, K.; Lee, S.; Qing, Y.; Wu, Y. Cellulose Nanopart Relationships. ACS Sustain. Chem. Eng. 2015, 3, 821–832. [CrossRef] 44. Li, M.; Wu, Q.; Song, K.; Lee, S.; Qing, Y.; Wu, Y. Cellulose Nanoparticles: Structure-morphology-rheology Relationships. ACS Sustain. Chem. Eng. 2015, 3, 821–832. [CrossRef] 5. Meudtner, R.M.; Hecht, S. Helicity Inversion in Responsive Foldamers Induced by Achiral Halide Ion Gu Angew. Chem. Int. Ed. 2008, 47, 4926–4930. [CrossRef] 46. Zhao, Z.; Shklyaev, O.; Nili, A.; Mohamed, M.; Kubicki, J.; Crespi, V.; Zhong, L. Cellulose Microﬁbril Twist, Mechanics, and Implication for Cellulose Biosynthesis. J. Phys. Chem. A. 2013, 117, 2580–2589. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4220904206	https://www.frontiersin.org/articles/10.3389/fnana.2022.795231/pdf	English	null	Brain Changes Associated With Long-Term Ketamine Abuse, A Systematic Review	Frontiers in neuroanatomy	2,022	cc-by	11,739	SYSTEMATIC REVIEW published: 18 March 2022 doi: 10.3389/fnana.2022.795231 SYSTEMATIC REVIEW Edited by: Gert Holstege, University of Groningen, Netherlands Reviewed by: Keith Trujillo, California State University San Marcos, United States Valerio Ricci, San Luigi Gonzaga University Hospital, Italy Correspondence: Jurriaan F. M. Strous j.f.m.strous@umcg.nl Jurriaan F. M. Strous 1†, Cees J. Weeland 2†, Femke A. van der Draai 3, Joost G. Daams 4, Damiaan Denys 4,5, Anja Lok 4, Robert A. Schoevers 1 and Martijn Figee 6 1 Department of Psychiatry, University Medical Center Groningen, Groningen, Netherlands, 2 Amsterdam University Medical Center, Location Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 3 Arkin, Mental Health Service, Amsterdam, Netherlands, 4 Amsterdam University Medical Center, Location Academic Medical Center, Amsterdam, Netherlands, 5 Netherlands Institute for Neuroscience, Royal Academy of Arts and Sciences, Amsterdam, Netherlands, 6 Icahn School of Medicine at Mount Sinai, New York, NY, United States Recently, the abuse of ketamine has soared. Therefore, it is of great importance to study its potential risks. The effects of prolonged ketamine on the brain can be observationally studied in chronic recreational users. We performed a systematic review of studies reporting functional and structural brain changes after repeated ketamine abuse. We searched the following electronic databases: Medline, Embase and PsycINFO We screened 11,438 records and 16 met inclusion criteria, totaling 440 chronic recreational ketamine users (2–9.7 years; mean use 2.4 g/day), 259 drug-free controls and 44 poly-drug controls. Long-term recreational ketamine use was associated with lower gray matter volume and less white matter integrity, lower functional thalamocortical and corticocortical connectivity. The observed differences in both structural and functional neuroanatomy between ketamine users and controls may explain some of its long-term cognitive and psychiatric side effects, such as memory impairment and executive functioning. Given the effect that long-term ketamine exposure may yield, an effort should be made to curb its abuse. Received: 14 October 2021 Accepted: 10 January 2022 Published: 18 March 2022 Received: 14 October 2021 Accepted: 10 January 2022 Published: 18 March 2022 Keywords: ketamine, drug abuse, side effects, gray matter volume, white matter volume, connectivity INTRODUCTION Ketamine is an anesthetic agent acting as an uncompetitive antagonist at the N-Methyl-D-Aspartate (NMDA) receptor. More recently, ketamine has emerged as a promising antidepressant (Berman et al., 2000; Zarate et al., 2012; DeWilde et al., 2015; Albuquerque et al., 2016; Singh et al., 2016; Daly et al., 2018). In a meta-analysis including 201 patients with major depressive disorder (MDD), a single intravenous administration of ketamine was associated with a marked reduction in depression severity compared to placebo within 4 hours (Xu et al., 2016), and recently esketamine has been approved as a treatment for depression in both the United States and Europe (EMA, 2019; FDA, 2019). Brain Changes Associated With Long-Term Ketamine Abuse, A Systematic Review Jurriaan F. M. Strous 1*†, Cees J. Weeland 2†, Femke A. van der Draai 3, Joost G. Daams 4, Damiaan Denys 4,5, Anja Lok 4, Robert A. Schoevers 1 and Martijn Figee 6 Citation: Strous JFM, Weeland CJ, van der Draai FA, Daams JG, Denys D, Lok A, Schoevers RA and Figee M (2022) Brain Changes Associated With Long-Term Ketamine Abuse, A Systematic Review. Despite the promising short-term eﬀects, ketamine recently has been emerging as a drug of abuse. The prevalence of ketamine abuse was 1.7% in the United Kingdom in 2008/2009 (4% lifetime) (Murphy and Roe, 2010; Zou and Tan, 2016), and around 1% in American college students (Maxwell, 2005). Chronic ketamine abuse in these countries was associated with long-term cognitive impairment, mood disorders, psychotic and dissociative symptoms, suggesting Front. Neuroanat. 16:795231. doi: 10.3389/fnana.2022.795231 March 2022 \| Volume 16 \| Article 795231 Frontiers in Neuroanatomy \| www.frontiersin.org 1 Brain Changes After Long-Term Ketamine Strous et al. exist between ketamine users and control subjects, including use of other drugs. that prolonged ketamine use may indeed negatively aﬀect brain structure and functioning. An alternative explanation is that primary depressive, psychotic or dissociative symptoms are reasons for ketamine self-medication rather than long-term side- eﬀects. It should be noted that recreational dosages are much higher than clinical dosages, both per dose and cumulatively. A scoping search identiﬁed key articles and search concepts. All key articles had to be retrieved by the systematic search strategy. The search concept combination can be displayed as ketamine AND (chronic OR long term OR abuse OR dependence OR known long term use eﬀects OR induced adverse eﬀects). To date, the safety of prolonged ketamine administration has sparsely been investigated in humans in a prospective manner. The studies that have been done, have been conducted in clinical setting, with a much lower dose than the doses that are used recreationally. However, given the scarcity of research on the topic, these ﬁndings are worth mentioning. The most frequently reported side eﬀects of short term ketamine (hours/days) are related to the nervous system, such as dissociation, sedation, headache, dizziness, blurred vision and memory impairment (Short et al., 2017). Small case series of ketamine administration in various doses for up to one year in patients with MDD or chronic pain suggest that some of these neural side eﬀects may remain with prolonged ketamine use (Cvrcek, 2008; Szymkowicz et al., 2013). Data Extraction Three of us (JS, FD, CW) independently extracted data from the retrieved articles: the N, characteristics of the ketamine consuming subjects, characteristics of the controls, signiﬁcant diﬀerences between the ketamine consuming subjects compared to controls, the statistic measure and correlations (Tables 1–4). The quality of the studies was assessed using the Sackett Scale and the Oxford CEBM levels of evidence scale (Sackett, 1989; Howick et al., 2018). Analysis Search Strategy and Selection Criteria Preferred Reporting Items for Systematic Reviews and Meta- analyses (PRISMA) guidelines were followed during the writing process (Moher et al., 2009). We formulated the following PICO: what neuroanatomical diﬀerences exist between long- term ketamine users compared to non-ketamine using controls. First, we performed a broad search to include all relevant studies investigating the eﬀects of long-term ketamine use on all organ systems, for possible future reports. For this review, we focused on gray matter volume changes, loss of white matter integrity, diﬀerences in functional connectivity and activation patterns and receptor-binding after long-term recreational ketamine use in adults. The intervention of interest was repeatedly-dosed ketamine with a minimum duration of more than 14 days. The use of ketamine in recreational users was compared to non-drug- using controls or poly-drug users. Although we did not exclude studies in which subjects also used other drugs, we considered the limitation that diﬀerences other than ketamine use alone could The results were subdivided into structural diﬀerences in gray and white matter, functional diﬀerences and eﬀects on neurotransmission. Given the limited number of included studies and diversity of outcome measures in the studies, the data was deemed not suitable for meta-analysis. Therefore, we performed a conceptual synthesis of these heterogenous results. Because of these heterogenous results, we could not perform a quantitative bias analysis. Bias could play a role, since the 5 studies by Liao et al. (2010, 2011, 2012, 2016, 2018) were based on the same sample. For that reason, we compared the results if four of these ﬁve studies were left out of the analysis, to the situation in which all ﬁve studies were included. Citation: Contrastingly, another study suggests that prolonged add-on treatment with intranasal esketamine, which has recently been approved for treatment of treatment resistant depression (TRD) twice or thrice weekly did not worsen cognitive performance after 44 weeks of maintenance treatment compared to baseline in patients with treatment resistant depression (TRD) (EMA, 2019; FDA, 2019; Wajs et al., 2020). g Medline, Embase and PsycINFO were systematically searched (JD JS, FD) from inception until February 2021 using the Ovid interface. The search strategy was designed by JD. A detailed search strategy can be obtained from the corresponding author. After removal of duplicates, 11,438 records were retrieved. The titles and abstracts of these records were independently screened by authors JS, CW and FD based on the selection criteria. Afterwards, conﬂicting results between the screeners were resolved by consensus. Original studies about recreative ketamine use in which neuroanatomical measurements were performed, either structural or functional, were included. To obtain the articles meeting this inclusion criterion we ﬁrst excluded all articles that were not about ketamine. Then we excluded all articles that were not about the brain. Subsequently we excluded articles that were only about brain function and not about neuro-anatomical outcomes (e.g., performance on cognitive tests). Lastly, we excluded papers that were about animals or were no original investigations. Then, we sorted the articles levels of evidence. Of all remaining articles, full-texts and one congress abstract were read. Finally, we found our complete dataset consisting of 16 studies (see Figure 1) for the inclusion ﬂowchart. Despite the absence of prospective studies, some ﬁrst insights might be gained from retrospective cohort studies in recreational ketamine users (UNODC, 2016). We sought to systematically review all available studies measuring long-term structural and functional neuroanatomical diﬀerences between long-term recreational ketamine users and controls. The results of this review could provide some ﬁrst insights into potential eﬀects of prolonged recreational ketamine use which may inform clinicians better about the risks associated with ketamine abuse. Frontiers in Neuroanatomy \| www.frontiersin.org RESULTS We included 16 studies in our review, totaling 440 chronic ketamine users with a mean ketamine use of 2–9.7 years and March 2022 \| Volume 16 \| Article 795231 Frontiers in Neuroanatomy \| www.frontiersin.org 2 Brain Changes After Long-Term Ketamine Strous et al. the study. Subjects consumed the ketamine by snorting ketamine powder. The duration of ketamine use was negatively correlated with the gray matter volume in the left superior frontal gyrus (SFG) and right middle frontal gyrus (MFG) (Liao et al., 2011). Also, estimated total lifetime consumption of ketamine was negatively correlated with gray matter volume in the left SFG, but not in the right MFG (Liao et al., 2011). In a second structural MRI study, smaller cortical thickness in several regions in the right frontal area was observed in 14 chronic ketamine users compared to 13 poly-drug controls (Chesters et al., 2016). The route of ketamine administration was not mentioned. To assess the possible progression of brain changes following ketamine use over time, a third structural MRI study analyzed scans of 21 chronic ketamine users with varying durations of drug addiction, ranging from 0.5 to 12 years (Wang et al., 2013). Changes were observed in both white and gray matter across the internal capsule, basal forebrain, cerebellum and diencephalon. Longer use of ketamine was associated with more extensive cortical atrophy in the parahippocampal gyrus and frontal, parietal and occipital cortex. Interestingly, subjects that had been addicted to ketamine for 3 years or less showed less atrophy than subjects that were addicted more than 3 years. For example, the cortex was not aﬀected in subjects with a ketamine addiction of < 3 years, and the limbic system was not aﬀected in subjects with an addiction of < 4 years. However, cortical atrophy occurred earlier than 3 years in a patient who had been using a high dose of 3 grams ketamine per day (Wang et al., 2013). FIGURE 1 \| Inclusion ﬂowchart. A structural MRI study with 124 ketamine chronic ketamine users (dose and duration of abuse not reported) found lower gray matter volume in the right orbitofrontal cortex (rOFC), the right medial prefrontal cortex (rmPFC), the left globus pallidus (lGP), the left hippocampus (lH) and the right nucleus accumbens (rNAC) in the ketamine group compared to 57 controls. RESULTS Gray matter volumes in rOFC, rMPFC and rNAC were negatively correlated with ketamine dependence severity and gray matter volumes of the rOFC, rmPF, lCN, lGP, lH, and rNAC negatively correlated with cognitive performance (Liu et al., 2016; Tang et al., 2016). Diﬀerent from other studies, this study also found higher gray matter volume in ketamine users compared to controls, i.e., in the left caudate nucleus. FIGURE 1 \| Inclusion ﬂowchart. 2.4 grams per day, compared to 259 drug-free controls and 44 poly-drug controls. Five studies were based on the same sample (Liao et al., 2010, 2011, 2012, 2016, 2018). The included studies described structural gray matter and white matter diﬀerences, diﬀerences in brain functionality and diﬀerences in neurotransmitter receptor binding. All retrieved studies were retrospective cohort studies, level IV on the Sackett scale or level 2b on the Oxford CEBM levels of evidence scale (Sackett, 1989; Howick et al., 2018). A study with 34 chronic ketamine users and 19 healthy controls found lower gray matter volume in the right insula, the left dorsolateral prefrontal cortex (DLPFC), the rOFC and the left inferior parietal cortex in ketamine users compared to controls (Hung et al., 2020a). Within the ketamine users group, adolescent onset users were compared to adult-onset users. Adolescent- onset users showed a signiﬁcantly smaller left precuneus volume than the adult-onset group and the healthy control group. Structural Differences: Gray Matter Structural Differences: White Matter The results are shown in Table 2. Using diﬀusion-weighted MRI scans, fractional anisotropy (FA) can be used for estimating white matter ﬁber density, myelination and axonal diameter. FA reductions were found in bilateral frontal and left temporoparietal white matter in 41 ketamine users with a mean use of 2 grams/day for 3.4 years, in comparison with 44 drug-free controls (Liao et al., 2010). FA in the left and right The results are shown in Table 1. A structural MRI study in 41 chronic ketamine users and 44 drug-free controls, found smaller gray matter volume in the left superior frontal cortex and the right middle frontal cortex in the ketamine group compared to controls (Liao et al., 2011). Subjects in this study used on average 2 grams of ketamine per day for a mean duration of 3.4 years from the start of ketamine use until the subject was included in March 2022 \| Volume 16 \| Article 795231 Frontiers in Neuroanatomy \| www.frontiersin.org 3 ubjects (ketamine use-other se-comorbid disorders) Controls Signiﬁcant differences ketamine subjects compared to controls Statistic measure Correlation sorder, 15.3% anxiety disorder. mine users used other drugs like ocaine. ne use: not mentioned ministration: d. drugs: not mentioned. nd/or somatic illnesses: not not mentioned ers performed worse than telligence and cognitive tasks. psychiatric or somatic disorder is d. Healthy controls Smaller gray matter volume in: -right orbitofrontal cortex (rOFC) -right medial prefrontal cortex (rMPFC) -left globus pallidus (lGP) -left hippocampus (lH) -right nucleus accumbens (rNAC) Higher gray matter volume in: -left caudate nucleus (lC) Not reported -Gray matter volumes in rOFC, rMPFC, rNAC were negatively correlated with ketamine dependence severity. -rOFC, rMPFC, lC, lGP, lH, rNAC volumes correlated with performance in cognitive tests. .9 years 33/8 ne use: 2 g/day s consumed by snorting used in users: co amine+ phetamine na azepines neurologic disorder or nesses Drug-free controls Mean age 26.3 years Male/female 34/10 0% Tobacco 20% Alcohol No history of neurologic disorder or psychiatric illnesses Smaller gray matter volume in 1) left superior frontal cortex 2) right middle frontal cortex Gray matter volume 1) Left superior frontal cortex: Voxel Z 4.48 2) Right middle frontal cortex voxel Z 4.81 P-cluster level <0.05 Negative correlation with lower gray matter in left superior frontal cortex (p = 0.011) and right middle frontal cortex (p = 0.009) and duration of ketamine use (months). Structural Differences: Gray Matter Negative correlation with lower left superior frontal cortex gray matter volume and estimated lifetime consumption (p = 0.022) (C ge not menti ale/female n sed drugs: DMA: 46% ocaine: 38% mphetamine eroin: 0% ther psychia sorders not + mine es ogic diso s Signiﬁcant differences ketamine subjects compared to controls Statistic measure Correlation rols After ketamine addiction duration 1 yr: atrophy spots in superﬁcial white matter of cortex 3 yrs: extension to internal capsule 4 yrs: atrophy spots in basal forebrain, cerebellum, pons and diencephalon; atrophy in small region in frontal, parietal and occipital cortex 5 yrs: atrophy of parahippocampal gyrus 6 yrs: atrophy lesions in corpus striatum. 7 yrs: extension of atrophy in frontal, parietal and occipital cortex. Lesions in midbrain. 10–12 yrs: all lesions as above. Lesions were not quantifeid In 1 subject, increased quantity (grams per year) ketamine use correlated with acceleration of cortical atrophy. In 1 subject, high amounts of polydrug (ecstasy, amphetamine, ketamine) correlated with acceleration of atrophy in basal prefrontal gyrus rectus 1) Lower gray matter volume (GMV) in the left precuneus of ketamine users. The volume was 1) GMV 0.412 cm3 (adolescent), 0.48 cm3 (late onset) 0.51 cm3 (HC) 26 years male 11/8 ants had no major or neurological illness. lower in the adolescent onset group than in the adult onset group. Lower GMV in the 2) right insula, 3) left inferior parietal lobule, 4) left DLPFC 5) lmOFC p < 0.001 2) GMV 0.87 cm3 SD vs 1.11 cm3 3) GMV 0.70 cm3 SD = 0.02 vs. 0.82 cm3 SD = 0.03 (p < 0.001) 4) 0.90 cm3 vs. 1.04 cm3 p < 0.001) 5) 0.63 cm3 vs. 0.73 cm3 trols Signiﬁcant differences ketamine subjects compared to controls Statistic measure Correlation g-free controls e/female 34/10 n age 26.3 years Alcohol Lower FA in 1) left frontal cortex 2) right frontal cortex 3) left temporoparietal cortex white matter. 1) z= 4.21 (p = 0.014) 2) z=3.96 (p = 0.014) 3) z=3.85 (p = 0.014) Negative correlation between FA in left (p = 0.015) and right (p = 0.003) frontal white matter and total lifetime consumption(g) drug controls n age 28.5 years e/female 11/5 ular use: Alcohol MDMA Cannabis Benzodiazepines Cocaine ence of psychiatric or atic disorder is mentioned. Less axial diffusivity in Eight right hemisphere clusters 1) anterior corona radiata and inferior fronto-occipital fasciculus (IFOF) 2) forceps minor 3) anterior forceps minor Adjacent to parietal cortex: 4) close to the intraparietal sulcus 5) close to posterior thalamic radiation and IFOF Near SLF 6) and somatosensory cortex 7) and corticospinal tract Other areas 8) sagittal stratum, inferior longitudinal fasciculus (IFL) and IFOF 1) T=3.972 671 voxels 2) T=3.966 177 voxels 3) T=4.345 34 voxels 4) T=4.71 173 voxels 5) T=3.482 165 voxels 6) T=3.256 22 voxels 7) T=3.637 92 voxels 8) T=2.49 38 voxels (Continued) Negative correlation between FA in left (p = 0.015) and right (p = 0.003) frontal white matter and total lifetime consumption(g) (Continued) her oned order is Polydrug controls Mean age 28.5 years Male/female 11/5 Regular use: 75% Alcohol 0% MDMA 50% Cannabis 6% Benzodiazepines 44% Cocaine Presence of psychiatric or somatic disorder is not mentioned. Less axial diffusivity in Eight right hemisphere clusters 1) anterior corona radiata and inferior fronto-occipital fasciculus (IFOF) 2) forceps minor 3) anterior forceps minor Adjacent to parietal cortex: 4) close to the intraparietal sulcus 5) close to posterior thalamic radiation and IFOF Near SLF 6) and somatosensory cortex 7) and corticospinal tract Other areas 8) sagittal stratum, inferior longitudinal fasciculus (IFL) and IFOF 1) T=3.972 2) T=3.966 3) T=4.345 4) T=4.71 5) T=3.482 6) T=3.256 7) T=3.637 8) T=2.49 Signiﬁcant differences ketamine subjects compared to controls Statistic measure Correlation 1) Both primarily ketamine users and ketamine + polysubstance users had larger caudate nuclei than the non-drug controls. 2) Both ketamine groups had larger white matter volumes throughout the whole brain. 3) The K+polysubstance abuse showed even higher white matter volumes. White matter volume was measured as a percentage of total intracranial volume. 1) (p = 0.030) 2) (p = 0.046) 3) (p = 0.011 compared to controls) Earlier age of ketamine (both the primarily K and K+polysubstance users) use predicted larger white matter volumes. iculus. e matter volume was ured as a percentage of ntracranial volume. = 0.030) = 0.046) = 0.011 compared ntrols) Earlier age of ketamine (both the primarily K and K+polysubstance users) use predicted larger white matter volumes. gniﬁ tam con Both d ke ers h n th Both ger w oug The owe ume us. s (ketamine use-other omorbid disorders) Controls Signiﬁcant differences ketamine subjects compared to controls Statistic measure Correlation r, 15.3% anxiety disorder. sers used other drugs like e. : not mentioned entioned formed worse than nce and cognitive tasks atric or somatic disorder Healthy controls -Less functional connectivity in orbital part of right inferior gyrus, left anterior cingulate and paracingulate gyri, right superior temporal gyrus and bilateral vermic lobule VI of cerebellum. -More functional connectivity left middle occipital gyrus. No test statistic reported : rs umed by snorting mine +caffeine ogic disorder or other Excluded if other ence (excl. nicotine) Drug-free controls Mean age 27.1 years Male female 70/18 50% Tobacco 55% Alcohol No personal history of neurologic or psychiatric illnesses Less thalamocortical connectivity between ketamine users and healthy controls. connectivity of thalamic nuclei with: prefrontal cortex 1) left medial dorsal nucleus motor/supplementary motor area 2) left ventral posterior lateral nucleus 3) right ventral lateral nucleus posterior parietal cortex 4) right pulvinar nucleus 5) left pulvinar n. 6) left medial dorsal n. 7) right ventral lateral n. 8) right lateral dorsal n. 1) T=3.72, voxel 270 mm3 2) T=3.41 v=459 3) T=3.34 v=1354 4) T=3.72 v=243 5) T=2.76 v=108 6) T=4.32 v=108 7) T=3.20 v=162 8) T=3.53 v=405 Less thalamocortical connectivity in posterior parietal cortex and individual craving scores. (p < 0.05) A correlation between total ketamine intake and craving scores. (p = 0.003) e use-other sorders) Controls Signiﬁcant differences ketamine subjects compared to controls Statistic measure Correlation xiety disorder. her drugs like oned se than gnitive tasks atic disorder Healthy controls -Less functional connectivity in orbital part of right inferior gyrus, left anterior cingulate and paracingulate gyri, right superior temporal gyrus and bilateral vermic lobule VI of cerebellum. -More functional connectivity left middle occipital gyrus. No test statistic reported norting er or other f other icotine) Drug-free controls Mean age 27.1 years Male female 70/18 50% Tobacco 55% Alcohol No personal history of neurologic or psychiatric illnesses Less thalamocortical connectivity between ketamine users and healthy controls. connectivity of thalamic nuclei with: prefrontal cortex 1) left medial dorsal nucleus motor/supplementary motor area 2) left ventral posterior lateral nucleus 3) right ventral lateral nucleus posterior parietal cortex 4) right pulvinar nucleus 5) left pulvinar n. 6) left medial dorsal n. 7) right ventral lateral n. 8) right lateral dorsal n. 1) T=3.72, voxel 270 mm3 2) T=3.41 v=459 3) T=3.34 v=1354 4) T=3.72 v=243 5) T=2.76 v=108 6) T=4.32 v=108 7) T=3.20 v=162 8) T=3.53 v=405 Less thalamocortical connectivity in posterior parietal cortex and individual craving scores. (p < 0.05) A correlation between total ketamine intake and craving scores. (p = 0.003) norting er or other f other icotine) Drug-free controls Mean age 26.3 No history of neurologic or psychiatric illnesses 1) Lower ReHo in right anterior cingulate cortex. 2) More ReHo in left precentral frontal gyrus. 1) peak T=4.32 p < 0.001 voxel Z = 4.09 2) peak T=4.85 p < 0.001 Z = 4.54 Higher ReHo in left precentral gyrus negatively correlated with estimated total lifetime consumption (p = 0.035) and ketamine craving (p = 0.007). (Continued) ntinued) or Mean tobacco use: 2.5 years (M) 0 years (F) Mean alcohol use: 5.2 years (M) 1.9 years (F) Subjects had no major medical or neurological illness orbitofrontal cortex, bilateral temporal poles/superior temporal gyri, bilateral caudate head/ventral striatum/anterior thalamus and negative connectivity to bilateral occipital cortex, bilateral inferior frontal cortex/anterior insula (rIFC/AI), bilateral middle frontal gyrus and left angular gyrus positively correlated with CES-D both in KU (p = 0.00002, r = 0.74) and in HC (p = 0.009, r = 0.74), men only - sgACC-dmPFC connectivity positively correlated with the CES-D in KU (p = 0.00003, r = 0.97), not in HC (p = 0.69, r = −0.16), female only illnesses Polydrug controls Mean age 26.13 years Male/female 10/5 87% Tobacco 100% Alcohol 6.7% Cocaine 0% Ecstasy 27% Cannabis 1/15 personal history of mental illnesses somatic illness is not mentioned 1) Less activity of right hippocampus and 2) Left parahippocampal gyrus in spatial memory task. 3) Less left caudate activation during memory updating. 1) t=2.77 p = 0.03 controls>ketamine users 2) t=2.81 p = 0.03 3) t=2.6, p = 0.048 mentioned atic disease Drug-free controls Mean age not mentioned Male/female not mentioned Presence of psychiatric or somatic disease not mentioned Lower number of activated areas in cerebellum during simple motor activities. Consecutively, subject drank 200 ml of red wine and repeated the motor task. In controls, 55.7% of cerebellar volume was activated vs. 27.7% in ketamine users, and 21.1% in ketamine users + red wine condition. ntinued) (Continued) use-other rders) Controls Signiﬁcant differences ketamine subjects compared to controls Statistic measure Correlation ed) Drug-free controls Mean age 25.3 (M) 25.1 (F) Male/female 11/9 Mean tobacco use: 2.5 years (M) 0 years (F) Mean alcohol use: 5.2 years (M) 1.9 years (F) Subjects had no major medical or neurological illness 1) No difference in sgACC connectivity 2) Positive connectivity between sgACC and rostral ACC/medial prefrontal cortex/medial orbitofrontal cortex, bilateral temporal poles/superior temporal gyri, bilateral caudate head/ventral striatum/anterior thalamus and negative connectivity to bilateral occipital cortex, bilateral inferior frontal cortex/anterior insula (rIFC/AI), bilateral middle frontal gyrus and left angular gyrus 1) A two sample t-test was performed 2) A one sample t-test was performed - sgACC-OFC connectivity negatively correlated with CES-D (p = 0.00001, r =-0.66) in KU (M & F) - sgACC-STG connectivity positively correlated with CES-D both in KU (p = 0.00002, r = 0.74) and in HC (p = 0.009, r = 0.74), men only - sgACC-dmPFC connectivity positively correlated with the CES-D in KU (p = 0.00003, r = 0.97), not in HC (p = 0.69, r = −0.16), female only nesses Polydrug controls Mean age 26.13 years Male/female 10/5 87% Tobacco 100% Alcohol 6.7% Cocaine 0% Ecstasy 27% Cannabis 1/15 personal history of mental illnesses somatic illness is not mentioned 1) Less activity of right hippocampus and 2) Left parahippocampal gyrus in spatial memory task. 3) Less left caudate activation during memory updating. 1) t=2.77 p = 0.03 controls>ketamine users 2) t=2.81 p = 0.03 3) t=2.6, p = 0.048 mentioned c disease Drug-free controls Mean age not mentioned Male/female not mentioned Presence of psychiatric or somatic disease not mentioned Lower number of activated areas in cerebellum during simple motor activities. Consecutively, subject drank 200 ml of red wine and repeated the motor task. In controls, 55.7% of cerebellar volume was activated vs. 27.7% in ketamine users, and 21.1% in ketamine users + red wine condition. ntinued) (Continued) 8) right frontal cortex (precentral gyrus) Sexual cues 9) left cerebellum 10) middle temporal cortex Ketamine cues minus smoking cue 11) left inferior parietal cortex 12) posterior cingulate/precuneus 13) left middle temporal cortex lower activation in left cerebellum and middle temporal cortex in response to sexual cues 8) peak T=4.20 voxel z = 4.06 Voxel size=33 mm3 9) peak T=4.65, voxel z = 4.46 Voxel size=123 mm3 10) peak T=4.33, voxel z = 4.17 Voxel size=80 mm3 11) peak T=4.22, voxel z = 4.14 Voxel size=130 mm3 12) peak T=3.74, voxel z = 3.68 Voxel size=81 mm3 13) peak T=3.58, voxel z = 3.53 Voxel size = 361 mm3 d between ge 20) onset users et after age 20). ntioned , ecstasy and medical or tamine users was e control group. Participants had no major medical or neurological illness. Age: 25.26 years Male/female 11/8 Both 1) adolescent onset ketamine users and 2) adult onset ketamine users had higher functional connectivity between the left and right precuneus than 3) controls 1) Z = 0.21 SD = 0.03 2) Z = 0.25 SD = 0.03 3) Z = −0.02 SD = 0.03 P < 0.001 (Continued) tamine u e contro Brain Changes After Long-Term Ketamine Strous et al. frontal white matter was negatively correlated with the total lifetime consumption of ketamine. Diﬀusivity can be divided in axial and radial diﬀusivity. Axial diﬀusivity is thought to be a measure of axonal density and radial diﬀusivity is thought to be related to the degree of myelination (Liao et al., 2010). In 16 ketamine users averaging 2.4 grams/day for 7.3 years, a lower level of axial diﬀusivity was found compared to 16 polydrug controls, especially in the frontal part of the right hemisphere (Edward Roberts et al., 2014). Axial diﬀusivity was signiﬁcantly lower in eight white matter clusters in the right hemisphere in the ketamine group compared to the control group, the three largest being located in the frontal cortex (Edward Roberts et al., 2014). Also, probabilistic tractography was performed to investigate cortico-subcortical white matter connectivity proﬁles, which revealed that white matter connectivity between the caudate nucleus and the lateral prefrontal cortex was positively correlated with severity of long-term dissociative symptoms (Edward Roberts et al., 2014). References N (ketamine vs controls) Ketamine subjects (ketamine use-other substance use-comorbid disorders) Controls Signiﬁcant differences ketamine subjects compared to controls Statistic measure Correlation Hung et al. ntinued) (2020b) 56 (36 ketamine users, 20 healthy controls Age 25.2 years (M) 27.5 years (F) Male/female 26/10 Mean ketamine use: dose not recorded 59.4 months (M) 59.0 months (F) None of the ketamine users had a major medical, neurological illness. Age 25.3 years (M) 25.1 years (F) Male/female 11/9 Ketamine users showed higher connectivity between 1) caudate and the dorsal anterior cingulate cortex 2) pallidum and bilateral cerebellum 1) voxel z=4.24 2) voxel z=5.28 The connectivity between putamen and lOFC correlated with months of ketamine use and BIS impulsivity scores (mediation analyes p = 0.00007–0.007) rsfMRI, resting-state functional magnetic resonance imaging; ReHo, regional homogeneity; KU, ketamine user; M, male; F, female; CES-D, Center of Epidemiological Study-Depression score; sgACC, subgenual anterior cingulate cortex; OFC, orbitofrontal cortex; STG, superior temporal gyrus; dmPFC, dorsomedial prefrontal cortex; fMRI, functional magnetic resonance imaging. In a study by Liang et al. (2020), ketamine users had larger caudate volume and total white matter volume than non-drugs controls. Ketamine users that also used stimulants had even larger white matter structures, suggesting an additive eﬀect of ketamine and these stimulants. Participants in this study were asked to refrain from drug use 4–7 days before imaging was performed (Liang et al., 2020). The authors of this study hypothesize that the higher gray and white matter volumes reported in this study in ketamine users may not necessarily reﬂect neurotoxicity but rather represent positive compensatory changes as they were associated with less long-term cognitive impairments and depression. Opposingly, one could hypothesize that increased white matter volume may imply that an underlying low grade inﬂammatory edematous process exists, comparable to edema observed after brain injury (Weimer et al., 2017). Frontiers in Neuroanatomy \| www.frontiersin.org Functional Differences The results are shown in Table 3. Using resting-state functional MRI (fMRI), Liao al. investigated the functional connectivity between the thalamus and speciﬁc cortical regions in 40 chronic ketamine users with a mean use of 2 grams/day for 3.4 years, compared to 88 drug-free controls (Liao et al., 2016). Lower functional connectivity was found between the thalamus and the motor-, posterior parietal- and prefrontal cortex. Functional connectivity between the posterior parietal cortex and right lateral dorsal nucleus was signiﬁcantly correlated to individual ketamine craving scores (Liao et al., 2016). Resting-state fMRI can also be used to measure regional homogeneity (ReHo). ReHo describes the summarized local functional connectivity between a voxel and its neighboring voxels. This is an index of network centrality, showing the importance of a voxel in a functional network. In 41 chronic ketamine users with a mean use of 2 grams/day for 3.4 years compared to 44 drug-free controls, lower ReHo in the right anterior cingulate cortex and higher ReHo in the left precentral frontal gyrus were found (Liao et al., 2012). The higher ReHo in the left precentral frontal gyrus was negatively correlated with estimated total lifetime ketamine consumption and ketamine craving (Liao et al., 2012). This may suggest that ReHo is initially increased more by ketamine use but that this March 2022 \| Volume 16 \| Article 795231 11 Brain Changes After Long-Term Ketamine Strous et al. they found higher functional connectivity in the left middle occipital gyrus. increase eventually decreases with more prolonged and intensive use, which may alter functional organization in frontal networks. However, since subjects had to be abstinent from ketamine for only 48 hours, and since the direct eﬀect of ketamine can last for more than 48 hours, the altered frontal network organization might also be a direct result of ketamine instead of a long-term side eﬀect (Zarate et al., 2012). In the same sample, the authors compared smoking chronic ketamine users with non-ketamine smokers and with non-ketamine, non-smokers by performing fMRI. They found a higher activation in the anterior cingulate cortex (ACC) in response to ketamine cues. Also, ketamine subjects showed lower activation in the cerebellum and the middle temporal cortex in response to natural rewarding (sexual) cues (Liao et al., 2018). In a study by Hung et al. Frontiers in Neuroanatomy \| www.frontiersin.org Dopamine D1 Receptors p 1 p The results are shown in Table 4. One study investigated how long-term ketamine use aﬀected neurotransmitter systems (Narendran et al., 2005). Dopamine D1 binding potential was studied using positron emission tomography (PET) imaging after intravenously injecting the selective D1 receptor radio ligand [11C]NNC 112 in 14 ketamine users with a mean use of 0.75 gram/week for 4.1 years and 14 drug-free controls. D1 receptor availability was signiﬁcantly upregulated in the dorsolateral prefrontal cortex of ketamine users compared to controls, which could result from increased receptor density or aﬃnity. D1 binding potential correlated with the total amount of ketamine consumption (Narendran et al., 2005). Morgan et al. (2014) used fMRI to compare brain activity of 11 ketamine users with a mean use of 1.5 grams/day for 9.7 years, to 15 polydrug controls during a spatial memory task. Ketamine users showed lower activity in the right hippocampus and left parahippocampal gyrus compared to controls. Left caudate activity was greater in polydrug controls. These ﬁndings suggest an impact of long-term ketamine abuse on spatial memory processes associated with impaired (para)hippocampal activation. Lastly, in a small fMRI study using a motor task in which subjects had to ﬂex and extend their upper limbs, three long-term ketamine users with a mean use of 1–2 grams/day for 2 years demonstrated less cerebellar activity compared to 3 drug-free controls (Chan et al., 2012). Being under the inﬂuence of ketamine was no exclusion criterion. Functional Differences (2020a), chronic ketamine users compared to healthy controls showed higher functional connectivity between the left DLPFC and the right inferior frontal/superior temporal gyrus and the left OFC and the right insula/inferior temporal gyrus. Within the ketamine users group, adolescent onset users were compared to adult onset users. Both the adult and the adolescent groups had higher functional connectivity between the left and right precuneus (Hung et al., 2020a). In a pilot that studied white matter connectivity, chronic ketamine users showed higher connectivity between caudate nuclei and the dorsal anterior cingulate cortex (dACC). Ketamine users also showed a higher connectivity between the pallidum and the bilateral cerebellum. Furthermore, in ketamine users, the putamen showed higher connectivity to the OFC, which correlated with duration of ketamine use. Also, the ventral striatum (VS) showed lower connectivity with the right superior temporal sulcus (STS) and the left superior frontal gyrus (SFG) which was mediated by higher scores on the Barratt Impulsiveness Scale (BIS-11) (Hung et al., 2020b). Li et al. (2017) assessed resting-state functional connectivity of the subgenual anterior cingulate cortex (sgACC) in relation to depression scores of 36 chronic ketamine users with an average ketamine use of 4.9 years (dose not reported) compared to 20 drug-free controls. Overall, no diﬀerence in sgACC connectivity was found between groups, but in ketamine users higher depression scores correlated with lower sgACC connectivity to the right lateral and bilateral medial OFC. Further analysis revealed functional connectivity changes, with male and female ketamine users showing higher sgACC connectivity than controls to the bilateral superior temporal gyrus or dorsomedial prefrontal cortex (dmPFC), respectively (Li et al., 2017). Also, they found a correlation between higher sgACC connectivity with the dmPFC and higher depression scores in women, but not in men. Although ketamine has strong short-term antidepressant eﬀects, the current data would suggest that chronic ketamine use may actually induce depression via sex-speciﬁc dysregulation of brain networks for positive and negative emotions. It remains unclear whether the altered connectivity patterns found in this study could be a direct result of ketamine. Being under the inﬂuence of ketamine was not an exclusion criterion for participation in this study. DISCUSSION We systematically reviewed structural and functional brain changes after prolonged recreational ketamine use. Long-term ketamine abusers compared to controls displayed: (1) lower gray matter volume or cortical thickness in primarily the frontal, parietal and occipital lobes (2) lower white matter integrity in frontal and temporoparietal lobes (3) lower functional thalamocortical and corticocortical connectivity and abnormal frontal network organization (4) lower activity of brain regions for spatial memory and motor execution (5) higher functional connectivity between the DLPFC and the OFC and larger total white matter volume (6) higher dopamine D1 binding potential in the dorsolateral prefrontal cortex. Lin et al. used resting state fMRI to compare a group of chronic ketamine users, many of which also used other drugs like cannabis or cocaine, to healthy controls (Liu et al., 2016). They found lower functional connectivity of the default mode network in the orbital right inferior frontal gyrus, left anterior cingulate gyrus, paracingulate gyri, right superior temporal gyrus and bilateral vermic lobule VI of the cerebellum. In contrast, Including all ﬁve studies by Liao et al. could be a confound. Therefore, we also analyzed the results after excluding four of these ﬁve studies. As a result of this, ﬁnding (2) would change to “lower white matter integrity in right frontal and temporoparietal lobes” and/or ﬁnding (3) would not stand, depending on which articles were left out. March 2022 \| Volume 16 \| Article 795231 12 Brain Changes After Long-Term Ketamine Strous et al. TABLE 4 \| D1 receptor. References N (ketamine vs. controls) Ketamine subjects (ketamine use- other substance use-comorbid disorders) Controls Signiﬁcant differences ketamine subjects compared to controls Statistic measure Correlation Narendran et al. (2005) 28 (14–14) PET-scan with radio-active ligand. Mean ketamine use: 0.75 g/week 4.1 years Mean age 25 years Male/female 10/4 Route of administration was not mentioned 71% smokers. Absence of DSM-IV axis I diagnosis other than ketamine or cannabis abuse or dependence. Drug-free controls Mean Age 25 years Male/female 9/5 43% smokers No history of neurologic or psychiatric illnesses incl. substance abuse. Higher binding potential for D1 receptor in dorsolateral prefrontal cortex. [11C]NNC 112 binding potential ml/g Positive correlation between higher D1 binding potential and amount of consumed ketamine (p = 0.005). PET, positron-emitted tomography; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, 4th Edition. PET, positron-emitted tomography; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, 4th Edition. DISCUSSION of glutamatergic receptor units GluA1, GluA2, GluN2A, and GluN2B, as well as a lower expression of synaptic proteins Syn and PSD-95, deteriorated cognitive skills, lower spine density, impairments in long-term potentiation and hampered transmission in the hippocampal CA1 area (Luo et al., 2020). Accordingly, in another study, the amount of parvalbumine positive interneurons in the CA1 region of mice hippocampi was reduced after 1 month of subcutaneous 16 mg/kg ketamine injections (Koh et al., 2016). These mice showed memory impairments, which is in line with a study in which parvalbumine positive interneurons are shown to play a role in hippocampal memory consolidation (Ognjanovski et al., 2017). Brains of mice and cynomolgus monkeys treated with 3 months of daily 30 mg/kg intraperitoneal ketamine (mice) or 1 mg/kg intravenous ketamine (monkeys) showed hyperphosphorylation of the tau protein, which could be interpreted as a neurotoxic eﬀect since phosphorylation of tau protein is perceived as an aging marker (Yeung et al., 2010). Mice treated with 3 or 6 months daily subanesthetic doses (30 or 60 mg/kg) of intraperitoneal ketamine showed hyperphosphorylation of tau- protein hampering traﬃcking of AMPA-receptors, which in turn worsened signal transduction (Li et al., 2019). Another study suggested that impaired working memory after chronic exposure to ketamine in mice was mediated by upregulation of Gaba-5 subunit of the GABAA receptor in the prefrontal cortex (Tan et al., 2011). The same group found that a 3-month ketamine treatment in mice was associated with upregulation of tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine- synthesis. The upregulation of TH might in turn be caused by upregulation of BDNF, which was also found after long-term ketamine use. Many of the observed changes were correlated with the amount and duration of ketamine consumption, suggesting a possible dose dependent eﬀect of prolonged ketamine on brain structure and function. Although the identiﬁed lower gray and white matter volumes or integrity could suggest direct neurotoxic eﬀects of ketamine, the observed higher structural and functional connectivity and dopamine binding may suggest indirect compensatory eﬀects. Together, these ﬁndings suggest that long-term intensive ketamine use may aﬀect the structure and function of cortical gray and white matter, especially in frontoparietal regions. It must be noted that the reported changes were dependent on the dosage and duration of ketamine use which were substantially higher than for clinical use, so our ﬁndings cannot be translated to clinical ketamine use. Frontiers in Neuroanatomy \| www.frontiersin.org DISCUSSION It needs to be considered however, that there may be a U-shaped dose-eﬀect relation between ketamine and cognitive changes. In rats, diﬀerent 5–7-days dosing regimens of ketamine yielded opposite eﬀects on cognitive tasks in which the rats had to detect novel objects, or novel placement of objects. Whereas, low ketamine enhanced novelty detection compared to controls, higher doses impaired novelty detection (Schumacher et al., 2016). Even though this review shows associations between long- term ketamine and structural and functional neuroanatomical diﬀerences, it holds important limitations. The results have been obtained from recreational ketamine users for whom we do not precisely know what dose of ketamine they used, which type of ketamine (racemic or esketamine) and whether they consumed pure ketamine or ketamine contaminated with other substances. This review shows that several functional and structural changes appear to correlate with duration and dose of ketamine consumption and are most striking after more than 3 years of high doses. The results of this review are not translatable to clinical ketamine regimens, since therapeutic dosages may lead to signiﬁcantly less brain changes than recreationally dosed ketamine, or that it may take much longer to develop brain changes with therapeutic regimens. However, studies on long-term brain eﬀects of therapeutic ketamine are lacking. Nonetheless, evidence exists for opposite clinical eﬀects of low dose vs. high dose ketamine. Low dose, twice weekly ketamine schedules have strong antidepressant eﬀects in depressed patients (Singh et al., 2016), whereas high dose daily schedules have been associated with depression in ketamine abusers (Morgan et al., 2009). Furthermore, low dose ketamine schedules in mice The observed upregulation of dorsolateral prefrontal D1 receptors in ketamine users might be a compensatory mechanism for deﬁcient prefrontal dopamine function underlying impaired working memory function (Narendran et al., 2005). Consistent with this, monkeys treated with another non-competitive NMDA antagonist, MK-801, showed lower performance on working memory tasks and lower prefrontal dopamine levels (Kakiuchi et al., 2001). DISCUSSION All subjects in these studies used at least 0.2 grams of ketamine twice weekly, which is around two to four times the recommended clinical dosage (25–50 mg intravenously twice or thrice weekly, equivalent to 50–100 mg intranasal ketamine, the most common route of administration in recreational use), with most subjects even consuming more than 1 gram daily which is equivalent to 25–70 times the clinical dose (Berman et al., 2000; Xu et al., 2016; Sanacora et al., 2017). Nevertheless, this review does suggest that prolonged high dose ketamine use may have the potential to alter brain structure and function. Mechanistic support for such diﬀerences observed in long-term high dose ketamine comes from studies suggesting that exposure to ketamine induces apoptosis in adolescent primates and human brain cell cultures (Mak et al., 2010; Sun et al., 2014). In several of these preclinical studies chronic dosing regimens have been comparable to the regimens of chronic ketamine abusers. For example, 6 months of daily 60 mg intraperitoneal ketamine in mice was associated with reduced expression of GluA1-containg AMPA receptors and memory impairments (Ding et al., 2016) and 28 days of daily 30 mg/kg intraperitoneal ketamine was associated with reduced expression In conclusion, these animal studies may provide important clues for the potential neurotoxic eﬀects of prolonged ketamine use. Prolonged ketamine may either up- or downregulate important regulatory neuronal proteins, March 2022 \| Volume 16 \| Article 795231 13 Brain Changes After Long-Term Ketamine Strous et al. potentially resulting in impaired neuronal functioning and cognitive performance. hippocampal functioning is associated with the transition from a prodromal psychotic state to acute psychosis and repeated ketamine administration was able to induce these hippocampal impairments during progression to psychosis in a mouse model (Schobel et al., 2013). The dissociative symptoms after chronic ketamine use have been associated with diﬀerences in corticostriatal connectivity (Edward Roberts et al., 2014). Finally, despite the short-term antidepressant eﬀects of ketamine, chronic ketamine use is associated with depressive symptoms (Morgan et al., 2009; Li et al., 2017), which correlates with altered functional connectivity between sgACC, OFC and ventromedial prefrontal cortex (Li et al., 2017), which are all areas involved in emotion regulation (Laitinen and Vilkki, 1973; Meyer et al., 1973; Talairach et al., 1973). A possible mechanism for the white matter changes identiﬁed in the reviewed recreational ketamine studies could be AMPA- receptor mediated excitotoxicity. DISCUSSION In rats, ketamine was found to acutely elevate presynaptic glutamate in the prefrontal cortex at AMPA/kainite receptors (Moghaddam et al., 1997), and prolonged ketamine exposure may provoke cell death by regional glutamate-induced excitotoxicity. Excitation of AMPA receptors speciﬁcally induces axonal damage (Fowler et al., 2003), which could provide a potential mechanism for the prominent white matter changes observed after sustained ketamine exposure in three of the reviewed studies. Also, white matter changes in one of these studies preceded more widespread cortical atrophy with longer ketamine use, supporting that axonal cells are most vulnerable for glutamate-induced excitotoxicity by ketamine. However, these observations are still based on comparison between subjects rather than longitudinal data. Caution is required in deeming prolonged ketamine use causal to the observed brain diﬀerences for several reasons. The reviewed brain diﬀerences might have been pre-existing and may have predisposed subjects to ketamine dependence. This is plausible considering that many of the observed brain diﬀerences concerned prefrontal regions that are crucial for inhibiting addictive behaviors. On the other hand, prefrontal gray matter reductions may have been initiated by ketamine use, further impairing inhibition and facilitating ketamine dependence. Similar mechanisms have been demonstrated for prefrontal changes associated with other types of drug abuse, including MDMA and cocaine (Cowan et al., 2003; Lim et al., 2008). Another ﬁnding that could explain transition to ketamine addiction in recreational users is lower thalamocortical connectivity, which may impair control over cognitive and emotional processes involved in drug-seeking behavior (Balleine et al., 2015). Although only a subset of recreational ketamine users are reported to develop dependence (Muetzelfeldt et al., 2008) and tolerance (Bonnet, 2015), the current review suggests several potential mechanisms for addiction that should be further explored to gain an understand of ketamine abuse. Our reviewed ketamine-associated brain changes might also explain some of the cognitive impairments and psychiatric symptoms observed in chronic ketamine users (Morgan et al., 2009, 2014). Impairments of working-, semantic-, spatial- and episodic memory in chronic ketamine users (Morgan and Curran, 2006; Chan et al., 2013) may be related to the observed atrophy and impaired function of brain regions underlying these memory functions, including the hippocampal complex, prefrontal and temporoparietal cortex. Findings of impaired executive functioning in chronic ketamine users (Narendran et al., 2005; Morgan et al., 2009) align with the reviewed frontostriatal impairments. Frontiers in Neuroanatomy \| www.frontiersin.org DISCUSSION Regarding psychotic symptoms, the observation of higher D1 binding potential in the dorsal prefrontal cortex after chronic ketamine use, may mirror similar ﬁndings in schizophrenia, as a compensation mechanism for prefrontal dopamine dysfunction and potentially explaining the high incidence of psychosis in chronic ketamine users (Abi-Dargham et al., 2002; Narendran et al., 2005; Uhlhaas et al., 2007; Morgan et al., 2009, 2010), although it might also be an epiphenomenon of an inﬂammatory pathway induced by ketamine (Wersinger et al., 2004; Lud Cadet et al., 2010). In addition, the observed hippocampal impairments in ketamine users may also have contributed to psychotic symptoms (Morgan et al., 2014). Altered March 2022 \| Volume 16 \| Article 795231 Frontiers in Neuroanatomy \| www.frontiersin.org Frontiers in Neuroanatomy \| www.frontiersin.org 14 Brain Changes After Long-Term Ketamine Strous et al. increased sprouting in the medial prefrontal cortex compared to saline injections (Li et al., 2010), whereas chronic self- administration of high dose ketamine in rats reduced glutamate receptor expression in the medial prefrontal cortex (Caﬃno et al., 2017). This may be in line with brain volume loss and reduced connectivity after high dosing recreational ketamine schedules in humans (Liao et al., 2010, 2011, 2012, 2016; Wang et al., 2013; Edward Roberts et al., 2014; Chesters et al., 2016), but enhanced brain connectivity after lower dose ketamine (Abdallah et al., 2017). Furthermore, long-term uncontrolled dosing may lead to ketamine-tolerance, as illustrated by a case report of a woman who became more depressed after administering herself increasing doses of ketamine (Bonnet, 2015), while short term controlled regimens in rats led to ketamine sensitization, as shown by increased locomotor activity after two ketamine injections compared to one injection (Rocha et al., 2017). Lastly, it should be noted that racemic ketamine and esketamine may have distinct toxicity proﬁles. Recreational users may mainly use racemic ketamine, whereas long-term treatment of depression mainly concerns esketamine which could be less toxic to the brain (Wajs et al., 2020). for ketamine metabolism do not vary signiﬁcantly between people with Asian or Caucasian ancestry (Mizutani, 2003; Peltoniemi et al., 2016). We consider a few methodological limitations as well. The included studies followed a cross-sectional and retrospective design with considerable variability among studies in terms of subject age, ketamine type and dosage. The data was insuﬃcient to perform a meta-analysis. Additionally, ﬁve of the 16 studies were based on the same sample. DISCUSSION It should be noted that in some studies, ketamine users had a mood disorder and for many of the studies it was unclear whether the ketamine users were diagnosed with another substance use disorder or another psychiatric illness. Part of the structural and functional neuroanatomical diﬀerences could therefore be attributed to these concomitant conditions. Finally, we were unable to receive a few records containing potentially relevant data. In conclusion, prolonged high-dosed recreational ketamine use is associated with structural and functional brain diﬀerences. Why these diﬀerences exist, has not been established yet, but may follow patterns observed in animals, i.e., hyperphosphorylation of tau-protein and disrupting expression of several receptor subunits. A second limitation of the reviewed studies is that use of other substances including tobacco was more prevalent among ketamine users compared to the drug-free controls, although several studies included polydrug users as a control group. Therefore, the observed brain changes cannot indisputably be ascribed to ketamine alone. In addition, street ketamine might not be pure ketamine but could be contaminated with other drugs, which would strengthen this confounding. Also, ketamine abuse itself might give rise to abuse of other substances. For example, ketamine has an eﬀect on mu-opioid receptors and its antidepressant eﬀect might even be partially mediated by this receptor (Smith et al., 1980; Gupta et al., 2011; Williams et al., 2018). This eﬀect on the mu-opioid receptor might give rise to opioid abuse. In one of the included samples, 50% of ketamine users had also been using heroin, which could have contributed to the observed brain changes (Sanacora and Schatzberg, 2015; Chesters et al., 2016). This interplay between ketamine and mu- opioid receptors might also apply to other neurotransmitter systems and associated drugs of abuse. Despite its limitations, this review underscores the need for further study of potential risks associated with repeated administration of ketamine. To elucidate causal associations between brain changes and prolonged ketamine use, prospective and longitudinal imaging studies with controlled low- dose administrations are needed, ideally combined with cognitive tasks. FUNDING Fourth, most of the included subjects were of Asian ethnicity, which might have inﬂuenced outcomes for instance through genetic diﬀerences in drug metabolism. However, it has been shown that frequencies of cytochrome P450 variants responsible This study was funded by a suicide prevention grant from ZonMw, the Netherlands Organization for Health Research and Development, Grant No. 537001004. AUTHOR CONTRIBUTIONS JS, CW, FD, and JD performed the literature search. JS, CW, and FD collected and interpreted the data. JS, CW, FD, JD, AL, DD, RS, and MF wrote this paper. All authors provided their consent for publication of this paper. Third, since subjects were mostly recreational users, they might have used ketamine shortly before data were obtained. Therefore, the diﬀerent functional connectivity patterns could in part be caused or inﬂuenced by the direct, short term eﬀects of ketamine. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. Frontiers in Neuroanatomy \| www.frontiersin.org REFERENCES Abi-Dargham, A., Mawlawi, O., Lombardo, I., Gil, R., Martinez, D., Huang, Y., et al. (2002). Prefrontal dopamine D1 receptors and working memory in schizophrenia. J. Neurosci. 22, 3708–3719. doi: 10.1523/JNEUROSCI.22-09-03708.2002 Abdallah, C. G., Averill, C. L., Salas, R., Averill, L. A., Baldwin, P. R., Krystal, J. H., et al. (2017). Prefrontal connectivity and glutamate transmission: relevance to depression pathophysiology and ketamine treatment. Biol. Psychiatry Cogn. Neurosci. Neuroimaging 2, 566–574. doi: 10.1016/j.bpsc.2017. 04.006 Abdallah, C. G., Averill, C. L., Salas, R., Averill, L. A., Baldwin, P. R., Krystal, J. H., et al. (2017). Prefrontal connectivity and glutamate transmission: relevance to depression pathophysiology and ketamine treatment. Biol. Psychiatry Cogn. Neurosci. Neuroimaging 2, 566–574. doi: 10.1016/j.bpsc.2017. 04.006 Albuquerque, E. X., Thompson, S. M., Thomas, C. J., Zarate, C. A., and Gould, T. D. (2016). NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature 533, 481–486. doi: 10.1038/nature17998 March 2022 \| Volume 16 \| Article 795231 Frontiers in Neuroanatomy \| www.frontiersin.org 15 Brain Changes After Long-Term Ketamine Strous et al. Kakiuchi, T., Nishiyama, S., Sato, K., Ohba, H., Nakanishi, S., and Tsukada, H. (2001). Eﬀect of MK801 on dopamine parameters in the monkey brain. Neuroimage 16, 110. Balleine, B. W., Morris, R. W., and Leung, B. K. (2015). Thalamocortical integration of instrumental learning and performance and their disintegration in addiction. Brain Res. 1628, 104–116. doi: 10.1016/j.brainres.2014.12.023 Koh, M. T., Shao, Y., Sherwood, A., and Smith, D. R. (2016). Impaired hippocampal-dependent memory and reduced parvalbumin-positive interneurons in a ketamine mouse model of schizophrenia. Schizophr. Res. 171, 187–194. doi: 10.1016/j.schres.2016.01.023 Berman, R. M., Cappiello, A., Anand, A., Oren, D. A., Heninger, G. R., Charney, D. S., et al. (2000). Antidepressant eﬀects of ketamine in depressed patients. Biol. Psychiatry 47, 351–354. doi: 10.1016/S0006-3223(99)00230-9 Bonnet, U. (2015). Long-term ketamine self-injections in major depressive disorder: focus on tolerance in ketamine’s antidepressant response and the development of ketamine addiction. J. Psychoactive Drugs 47, 276–285. doi: 10.1080/02791072.2015.1072653 Laitinen, L. V., and Vilkki, J. (1973). Observations on the Transcallosal Emotional Connections. Baltimore, MD: University Park Press. Li, C. R., Zhang, S., Hung, C. C., Chen, C. M., Duann, J. R., Lin, C. P., et al. (2017). Depression in chronic ketamine users: Sex diﬀerences and neural bases. Psychiatry Res. 269, 1–8. doi: 10.1016/j.pscychresns.2017.09.001 Caﬃno, L., Piva, A., Giannotti, G., Di Chio, M., Mottarlini, F., Venniro, M., et al. (2017). Ketamine self-administration reduces the homeostasis of the glutamate synapse in the rat brain. Mol. Neurobiol. REFERENCES Lett. 522, 36–40. doi: 10.1016/j.neulet.2012.06.009 Daly, E. J., Singh, J. B., Fedgchin, M., Cooper, K., Lim, P., Shelton, R. C., et al. (2018). Eﬃcacy and safety of intranasal esketamine adjunctive to oral antidepressant therapy in treatment-resistant depression: a randomized clinical trial. JAMA Psychiatry 75, 139–148. doi: 10.1001/jamapsychiatry.2017.3739 Liao, Y., Tang, J., Liu, J., Xie, A., Yang, M., Johnson, M., et al. (2016). Brain Damage in Chronic Ketamine Users: A Multi-modal Imaging study. Thesis, Chinese University of Hong Kong, China. DeWilde, K. E., Levitch, C. F., Murrough, J. W., Mathew, S. J., and Iosifescu, D. V. (2015). The promise of ketamine for treatment-resistant depression: current evidence and future directions. Ann. N. Y. Acad. Sci. 1345, 47–58. doi: 10.1111/nyas.12646 Liao, Y., Tang, J., Ma, M., Wu, Z., Yang, M., Wang, X., et al. (2010). Frontal white matter abnormalities following chronic ketamine use: a diﬀusion tensor imaging study. Brain 133, 2115–2122. doi: 10.1093/brain/awq131 Ding, R., Li, Y., Du, A., Yu, H., He, B., Shen, R., et al. (2016). Changes in hippocampal AMPA receptors and cognitive impairments in chronic ketamine addiction models: another understanding of ketamine CNS toxicity. Sci. Rep. 6:38771. doi: 10.1038/srep38771 Lim, K. O., Wozniak, J. R., Mueller, B. A., Franc, D. T., Specker, S. M., Rodriguez, C. P., et al. (2008). Brain macrostructural and microstructural abnormalities in cocaine dependence. Drug Alcohol Depend. 92, 164–172. doi: 10.1016/j.drugalcdep.2007.07.019 Edward Roberts, R., Curran, H. V., Friston, K. J., and Morgan, C. J. (2014). Abnormalities in white matter microstructure associated with chronic ketamine use. Neuropsychopharmacology 39, 329–338. doi: 10.1038/npp.2013.195 Liu, T., Chen, X., Song, M., and Hao, W. (2016). Decreased thalamocortical connectivity in chronic ketamine users. PLoS ONE 11, e0167381. doi: 10.1371/journal.pone.0167381 Lud Cadet, J., Jayanthi, S. T., McCoy, M., Beauvais, G., and Sheng Cai, N. (2010). Dopamine D1 receptors, regulation of gene expression in the brain, and neurodegeneration. CNS Neurol. Disord. Drug Targets 9, 526–538. doi: 10.2174/187152710793361496 EMA (2019). Assessment Report Spravato. Amsterdam. FDA (2019). FDA Approves New Nasal Spray Medication for Treatment-Resistant Depression Available Onlt a Certiﬁed Doctor’s Oﬃce or Clinic. Washington, DC. Luo, Y., Yu, Y., Zhang, M., He, H., and Fan, N. (2020). Chronic administration of ketamine induces cognitive deterioration by restraining synaptic signaling. Mol. Psychiatry 26, 4702–4718. doi: 10.1038/s41380-020-0793-6 Fowler, J. H., McCracken, E., Dewar, D., and McCulloch, J. (2003). Intracerebral injection of AMPA causes axonal damage in vivo. Brain Res. 991, 104–112. doi: 10.1016/j.brainres.2003.08.004 Mak, Y. T., Lam, W. REFERENCES 54, 7186–7193. doi: 10.1007/s12035-016-0231-6 Li, N., Lee, B., Liu, R. J., Banasr, M., Dwyer, J. M., Iwata, M., et al. (2010). mTOR-dependent synapse formation underlies the rapid antidepressant eﬀects of NMDA antagonists. Science 329, 959–964. doi: 10.1126/science.1190287 Chan, K. W., Lee, T. M., Siu, A. M., Wong, D. P., Kam, C. M., Tsang, S. K., et al. (2013). Eﬀects of chronic ketamine use on frontal and medial temporal cognition. Addict. Behav. 38, 2128–2132. doi: 10.1016/j.addbeh.2013.01.014 g Li, Y., Ding, R., Ren, X., Wen, G., Dong, Z., Yao, H., et al. (2019). Long-term ketamine administration causes Tau protein phosphorylation and Tau protein- dependent AMPA receptor reduction in the hippocampus of mice. Toxicol. Lett. 315, 107–115. doi: 10.1016/j.toxlet.2019.08.023 Chan, W. M., Xu, J., Fan, M., Jiang, Y., Tsui, T. Y., Wai, M. S., et al. (2012). Downregulation in the human and mice cerebella after ketamine versus ketamine plus ethanol treatment. Microsc. Res. Tech. 75, 258–264. doi: 10.1002/jemt.21052 Liang, H., Tang, W. K., Chu, W. C. W., Ernst, T., Chen, R., and Chang, L. (2020). Striatal and white matter volumes in chronic ketamine users with or without recent regular stimulant use. Drug Alcohol Depend. 213:108063. doi: 10.1016/j.drugalcdep.2020.108063 Chesters, R. A., Stone, J. M., Hughes, E., Howes, O., Cooper, J. D., and Vernon, A. C. (2016). Translational neuroimaging reveals diﬀerential eﬀects of chronic ketamine on brain macrostructure in humans and mice. Eur. Neuropsychopharmacol. 26, S67–S68. doi: 10.1016/S0924-977X(16)70074-5 Liao, Y., Johnson, M., Qi, C., Wu, Q., Xie, A., Liu, J., et al. (2018). Cue-induced brain activation in chronic ketamine-dependent subjects, cigarette smokers, and healthy controls: a task functional magnetic resonance imaging study. Front. Psychiatry 9, 88. doi: 10.3389/fpsyt.2018.00088 Cowan, R. L., Lyoo, I. K., Sung, S. M., Ahn, K. H., Kim, M. J., Hwang, J., et al. (2003). Reduced cortical gray matter density in human MDMA (Ecstasy) users: a voxel-based morphometry study. Drug Alcohol Depend. 72, 225–235. doi: 10.1016/j.drugalcdep.2003.07.001 Front. Psychiatry 9, 88. doi: 10.3389/fpsyt.2018.00088 Liao, Y., Tang, J., Corlett, P. R., Wang, X., Yang, M., Chen, H., et al. (2011). Reduced dorsal prefrontal gray matter after chronic ketamine use. Biol. Psychiatry 69, 42–48. doi: 10.1016/j.biopsych.2010.08.030 j g Cvrcek, P. (2008). Side eﬀects of ketamine in the long-term treatment of neuropathic pain. Pain Med. 9, 253–257. doi: 10.1111/j.1526-4637.2007.00314.x Liao, Y., Tang, J., Fornito, A., Liu, T., Chen, X., Chen, H., et al. (2012). Alterations in regional homogeneity of resting-state brain activity in ketamine addicts. Neurosci. REFERENCES doi: 10.1038/npp.2014.338 Zou, J., and Tan, S. (2016). Emerging trends in the abuse of ketamine and its side eﬀects on health: toxicology and addiction potential. Adv. Clin. Toxicol. 1:1. Zou, J., and Tan, S. (2016). Emerging trends in the abuse of ketamine and its side eﬀects on health: toxicology and addiction potential. Adv. Clin. Toxicol. 1:1. Schobel, S. A., Chaudhury, N. H., Khan, U. A., Paniagua, B., Styner, M. A., Asllani, I., et al. (2013). Imaging patients with psychosis and a mouse model establishes a spreading pattern of hippocampal dysfunction and implicates glutamate as a driver. Neuron 78, 81–93. doi: 10.1016/j.neuron.2013.02.011 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Schumacher, A., Sivanandan, B., Tolledo, E. C., Woldegabriel, J., and Ito, R. (2016). Diﬀerent dosing regimens of repeated ketamine administration have opposite eﬀects on novelty processing in rats. Prog. Neuropsychopharmacol. Biol. Psychiatry 69, 1–10. doi: 10.1016/j.pnpbp.2016.03.007 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Short, B., Fong, J., Galvez, V., Shelker, W., and Loo, C. K. (2017). Side-eﬀects associated with ketamine use in depression: a systematic review. Lancet Psychiatry 5, 65–78. doi: 10.1016/S2215-0366(17)30272-9 Singh, J. B., Fedgchin, M., Daly, E. J., De Boer, P., Cooper, K., Lim, P., et al. (2016). A double-blind, randomized, placebo-controlled, dose-frequency study of intravenous ketamine in patients with treatment-resistant depression. Am. J. Psychiatry 173, 816–826. doi: 10.1176/appi.ajp.2016.16010037 Psychiatry 173, 816–826. doi: 10.1176/appi.ajp.2016.16010037 Copyright © 2022 Strous, Weeland, van der Draai, Daams, Denys, Lok, Schoevers and Figee. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. REFERENCES The cingulate gyrus and human behaviour. Electroencephalogr. Clin. Neurophysiol. 34, 45–52 doi: 10.1016/0013-4694(73)90149-1 Morgan, C. J., Dodds, C. M., Furby, H., Pepper, F., Fam, J., Freeman, T. P., et al. (2014). Long-term heavy ketamine use is associated with spatial memory impairment and altered hippocampal activation. Front Psychiatry 5, 149. doi: 10.3389/fpsyt.2014.00149 Tan, S., Rudd, J. A., and Yew, D. T. (2011). Gene expression changes in GABA(A) receptors and cognition following chronic ketamine administration in mice. PLoS ONE 6, e21328. doi: 10.1371/journal.pone.0021328 Morgan, C. J., Muetzelfeldt, L., and Curran, H. V. (2009). Ketamine use, cognition and psychological wellbeing: a comparison of frequent, infrequent and ex-users with polydrug and non-using controls. Addiction 104, 77–87. doi: 10.1111/j.1360-0443.2008.02394.x Tang, W., Lin, Y., Zhang, C., Liang, H., and Lai, W. (2016). Evidence of Brain Damage in Chronic Ketamine Users: A Brain Imaging Study, Australian and New Zealand Journal of Psychiatry. Hong Kong: Sage Publications. Uhlhaas, P. J., Millard, I., Muetzelfeldt, L., Curran, H. V., and Morgan, C. J. (2007). Perceptual organization in ketamine users: preliminary evidence of deﬁcits on night of drug use but not 3 days later. J. Psychopharmacol. 21, 347–352. doi: 10.1177/0269881107077739 Morgan, C. J., Muetzelfeldt, L., and Curran, H. V. (2010). Consequences of chronic ketamine self-administration upon neurocognitive function and psychological wellbeing: a 1-year longitudinal study. Addiction 105, 121–133. doi: 10.1111/j.1360-0443.2009.02761.x UNODC (2016). World Drug Report (2016). United Nations Oﬃce on Drugs and Crime. Muetzelfeldt, L., Kamboj, S. K., Rees, H., Taylor, J., Morgan, C. J., and Curran, H. V. (2008). Journey through the K-hole: phenomenological aspects of ketamine use. Drug Alcohol Depend. 95, 219–229. doi: 10.1016/j.drugalcdep.2008.01.024 Wajs, E., Aluisio, L., Holder, R., Daly, E. J., Lane, R., Lim, P., et al. (2020). Esketamine nasal spray plus oral antidepressant in patients with treatment- resistant depression: assessment of long-term safety in a phase 3, open-label study (SUSTAIN-2). J. Clin. Psychiatry 81:12891. doi: 10.4088/JCP.19m12891 Murphy, R., and Roe, S. (2010). Drug Misuse Declared: Findings From the 2009/10 British Crime Survey. England and Wales: Home Oﬃce. p g y p p study (SUSTAIN-2). J. Clin. Psychiatry 81:12891. doi: 10.4088/JCP.19m12891 Narendran, R., Frankle, W. G., Keefe, R., Gil, R., Martinez, D., Slifstein, M., et al. (2005). Altered prefrontal dopaminergic function in chronic recreational ketamine users. Am. J. Psychiatry 162, 2352–2359. doi: 10.1176/appi.ajp.162.12.2352 Wang, C., Zheng, D., Xu, J., Lam, W., and Yew, D. T. (2013). Brain damages in ketamine addicts as revealed by magnetic resonance imaging. REFERENCES P., Lu, L., Wong, Y. W., and Yew, D. T. (2010). The toxic eﬀect of ketamine on SH-SY5Y neuroblastoma cell line and human neuron. Microsc. Res. Tech. 73, 195–201. doi: 10.1002/jemt.20774 Gupta, A., Devi, L. A., and Gomes, I. (2011). Potentiation of µ-opioid receptor-mediated signaling by ketamine. J. Neurochem. 119, 294–302. doi: 10.1111/j.1471-4159.2011.07361.x Maxwell, J. C. (2005). Party drugs: properties, prevalence, patterns, and problems. Subst. Use Misuse 40, 1203–1240. doi: 10.1081/JA-200066736 Howick, J., Chalmers, I., Glasziou, P., Greenhalgh, T., Heneghan, C., Liberati, A., et al. (2018). Explanation of the 2011 Oxford Centre for Evidence-Based Medicine (OCEBM) Levels of Evidence. Available online at: https://www.cebm.ox.ac.uk/ resources/levels-of-evidence/ocebm-levels-of-evidence Meyer, G., McElhaney, M., Martin, W., and McGraw, C. P. (1973). Stereotactic cingulotomy with results of acute stimulation and serial psychological testing. Surg. Approaches Psychiatry 1973, 39–58. Hung, C. C., Liu, Y. H., Huang, C. C., Chou, C. Y., Chen, C. M., Duann, J. R., et al. (2020a). Eﬀects of early ketamine exposure on cerebral gray matter volume and functional connectivity. Sci. Rep. 10:15488. doi: 10.1038/s41598-020- 72320-z Mizutani, T. (2003). PM frequencies of major CYPs in Asians and Caucasians. Drug Metab. Rev. 35, 99–106. doi: 10.1081/DMR-120023681 Moghaddam, B., Adams, B., Verma, A., and Daly, D. (1997). Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex. J. Neurosci. 17, 2921–2927. doi: 10.1523/JNEUROSCI.17-08-02921.1997 Hung, C. C., Zhang, S., Chen, C. M., Duann, J. R., Lin, C. P., Lee, T. S., et al. (2020b). Striatal functional connectivity in chronic ketamine users: a pilot study. Am. J. Drug Alcohol Abuse 46, 31–43. doi: 10.1080/00952990.2019.16 24764 March 2022 \| Volume 16 \| Article 795231 Frontiers in Neuroanatomy \| www.frontiersin.org 16 Brain Changes After Long-Term Ketamine Strous et al. Moher, D., Liberati, A., Tetzlaﬀ, J., and Altman, D. G. (2009). Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. J. Clin. Epidemiol. 62, 1006–1012 doi: 10.1016/j.jclinepi.2009.06.005 Szymkowicz, S. M., Finnegan, N., and Dale, R. M. (2013). A 12-month naturalistic observation of three patients receiving repeat intravenous ketamine infusions for their treatment-resistant depression. J. Aﬀect. Disord. 147, 416–420. doi: 10.1016/j.jad.2012.10.015 Morgan, C. J., and Curran, H. V. (2006). Acute and chronic eﬀects of ketamine upon human memory: a review. Psychopharmacology 188, 408–424. doi: 10.1007/s00213-006-0572-3 Talairach, J., Bancaud, J., Geier, S., Bordas-Ferrer, M., Bonis, A., Szikla, G., et al. (1973). REFERENCES Front. Neuroanat. 7, 23. doi: 10.3389/fnana.2013.00023 Weimer, J. M., Jones, S. E., and Frontera, J. A. (2017). Acute cytotoxic and vasogenic edema after subarachnoid hemorrhage: a quantitative MRI study. Am. J. Neuroradiol. 38, 928–934. doi: 10.3174/ajnr.A5181 Ognjanovski, N., Schaeﬀer, S., Wu, J., Mofakham, S., Maruyama, D., Zochowski, M., et al. (2017). Parvalbumin-expressing interneurons coordinate hippocampal network dynamics required for memory consolidation. Nat. Commun. 8:15039. doi: 10.1038/ncomms15039 Wersinger, C., Chen, J., and Sidhu, A. (2004). Bimodal induction of dopamine- mediated striatal neurotoxicity is mediated through both activation of D1 dopamine receptors and autoxidation. Mol. Cell. Neurosci. 25, 124–137. doi: 10.1016/j.mcn.2003.10.002 Peltoniemi, M. A., Hagelberg, N. M., Olkkola, K. T., and Saari, T. I. (2016). Ketamine: A review of clinical pharmacokinetics and pharmacodynamics in anesthesia and pain therapy. Clin. Pharmacokinet. 55, 1059–1077. doi: 10.1007/s40262-016-0383-6 Williams, N. R., Heifets, B. D., Blasey, C., Sudheimer, K., Pannu, J., Pankow, H., et al. (2018). Attenuation of Antidepressant Eﬀects of Ketamine by Opioid Receptor Antagonism. Am. J. Psychiatry 175, 1205–1215. doi: 10.1176/appi.ajp.2018.18020138 Rocha, A., Hart, N., and Trujillo, K. A. (2017). Diﬀerences between adolescents and adults in the acute eﬀects of PCP and ketamine and in sensitization following intermittent administration. Pharmacol. Biochem. Behav. 157, 24–34. doi: 10.1016/j.pbb.2017.04.007 Xu, Y., Hackett, M., Carter, G., Loo, C., Galvez, V., Glozier, N., et al. (2016). Eﬀects of low-dose and very low-dose ketamine among patients with major depression: a systematic review and meta-analysis. Int. J. Neuropsychopharmacol. 19:124. doi: 10.1093/ijnp/pyv124 Sackett, D. L. (1989). Rules of evidence and clinical recommendations on the use of antithrombotic agents. Chest 108:227S−230S. doi: 10.1378/chest.95.2_Supplement.2S Yeung, L. Y., Wai, M. S., Fan, M., Mak, Y. T., Lam, W. P., Li, Z., et al. (2010). Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine. Toxicol. Lett. 193, 189–193. doi: 10.1016/j.toxlet.2010.01.008 Sanacora, G., Frye, M. A., McDonald, W., Mathew, S. J., Turner, M. S., Schatzberg, A. F., et al. (2017). A consensus statement on the use of ketamine in the treatment of mood disorders. JAMA Psychiatry 74, 399–405. doi: 10.1001/jamapsychiatry.2017.0080 Zarate, C. A., Brutsche, N. E., Ibrahim, L., Franco-Chaves, J., Diazgranados, N., Cravchik, A., et al. (2012). Replication of ketamine’s antidepressant eﬃcacy in bipolar depression: a randomized controlled add-on trial. Biol. Psychiatry 71, 939–946. doi: 10.1016/j.biopsych.2011.12.010 Sanacora, G., and Schatzberg, A. F. (2015). Ketamine: promising path or false prophecy in the development of novel therapeutics for mood disorders? Neuropsychopharmacology 40:1307. REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Smith, D. J., Pekoe, G. M., Martin, L. L., and Coalgate, B. (1980). The interaction of ketamine with the opiate receptor. Life Sci. 26, 789–795. doi: 10.1016/0024-3205(80)90285-4 Sun, L., Li, Q., Li, Q., Zhang, Y., Liu, D., Jiang, H., et al. (2014). Chronic ketamine exposure induces permanent impairment of brain functions in adolescent cynomolgus monkeys. Addict. Biol. 19, 185–194. doi: 10.1111/adb.12004 March 2022 \| Volume 16 \| Article 795231 Frontiers in Neuroanatomy \| www.frontiersin.org Frontiers in Neuroanatomy \| www.frontiersin.org 17
https://openalex.org/W2917576502	http://nora.nerc.ac.uk/id/eprint/522343/1/s41598-019-38714-4.pdf	English	null	Extreme spikes in DMS flux double estimates of biogenic sulfur export from the Antarctic coastal zone to the atmosphere	Scientific reports	2,019	cc-by	11,184	Extreme spikes in DMS flux double estimates of biogenic sulfur export from the Antarctic coastal zone to the atmosphere Received: 6 September 2018 Accepted: 8 January 2019 Published: xx xx xxxx A. L. Webb 1,2, M. A. van Leeuwe2, D. den Os2,3, M. P. Meredith4, H. J. Venables4 & J. S Biogenic dimethylsulfide (DMS) is a significant contributor to sulfur flux from the oceans to the atmosphere, and the most significant source of aerosol non sea-salt sulfate (NSS-SO4 2−), a key regulator of global climate. Here we present the longest running time-series of DMS-water (DMSW) concentrations in the world, obtained at the Rothera Time-Series (RaTS) station in Ryder Bay, West Antarctic Peninsula (WAP). We demonstrate the first ever evaluation of interseasonal and interannual variability in DMSW and associated flux to the atmosphere from the Antarctic coastal zone and determine the scale and importance of the region as a significant source of DMS. Impacts of climate modes such as El Niňo/Southern Oscillation are evaluated. Maximum DMSW concentrations occurred annually in January and were primarily associated with sea-ice break-up. These concentrations resulted in extremely high (up to 968 µmol m−2 d−1) DMS flux over short timescales, which are not parameterised in global-scale DMS climatologies. Calculated DMS flux stayed above the aerosol nucleation threshold of 2.5 µmol m−2 d−1 for 60% of the year. Overall, using flux determinations from this study, the total flux of DMS-sulfur from the Austral Polar Province (APLR) was 1.1 Tg sulfur yr−1, more than double the figure suggested by the most recent DMS climatologies. Dimethylsulfide (DMS) is a semi-volatile organic sulfur compound produced in surface waters around the globe, primarily from the breakdown of the algal osmolyte dimethylsulfoniopropionate (DMSP)1,2. Approximately 10% of total global DMS production ventilates through the sea-air interface3,4, where it accounts for approximately 50% of the natural global atmospheric sulfate burden5. Current estimates calculate a global DMS flux to the atmosphere of 28.1 (17.6–34.4) Tg S per year6, which is approximately half the anthropogenic global atmos- pheric sulfur input6,7. This makes DMS an important contributor to global sulfur fluxes. Once in the atmos- phere, DMS oxidation contributes to the formation of non sea-salt sulfate (NSS-SO4 2−), a cloud condensation nuclei (CCN) important for cloud formation and thereby reducing incoming solar radiation, and subsequently cooling the climate8. Charlson and co-authors postulated a negative feedback loop between global temperature and DMS production that would keep Earth’s temperature in homeostasis; this hypothesis is referred to as the CLAW-hypothesis. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 6 September 2018 Accepted: 8 January 2019 Published: xx xx xxxx Extreme spikes in DMS flux double estimates of biogenic sulfur export from the Antarctic coastal zone to the atmosphere The hypothesis has recently been questioned9 and model calculations on the future response of DMS to changes in global temperature vary widely: both increases10,11 and decreases12 in surface water DMS concentrations are predicted, with corresponding variation in Southern Ocean DMS flux between the models6,13. These results indicate large uncertainties in the processes surrounding DMS production, and emphasise the need to generate improved mechanistic understanding so to amend model parameterisation of the DMS flux.i g p g pl Global climatologies of DMS concentrations show that the polar regions are of significant importance to total global DMS production, in particular the Southern Ocean6,13. The total Southern Ocean (south of 40°S) DMS flux is calculated at approximately 3.4 Tg S during summer months (December to February)14, with an annual total flux estimated at 5.8 Tg S6,15. However, calculations of the DMS climatologies are still highly uncertain due to two factors. Firstly, the highest DMS concentrations (above 148 nmol L−1), as found in the marginal ice zone, are omitted because data are highly variable and scarce6. Secondly, availability of austral winter data is extremely 1School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK. 2GELIFES, University of Groningen, 9700 CC, Groningen, The Netherlands. 3Institute for Life Science and Technology, Hanze University of Applied Sciences, 9700 RM, Groningen, The Netherlands. 4British Antarctic Survey, High Cross, Cambridge, CB3 0ET, UK. Correspondence and requests for materials should be addressed to A.L.W. (email: alison.webb@warwick.ac.uk) Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 1 www.nature.com/scientificreports/ Figure 1. (a) Location of Rothera Research Station on the West Antarctic Peninsula, and (b) location of the Rothera Time Series (RaTS) sampling location. Figure 1. (a) Location of Rothera Research Station on the West Antarctic Peninsula, and (b) location of the Rothera Time Series (RaTS) sampling location. limited6,14. A recent update of the summertime climatology, however, indicated that extremely high DMS concen- trations might be a real feature in some areas of the Southern Ocean14; in the short, highly productive Antarctic spring and summer seasons, surface water DMS concentrations can exceed 50 nmol L−1 16–18. These high values are often associated with the release of ice algae, recognised as a significant source of DMSP19–21, from melted sea ice21–24. DMSP is an important osmolyte and cryoprotectant and may play a pivotal role in sea-ice algae surviving the extreme conditions of temperature and salinity that prevail in sea ice25. Extreme spikes in DMS flux double estimates of biogenic sulfur export from the Antarctic coastal zone to the atmosphere The release of DMSP during ice melt events may be the source of significant input of DMS into polar waters, potentially producing a short-term atmos- pheric flux of high DMS, although direct evidence for this pathway is limited26. DMS concentrations in Antarctic sea-ice leads have also been found to be an order of magnitude higher than in the underlying water column27,28.h g g y g The Western Antarctic Peninsula (WAP) is a climatically very sensitive region. During the second half of the twentieth century, a strong atmospheric warming trend was present, with a marked concurrent retreat in sea ice and warming of the upper ocean29–32. Over the same era, precipitation increased and winds strength- ened and veered to be more northerly33. These changes are known not to be monotonic, but to have significant interannual variability superposed34. Whereas the retreat in sea ice will directly impact on the release of DMSP and DMSP-producing algae, changes in the physical environment can also impact indirectly on phytoplankton productivity and composition through changes in light and nutrient availability32,35. p y p g g g y Developing a full understanding of the relevant processes that impact on DMSP production requires datasets that span the pertinent timescales. So far, data from the WAP on surface water DMS concentrations are extremely limited both spatially and temporally21,36–38, exceptions being two extensive datasets from the Palmer Long-Term Ecological Research (LTER) program, which show increasing DMS concentrations in December, reaching rel- atively stable concentrations in January between 5 and 15 nmol L−1 and occasional maxima exceeding 25 nmol L−1 39,40. To address this knowledge gap, we undertook a five-year study at the WAP that involved measuring water column DMS(P/O) concentrations to identify temporal variability over multiple consecutive years in both winter and summer and to associate these changes with trends in phytoplankton abundance and community compo- sition41. This study was carried out at Rothera Research Station (Fig. 1), as part of the year-round observational programme for water column measurements and sea-ice observations conducted by the British Antarctic Survey (BAS) since 1998. Here, we present the DMS data, and calculate fluxes from surface-water DMS concentrations, wind speed and oceanographic data. The results are analysed in the context of existing DMS climatologies of the area. We find significantly higher fluxes than previously reported, and postulate that these high numbers are more representative of the marginal ice zone than previous quantifications. Results O open water; breakup of fast ice to below 50% coverage, and ice movement due to wind and current, resulted in a mix of brash ice of both sea ice and glacial origin and icebergs. Over the five years, we observe a pattern of decreasing vertical mixing in the upper ocean in winter, in line with the increasing coverage and duration of sea ice. In all summers, the mixed layer depth (MLD) remained rela- tively shallow above 20 m (Fig. 2c). Wind at Rothera was highly variable within the range 2.2–40.7 m s−1 (Fig. 1d), with evidence of seasonality through stronger winds in winter. South-easterly winds were dominant, and through the five years there was no evidence of significant interannual differences. The wind speed on sampling days was open water; breakup of fast ice to below 50% coverage, and ice movement due to wind and current, resulted in a mix of brash ice of both sea ice and glacial origin and icebergs. Over the five years, we observe a pattern of decreasing vertical mixing in the upper ocean in winter, in line with the increasing coverage and duration of sea ice In all summers the mixed layer depth (MLD) remained rela open water; breakup of fast ice to below 50% coverage, and ice movement due to wind and current, resulted in a mix of brash ice of both sea ice and glacial origin and icebergs. O h fi b f d l h l Over the five years, we observe a pattern of decreasing vertical mixing in the upper ocean in winter, in line with the increasing coverage and duration of sea ice. In all summers, the mixed layer depth (MLD) remained rela- tively shallow above 20 m (Fig. 2c). Wind at Rothera was highly variable within the range 2.2–40.7 m s−1 (Fig. 1d), with evidence of seasonality through stronger winds in winter. South-easterly winds were dominant, and through the five years there was no evidence of significant interannual differences. The wind speed on sampling days was identified as being on average 3.9 m s−1 lower than the average over the entire 5 years, as identified in Fig. 2d. DMS Concentrations. Concentration of DMSW in surface waters of Ryder Bay was found to be extremely high, with concentrations exceeding 30 nmol L−1 in four out of five summer seasons and peaking at over 170 nmol L−1 in January 2015 (Fig. 3a). Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 Results O Oceanographic conditions. Marked seasonality in sea surface temperature (SST) in Ryder Bay was iden- tified over the five-year time-series ranging between minimum winter temperatures of −1.8 °C and a maximum summer temperature of 3.3 °C in 2012/13 (Fig. 2a), decreasing with each progressive year (2.5 °C in 2014, 1.3 °C in 2015 and 1.1 °C in 2016), followed by a markedly higher maximum summer SST in 2016/17 of 2.4 °C. The greatest variation in SST occurred during summer (November – March); temperatures above 0 °C were measured for the first time each year in mid to late December and declined to below 0 °C in late March/ April each year. The dura- tion of 100% sea ice cover increased over the five years of our survey commensurate with the decreasing summer SST, from 86 days in 2012 to 163 days in 2016 (Fig. 2b). Despite summer 2016/17 showing an increase in SST after the decreasing trend of the previous 4 years, ice cover was the highest with an average 62% ice cover, compared to approximately 35% ice cover in all previous summers. Ice type within the bay varied throughout the seasons, as did the extent of the coverage: periods of over 80% sea ice cover were comprised of fast ice with cracks and limited Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 2 www.nature.com/scientificreports/ Figure 2. Oceanographic conditions at the Rothera Time Series (RaTS) station in Ryder Bay at the West Antarctic Peninsula during Sep 2012–Mar 2017, showing (a) sea surface temperature (SST), (b) sea-ice cover given as a percentage coverage of Ryder Bay, (c) mixed layer depth (MLD) in the surface 100 m and d) mean daily wind speed (grey), mean daily wind speed on sampling days (black points), mean 5-year wind speed (dashed line) and mean sampling-day wind speed (solid line). Figure 2. Oceanographic conditions at the Rothera Time Series (RaTS) station in Ryder Bay at the West Antarctic Peninsula during Sep 2012–Mar 2017, showing (a) sea surface temperature (SST), (b) sea-ice cover given as a percentage coverage of Ryder Bay, (c) mixed layer depth (MLD) in the surface 100 m and d) mean daily wind speed (grey), mean daily wind speed on sampling days (black points), mean 5-year wind speed (dashed line) and mean sampling-day wind speed (solid line). Results O (a) DMS (nmol L−1) over the 5-year time-series in Ryder Bay at the West Antarctic Peninsula, presented as the mean over the surface 15 m and b) DMS flux (µmol m−2 d−1), calculated from DMS concentration, daily average wind speed and SST. Black solid lines represent summer (Nov–Mar) interpolated values calculated from in-situ measured DMS and red solid lines represent winter (Apr–Oct) interpolated values based on stored DMSPd samples analysed the following summer. Black circles represent the data points on which the interpolation was performed. Figure 3. (a) DMS (nmol L−1) over the 5-year time-series in Ryder Bay at the West Antarctic Peninsula, l Month Total No. Sampling days Mean Daily DMSw (nmol L−1) Mean Daily DMS Flux (µmol m−2 d−1) Total Monthly DMS Flux (µmol m−2 month−1) Mean Monthly Wind Speed (m s−1) Wind Speed Range (m s−1) Mean Monthly Ice Cover (%) Mean SST (°C) Jan 33 23.6 ± 35.3 51.2 ± 125.5 1588 9.3 ± 5.6 3.0–28.6 31 ± 11 0.99 ± 0.98 Feb 33 6.0 ± 5.8 26.3 ± 45.0 736 11.6 ± 6.3 2.7–28.1 28 ± 11 0.54 ± 0.79 Mar 20 2.6 ± 1.5 10.9 ± 9.7 338 13.3 ± 6.9 2.7–29.0 30 ± 12 −0.20 ± 0.65 Apr 11 1.2 ± 1.1 7.2 ± 9.8 217 14.7 ± 6.7 3.0–32.7 25 ± 13 −1.05 ± 0.51 May 13 0.4 ± 0.3 2.0 ± 2.8 61 13.0 ± 7.9 2.2–40.7 41 ± 18 −1.55 ± 0.25 Jun 8 0.1 ± 0.1 0.6 ± 1.0 18 13.6 ± 7.9 2.4–31.6 62 ± 33 −1.71 ± 0.09 Jul 6 0.1 ± 0.1 0.9 ± 1.9 29 14.2 ± 8.2 2.4–34.0 77 ± 24 −1.73 ± 0.11 Aug 10 0.2 ± 0.2 0.5 ± 0.8 15 13.9 ± 8.7 2.5–40.5 82 ± 25 −1.79 ± 0.05 Sep 2 0.5 ± 0.3 1.5 ± 3.4 46 15.1 ± 8.3 3.5–35.5 85 ± 21 −1.73 ± 0.05 Oct 2 1.7 ± 1.4 4.5 ± 6.2 140 15.2 ± 7.6 3.1–37.6 69 ± 30 −1.61 ± 0.12 Nov 8 3.2 ± 2.2 2.9 ± 6.3 176 12.3 ± 6.7 2.5–37.1 58 ± 29 −1.52 ± 0.21 Dec 21 5.1 ± 3.8 9.6 ± 17.6 296 8.7 ± 5.4 27.4–2.2 57 ± 29 −0.20 ± 0.77 TOTAL 167 3662 Table 1. Results O Mean monthly DMSw concentrations (nmol L−1; averaged over the top 15 m), sea surface temperature (SST; °C) and wind speed (m s−1), used to determine the total monthly DMS flux from Ryder Bay (µmol m−2). Values given are ± standard deviation. Also given is the mean monthly ice cover, given as a percentage. Table 1. Mean monthly DMSw concentrations (nmol L−1; averaged over the top 15 m), sea surface temperature (SST; °C) and wind speed (m s−1), used to determine the total monthly DMS flux from Ryder Bay (µmol m−2). Values given are ± standard deviation. Also given is the mean monthly ice cover, given as a percentage. Table 1. Mean monthly DMSw concentrations (nmol L−1; averaged over the top 15 m), sea surface temperature (SST; °C) and wind speed (m s−1), used to determine the total monthly DMS flux from Ryder Bay (µmol m−2). Values given are ± standard deviation. Also given is the mean monthly ice cover, given as a percentage. was above the nucleation threshold44 of 2.5 µmol m−2 d−1 for 63% of the time. The highest mean monthly fluxes occurred during January and February (43% and 20% of total annual DMS flux respectively; Table 1). The low- est monthly mean flux occurred in August (0.4% of annual total); the month with the highest mean ice cover (Table 1).ili was above the nucleation threshold44 of 2.5 µmol m−2 d−1 for 63% of the time. The highest mean monthly fluxes occurred during January and February (43% and 20% of total annual DMS flux respectively; Table 1). The low- est monthly mean flux occurred in August (0.4% of annual total); the month with the highest mean ice cover (Table 1).ili During the second, third and fourth years, at least one significant spike in DMS flux was identified each year, where flux exceeded 100 µmol m−2 d−1. These spikes were related to high DMSW concentrations and were important contributors to the flux totals, as each occurrence accounted for more than 20% of the mean monthly flux for the month of January. Indeed, a flux of 968 µmol m−2 d−1 was identified in January 2015 due to a combination of a mean daily wind speed greater than 20 m s−1 and the summer DMS peak where concentrations exceeded 80 nmol L−1. Results O Strong seasonality in DMSW was apparent, in addition to significant interannual var- iability. Peak DMS concentrations occurred mid to late January each year, except for 2017 where the maximum DMSW peak was identified during February with the lowest summertime DMSW maximum of 11.6 nmol L−1. There was no apparent relationship between DMSW concentrations and the trends identified in summer SST, sea ice cover and MLD. Wintertime (April to October) concentrations were distinctly lower than during summer, ranging between 0.1–7.1 nmol L−1 and averaging 0.5 nmol L−1 in 2013, 0.6 nmol L−1 in 2014 and 0.7 nmol L−1 in 2016. DMS Flux. With a 5-year averaged wind speed of 12.6 m s−1 (Fig. 2d), Ryder Bay is representative of areas with globally the highest wind speeds42. Calculating DMS transfer velocities at the sea-air interface are dependent on the gas-transfer coefficient and concentration differences at the interface (Eq. 1 in Methods). Determining the gas-transfer coefficient depends on the wind speed, but appears not to follow a simple relationship; especially at high wind speed suppression of the DMS flux is sometimes observed43. It was suggested that long waves suppress the water-side turbulence, thereby decreasing gas transfer of the relative soluble gases, such as DMS, whereas wave breaking and bubble-mediated exchange is of less importance for DMS exchange43. Given the fact that Ryder Bay is a relatively sheltered bay (Fig. 1) with limited fetch area, waves are modest. Therefore, the gas transfer ver- sus wind relationship as determined by Nightingale et al. (2000) was used with the current RaTS data.l p y g g DMS flux showed the same strong seasonality as the DMSW concentrations (Fig. 3b; Table 1), with summer uxes often several orders of magnitude higher than those in winter. Over the five-year time-series, DMS flux Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 3 www.nature.com/scientificreports/ Figure 3. (a) DMS (nmol L−1) over the 5-year time-series in Ryder Bay at the West Antarctic Peninsula, presented as the mean over the surface 15 m and b) DMS flux (µmol m−2 d−1), calculated from DMS concentration, daily average wind speed and SST. Black solid lines represent summer (Nov–Mar) interpolated values calculated from in-situ measured DMS and red solid lines represent winter (Apr–Oct) interpolated values based on stored DMSPd samples analysed the following summer. Black circles represent the data points on which the interpolation was performed. Figure 3. Discussionh The DMSW and subsequent DMS flux presented here from five years of sampling in Ryder Bay allow us to quantify the interseasonal and interannual variation in DMS emission to the atmosphere from a highly productive region, and evaluate the changes identified with variations in long-term climate trends and shorter-term climate variabil- ity, such as the El Niño event that occurred in 2016/17. The results motivate a re-evaluation of existing DMS flux models in the WAP region and have comparable implications for other regions of the Marginal Ice Zone. g p p g g In winter and early spring, DMSW concentrations were generally low in water under the sea ice, due to limited in-situ production from water-column phytoplankton. In winter, mixed layers at the WAP are typically deep (50–150 m) due to buoyancy loss through cooling, brine rejection from ice formation, and mechanical mixing due to wind stress45,46. These conditions and the low-light levels in winter are unfavourable for phytoplankton growth. During periods of fast-ice cover, water temperature below −1.8 °C and low light penetration, algal communities instead thrive in close association with sea ice, living in the brine channels, porous ice layers and along the ice-sea interface25,47. Starting in early spring, sea ice retreats southward along the WAP as insolation and temperatures increase. Sea-ice melt together with runoff from land-based glaciers release low salinity water at the surface, resulting in stratification of the upper water column and development of a shallow (<20 m) mixed layer, despite the exposure of the sea surface to wind-driven mixing48,49. During mid-to-late December, a steady increase of DMSW concentrations developed in all years except in 2016, when ice cover was still 100%.i p y p During the highest peak of DMSW observed during the five years at Ryder Bay in January 2015, Stefels et al.26 calculated similar concentrations as those presented here throughout the wider Marguerite Bay area and further onto the continental shelf and also identified a strong sea-ice influence on surface water DMS(P) concentrations. Occurring in four out of the five years of DMSW analysis, this January peak in DMSW is judged a key part of the annual DMSW cycle, covering a significant percentage of the annual DMSW production in the region. Results O It is estimated that the flux of sulfur in the form of DMS from Ryder Bay was 9.0 Mg S yr−1 (Table 2), with 89% (mean 22.7 ± 67.6 µmol m−2 d−1) produced during summer and 11% (mean 2.5 ± 5.3 µmol m−2 d−1) during the winter months. DMSW and DMS flux from Ryder Bay and the wider WAP region. It is estimated that the flux of sulfur in the form of DMS from Ryder Bay was 9.0 Mg S yr−1 (Table 2), with 89% (mean 22.7 ± 67.6 µmol m−2 d−1) produced during summer and 11% (mean 2.5 ± 5.3 µmol m−2 d−1) during the winter months. Results O Despite releasing 31 mg m−2 sulfur to the atmosphere from Ryder Bay in a single day, this high flux still only contributed 25% of the total January flux in 2015, due to multiple consecutive days of exceptionally high flux. Summer 2016/17 did not produce a high spike in DMS as the previous summer seasons, which corresponded to lower DMSW concentrations. The highest flux within this year occurred later in the season in February (77 µmol m−2 d−1 and at 16 m s−1 wind speed). These data were used to generate a year-round climatology of both surface Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 4 www.nature.com/scientificreports/ Location Time of Year Wind Speed Range (m s−1) DMSW Concentration (nmol L−1) DMS Flux (µmol m−2 d−1) Calculated Total Annual Sulfur Flux (Tg S yr−1) (area given in brackets) Ryder Bay (This Study) Year-round 2.2–40.7 0.05–170 0.01–968 9.02 × 10−6 (77 km2) West Antarctic Peninsula Sea Ice Zone (This Study) Year-round 0.06 (5.0 × 105 km2) Austral Polar Province (APLR) Zone (This Study) Year-round 1.08 (9.2 × 106 km2) Antarctic Sea Ice Zone (This Study) Year-round 2.18 (1.86 × 107 km2) Southern Ocean 60–70°S6 Sep-Apr (1972–2010) 0–50 0.9 (1.88 × 107 km2) Weddell Sea Leads27 Dec-Jan 2004 3.7–9.2 0.6–45.9 0.2–5.3 Eastern Antarctic Sea-Ice Zone72 Dec 1998 2.0–25.0 3–31 1–101 Eastern Antarctic Sea Ice Zone23 Sep-Mar (1991–1995) 11.7 7.9 49 2.7 Palmer Station, WAP36 Jan-Feb 1994 0.7–3.7 0.03–19.2 Australian Antarctic Divergence 63–68°S60,73 Nov 1988-Jan 1989 2.5–16.0 5.3–18.8 1.0 ± 1.6 Weddell Sea61 Nov-Dec 1990 2.3 ± 1.6 0.17 ± 0.09 Weddell Sea (ISPOL)19 Dec 2004 0.3–1.3 14.2 Ross Sea74 Nov-Dec 1.0–22.0 0.6–14.2 0.2–24.3 Ross Sea17 Dec 2004 – Jan 2005, Nov 2005 4.3–65.3 0.6–9.8 Southern Ocean75 Mar-Apr 2008 1.0–18.0 1.6 ± 0.7 2.9 ± 2.1 Palmer Station, WAP40 Oct 2012- Mar 2013 0–20 0.3–9.3 Table 2. Summary of Antarctic DMSW concentrations and associated fluxes (range or mean ± s.d.) in different areas of the Antarctic sea-ice zone as identified in the literature. Table 2. Summary of Antarctic DMSW concentrations and associated fluxes (range or mean ± s.d.) in different areas of the Antarctic sea-ice zone as identified in the literature. DMSW and DMS flux from Ryder Bay and the wider WAP region. Discussionh Monthly mean DMSW (nmol L−1) for this study (open bars) as presented in Table 1, compared with the Lana et al.6 sea surface DMS climatology for both the WAP area (grey filled bars) and the entire Longhurst Austral Polar Province (APLR; solid bars), with error bars displaying the standard deviation. the L11 climatology, DMSW concentrations for the WAP are often lower than for the entire APLR, suggesting it is an area of low DMS production. However, mean January DMSW concentrations in Ryder Bay were three times higher (23.6 nmol L−1) than the L11-WAP data (7.8 nmol L−1), which resulted in annual mean surface DMSW concentrations in Ryder Bay higher than the WAP (3.7 nmol L−1 and 2.8 nmol L−1 respectively). When comparing the standard deviations of the data, our data reveal a much higher variation with on average a 43% variation over the year, compared to a 6% variation for the L11-WAP data. This suggests that the L11 climatology is not accu- rately predicting WAP summer DMSW production, and in particular is missing peak-DMS production events. This motivates improved data coverage along the WAP and further development of long-term DMS monitoring programs in order to further determine the causal factors that drive such peak events. S i d l l bl k DMS fl lik ‘ ’ h f 6 A Sea ice cover was assumed to completely block DMS flux like a ‘cap’ over the water surface6. As no attempt was made to quantify DMS flux from sea ice or slush snow layers on top of sea ice, quantification of DMS flux in this study will underestimate the total Antarctic DMS-derived sulfur flux. Gas transfer from the water column can occur through partial ice cover, particularly in coastal environments such as Ryder Bay where movement of ice between the bay and the greater Marguerite Bay area results from wind, water and tidal action. Development of summer shallow MLD upon ice breakup initially suggests limited exchange to the atmosphere of DMSW produced below the immediate surface layers (Fig. 2c), however spatially and temporally localized mixing processes such as ice movements, wave action, bubble entrainment and internal waves will support DMS release from deeper waters53–55.l Interseasonal and interannual variations in the flux of DMS were strongly pronounced but not always related to the DMSW concentrations. Discussionh In general, the highest DMSW concentrations were associated with the summer break-up of sea ice, with the majority of high concentrations observed when the area coverage of ice was estimated between 10 and 40% (Fig. 4). The relatively high DMSW concentration in February (Fig. 4) appeared to be an anomaly that may have been trig- gered by the December 2015 El Niňo. Similar coverage data during other months of the year did not resolve such high DMSW concentrations, indicating that factors other than ice coverage play a role. Simo and Dachs (2002) identified a significant global relationship between DMSW and Chlorophyll a50. Chlorophyll a is determined by fluorimetry as part of the RaTS program, and is used as a proxy for water column primary production in Ryder Bay. No relationship between Chlorophyll a and DMSW could be identified during the five years studied, nor was there a relationship with MLD-normalised chlorophyll a concentration, as also proposed by Simo & Dachs (2002; Supplementary material). Further investigation into the relationship between the phytoplankton commu- nity structure, other biogeochemical parameters and subsequent DMSP, DMS and DMSO concentrations are currently undertaken41,51. y To examine the DMSW concentrations from Ryder Bay in a wider context, monthly mean DMSW concentra- tions (Table 1) were compared to data from the Lana 2011 (L11) climatology of the WAP area and the Longhurst Austral Polar Province (APLR), which covers all data south of 59°S6,52. Strong seasonal differences were observed over both the WAP and the APLR, with maximum DMS concentrations in December (Fig. 5). Overall, within Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 5 www.nature.com/scientificreports/ Figure 4. Pooled DMSW concentrations (nmol L−1) over the 5-year time-series as a function of ice coverage (%) and time of year. Figure 4. Pooled DMSW concentrations (nmol L−1) over the 5-year time-series as a function of ice coverage (%) and time of year. Figure 5. Monthly mean DMSW (nmol L−1) for this study (open bars) as presented in Table 1, compared with the Lana et al.6 sea surface DMS climatology for both the WAP area (grey filled bars) and the entire Longhurst Austral Polar Province (APLR; solid bars), with error bars displaying the standard deviation. Figure 5. Discussionh Whereas spikes of high flux corresponded closely to the peaks in surface DMSW concentration, the additional drivers – SST, wind speed and fraction of open water – resulted in a poor rela- tionship between DMSW concentration and flux over the entire five-year data set (linear regression, r2 = 0.371, p < 0.01). More specifically, the short-term high-flux peaks in January of each year resulted in flux rates of DMS Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 6 www.nature.com/scientificreports/ that exceeded DMSW production, and resulted in the rapid decrease in DMSW concentrations the following day. Interannual variation is especially visible when comparing the 2016–2017 period with previous years, and it is suggested that connections between interannual modes of climate variability (including the El Niño/Southern Oscillation (ENSO) phenomenon and the Southern Annular Mode (SAM)) and the DMS concentrations and flux are present. Firstly, the El Niño event in 2015/2016 coincided with a period of sustained positive SAM and relatively prolonged period of medium-high DMS concentrations during summer. Secondly, an anomalously long period of fast-ice cover in Ryder Bay the following winter, together with a strong increase of SST when ice cover broke-up (Fig. 2a,b), correlated with a lack of DMSW spikes in summer 2017 (Fig. 3a). External forcings are known to be significant in driving interannual changes in ocean temperature and sea ice at the WAP, and the role of SAM and ENSO has been highlighted previously56–59. The observed persistence of sea ice cover in Marguerite Bay noted here (Fig. 2; see also Fig. 3d of Turner et al.34) conflicts with the rapid retreat of sea ice else- where around Antarctica34, but is a known consequence of the directionality of the wind and local topography. The higher SST in Ryder Bay during the summer of 2017 (Fig. 2a) is at least partly due to reduced winter mixing and strong freshwater injection to the upper ocean upon eventual breakup and melt of the ice. This would retain incident heat from insolation in shallower layers and increase surface temperature. This resulted in a stable water column and shallow MLD. As a result, DMS flux in 2017 was still calculated to be of the same order of magnitude as the preceding four summer seasons (Fig. Discussionh 3b), despite the absence of large spikes.hl p g ( g ) p g p The flux values from Ryder Bay exceed those presented from the area of Palmer Research further north on the WAP (~64°S) by at least one order of magnitude36,40, as well as from other sites around Antarctica (Table 2). Of critical importance within the annual cycle were the high spikes of flux in January, potentially related to sea-ice derived DMS production. Given the short timescales over which these spikes occurred, it is highly likely that they were detected in this study only because of the high resolution of this time series; other DMS flux determinations have been from research cruises which are limited in temporal resolution17,23,60,61, or from studies only measuring a single summer season during which a spike may not occur40. High DMS fluxes above the acknowledged nucle- ation threshold of 2.5 µmol m−2 d−1 were observed during 63% of the year, suggesting significant periods of CCN development44. Within the relatively unpolluted environment of the Antarctic, nucleation from the breakdown of DMS within the Marine Boundary Layer (MBL) will be the primary source of aerosol particles44,62–64. A previous study demonstrated tight coupling between MBL DMS and NSS-SO4 2−, particularly during periods of intense DMS flux63 such as we identified, suggesting that these spikes in DMS flux will further drive significant produc- tion of NSS-SO4 2- and subsequent particle nucleation. Given that high particle concentrations from nucleation events have been shown to be an important contributor to the global atmospheric aerosol budget64, our data provide evidence that the Antarctic coastal zone particle production has a significant impact on global particle concentrations. Our data also indicates that pulses of DMS flux are an intricate feature of Antarctic coastal areas, which can easily be missed when the frequency of data collection is insufficiently high. l l fl f d d h fl ll l f h y q yfi y g In calculating DMS flux from Ryder Bay, we assumed the flux represents well a large proportion of the Antarctic coastal zone, and, in particular, that of the Marguerite Bay area, as has been demonstrated previously26. Therefore, we have used the interannual and interseasonal assessment of DMS flux to calculate the total atmos- pheric sulfur input from the coastal areas of the WAP and wider Antarctic marginal ice zone (Table 2). Discussionh p p g ( ) Given the area and scale of the Antarctic coastal sea-ice zone and APLR, temporal and spatial availability of data on DMSW concentrations and flux are low, with high variation between available measurements. In the L11 climatology6, surface DMSW concentrations were only available for 8 months of the year, and therefore include no winter data. Using the average annual flux from the five-year time series, 1.1 million tonnes of S are released to the atmosphere from the APLR (9.2 × 106 km2) compared to 0.9 million tonnes from the 60–70 °S latitude band (1.9 × 107 km2) as calculated by L116. Normalising for area, a twofold higher sulfur flux was calculated as our annual mean flux compared to L11, showing that the latter is significantly underestimating the Antarctic contribution to atmospheric NSS-SO4 2− input. In addition, we highlight the south-western side of the WAP as a significant source of sulfur. gi Jarníková and Tortell (2016)14 utilised a larger DMSW dataset in the Southern Ocean, including a better parameterisation of DMSW and region-specific parameters, to generate a new Southern Ocean DMS summer cli- matology (J16). During December to February, when our mean DMS flux from Ryder Bay was 30 µmol m−2 d−1, J16 calculated western WAP DMS flux was below 10 µmol m−2 d−1 14. It has been previously discussed26 that the basin-scale methodology used in the J16 climatology does not account for large and dynamic fluctuations in the DMSW concentration and DMS flux shown in the Ryder Bay time series. It is apparent that the important pulses of DMS, leading to potential nucleation events and high particle production, are missed if relatively small data sets are averaged over longer time periods. We show that the data points capped in climatologies should be considered in the model calculations, as these are recurring features of the Antarctic coastal zone. In a global atmospheric circulation model, based on the existing DMS climatologies, it was established that despite variation in spatial and temporal production of DMS, the global mean radiative effect of sulfate is linearly proportional to the global mean surface flux of DMS44,65. It remains an open question what the radiative effect of the high spikes of DMS and subsequent flux as found in this study will be if incorporated into climate models. Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 Methods l Sample collection and analysis. Samples for DMS and physical parameters were collected 2–3 times per week throughout five summer seasons (January 2013 through to March 2017) from the primary Rothera Time Series (RaTS) site at 67.570°S 68.225°W in south-facing Ryder Bay (Fig. 1, 520 m depth), approximately 4 km to the west of Rothera Research Station. During the winter seasons of 2013, 2014 and 2016, samples were taken and stored for analysis in the following summer. In general, during summer months, samples were taken from the surface, 5 m and 15 m, however during the 2014–15 summer season and adjacent winters, no surface samples were taken. When algal biomass was very low in winter, only 15m-depth samples were taken.hl g y y p p The surface-water sample was taken from a small boat using an 80 mL plastic syringe with teflon tubing from approximately 5 cm depth; deeper samples were collected using an 8 L Niskin bottle and hand-winch. In case of 100% sea-ice coverage, samples were collected through an ice-hole from a sledge-mounted winch. Two samples from each depth were collected in 70 mL amber glass vials, using silicone tubing from the outlet of the Niskin bottle; vials were filled from the bottom and overflowed for at least twice the volume of the vial before the tubing was carefully removed. Vials were sealed with Teflon-lined screw caps, with care taken to avoid bubble formation, and were protected from light and temperature changes in a cooler filled with surface water. i Alongside discrete water sampling, continuous data collection for physico-chemical properties of the RaTS sampling site was undertaken. A Seabird 19+ conductivity, temperature and depth (CTD) instrument, combined with a WetLabs in-line fluorometer for Chlorophyll a fluorescence and LiCOR photosynthetically active radiation (PAR) sensor, was deployed in a single 500 m cast. Data was collected on the down-cast of the instrument, ensur- ing the shadow of the boat was not interfering with the irradiance data. The surface 1 m measurements were used to determine SST and salinity for flux calculations. The mixed layer depth (MLD) was calculated as the depth at which the density difference relative to the surface is 0.05 kg m−3, consistent with previous RaTS studies48. Full details of RaTS sampling is given in Clarke et al.49. Wind speed at Rothera was obtained from meteorological sensors on station67, and averaged over 24 hours. Methods l Sea ice cover in the bay was evaluated visually on a daily basis by observers on station: 100% sea ice cover was characterised by zero visible open water, and zero percent was no visible ice. Sea Ice trends have previously shown good agreement with wider-scale satellite-derived meth- ods46,48 Fractional ice cover in between the two extremes was comprised of occasional large ice sheets, smaller floes, ice bergs and brash ice. The ice cover would move throughout the bay during the day with wind and water movements. Samples were delivered to the laboratory at Rothera within two hours of collection and were stored at 2 °C in the dark. During summer, 50 µL of a known solution of 13C-DMS was added to each amber sample vial, in order to quantify DMS loss during further processing: 8 mL was taken from the vial and stored in a teflon-stoppered vial for quantification of the total amount of 13C-DMS added. The remaining contents (62 mL) of the amber vial were gravity filtered through a 47 mm Whatmann glass microfiber GF/F filter under dim light conditions, with the process stopped after approximately 20 mL had filtered, prior to the filter being exposed to the atmosphere. Two 8 mL replicates were collected in 20 mL glass, Teflon-stoppered vials for immediate analysis of DMS. DMS was injected into a Proton-Transfer-Reaction Time-Of-Flight mass spectrometer (PTR-TOF-MS 8000, Ionicon Analytik GmbH, Innsbruck, Austria) by purging at a flow rate of 150 ml min−1 through the sample within the vial. A full description of the PTR-TOF-MS method is described elsewhere26. The instrument was calibrated every 10 samples using fresh ~6 µM working DMS standard made in Milli-Q from which ~300 pmol DMS was injected. DMS standards were regularly cross-referenced with deuterated D3-DMSP standards. The instrument has a detection limit of 1–2 pmol. Final DMS concentrations were calculated after correction for the amount of 13C-DMS lost during filtration. i During the Austral winter (Apr–Nov) of 2013, 2014 and 2016, water samples were collected and stored for DMS + DMSP (total and dissolved), with no direct analysis of in-situ DMS. Two 70 mL amber glass vials were filled from a Niskin bottle typically collected from 15 m depth, but also from 5 m depth when CTD-fluorescence measurements indicated increased algal biomass. The sample was gravity filtered in a similar way as described for the summer samples. www.nature.com/scientificreports/ www.nature.com/scientificreports/ particle formation. December was the month of maximum DMS concentrations in the L11-climatology (Fig. 5), whereas our Ryder-Bay data showed a persistent January maximum associated with the marginal ice zone. The L11-December maximum was driven by a very strong DMS signal along the north and east of the WAP and around the South Orkney Islands6. Given the seasonal retreat of sea ice southward along the Peninsula, it can be expected that springtime sea ice-derived DMS production will commence earlier in more northerly latitudes of the APLR compared to the more southerly Peninsula, and spread south, reaching Ryder Bay in January for peak DMSW concentrations. W Variations in winter sea ice coverage, SST and MLD within Ryder Bay were drivers of spring and summer surface water conditions, following the patterns previously identified by Venables and Meredith (2014)66. Of particular interest during this study was the significant shift in ENSO and SAM modes of climate variability in late 2015, which resulted in changes in SST and ice formation around the WAP during winter 2016 and further into the following summer. External forcings such as these have previously been significant in driving interannual changes in SST and sea ice56,57,59, and we show that the resulting changes have the potential to effect production of DMSW. Further long-term measurements and analysis of both DMSW and DMS flux are essential to improve mechanistic understanding of the changes within the sulfur cycle during long-term climate variability and to understand the driving forces of DMS spikes that drive an important part of the total flux. Conclusion We present here the DMSW concentrations from the longest running time-series studying organic sulfur cycling in the world, and demonstrate the first ever evaluation of interseasonal and interannual variability in DMSW and associated flux from Antarctica. In situ concentrations of DMSW and derived DMS flux were compared to those extracted for the WAP from existing DMS climatologies, and were significantly higher than models predict during January, the most productive time of the annual cycle. Our data also indicate that DMS flux is above the nucleation threshold of 2.5 µmol m−2 d−1 for >60% of the time. Together with extremely high spikes of DMS flux during the Austral summer, this would result in significant production of NSS-SO4 2− and subsequent Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 7 Methods l A 10 mL subsample of the filtrate for determination of DMS plus the dissolved DMSP fraction (DMS(P)d) was collected in a 20 mL glass vial, a pellet of NaOH was added and the vial crimp sealed. Samples were stored at −20 °C and analysed during the following summer season. For these winter measure- ments, the assumption was made that the DMS + DMSPd concentration was the absolute upper limit of potential winter DMS water concentrations and is therefore likely to be an overestimation of DMS. Over the course of the Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 8 www.nature.com/scientificreports/ five summer seasons, DMS was on average 2.5 times higher in concentration than DMSPd, suggesting that the inclusion of DMSPd in the winter values will overestimate winter DMS by a mean of 40%. During the winter of 2016, loss of DMS during storage was tested: all samples received an addition of ~200 pmol D3-DMSP standard, before adding the NaOH pellet. Ten individual D3-DMSP working standards were prepared in March 2016, ana- lysed to provide a baseline concentration (t0) and stored in crimp-sealed vials. Every month during winter, a new standard vial was opened for addition to the samples and control samples prepared and stored (t1). Samples from all remaining standards were again prepared upon return to summer-sampling protocol in November 2016 and immediately analysed (tend). This analysis was compared with the t0 and t1 analyses, to quantify loss during use of the standard. All samples collected for DMS(P)d during winter were compared to the t0 and tend standards, and the concentration of each sample adjusted for the percentage recovery of D3-DMS. Mean percentage recovery across the entire winter was 93 ± 14%. Concentrations were averaged over the upper 15 m, with the non-constant depth intervals between samples accounted for in the averaging. This integrated concentration was divided by the depth, to give the mean DMS concentration and was assumed to be the concentration available at the surface for flux. This mean concentration was preferred to a direct measurement of DMS at the surface to remove spatial variability in surface values due to ice cover, and also to provide a best comparison with existing literature. The 15 m cut-off point was selected to provide the best comparison between summer and winter. DMS concentrations for non-sampling days were interpolated from existing values, assuming a linear relationship between concentrations on each sampled day. Data Availabilityh y The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Methods l Sea-Air flux of DMS. The DMS flux (FDMS) to the atmosphere (µmol m−2 d−1) was calculated for each day, using Equation (1): F k A (DMS )(1 ) (1) DMS DMS W 0 4 = − . (1) where DMSW is the concentration of DMS (nmol L−1) and A is the fraction sea-ice cover. Due to the nature of sea-ice, it was expected that even during periods of extreme ice cover, flux would still be occurring through leads and brine channels68, and therefore the minimum open water fraction was set to 0.0169. The scaling of 0.4 allows for the determination of the effect of sea-ice cover on the flux, and is provided by the work of Loose et al. (2009)54. kDMS is the gas transfer velocity (cm−3 hr−1) as based on the model of Nightingale et al.70, according to the method given in Simó and Dachs50: = . + . −. u u k (5 88 1 49 )(Sc) (2) DMS 2 0 5 (2) where u is the wind speed (m s−1) and Sc is the Schmidt number (cm2 sec −1), which is dependent upon sea sur- face temperature (t) and salinity as described by the model of Saltzman et al.71: where u is the wind speed (m s−1) and Sc is the Schmidt number (cm2 sec −1), which is dependent upon sea sur- face temperature (t) and salinity as described by the model of Saltzman et al.71: t t t Sc 2674 0 147 12 3 72 0 038 (3) 2 3 = . − . + . − . (3) References References 1. Stefels, J. Physiological aspects of the production and conversion of DMSP in marine algae and higher plants. J. Sea Res. 43, 183 (2000). 1. Stefels, J. Physiological aspects of the production and conversion of DMSP in marine algae and higher plants. J. Sea Res. 43, 183–197 (2000). 2. Stefels, J. & van Boekel, W. H. M. Production of DMS from dissolved DMSP in axenic cultures of the marine phytoplankton specie Phaeocystis sp. Mar. Ecol. Prog. Ser. 97, 11–18 (1993). 2. Stefels, J. & van Boekel, W. H. M. Production of DMS from dissolved DMSP in axenic cultures of the marine phytoplankton species Phaeocystis sp. Mar. Ecol. Prog. Ser. 97, 11–18 (1993). y p g ( ) 3. Simó, R. & Pedrós-Alió, C. Short-term variability in the open ocean cycle of dimethylsulfide. Global Biogeochem. Cycles 13 1173–1181 (1999). 4. Simó, R. & Pedrós-Alió, C. Role of vertical mixing in controlling the oceanic production of dimethyl sulphide. Nature 402, 396–398 (1999). 4. Simó, R. & Pedrós-Alió, C. Role of vertical mixing in controlling the oceanic production of dimethyl sulphide. Nature 402, 396–398 (1999). ( ) 5. Simó, R. Production of atmospheric sulfur by oceanic plankton: Biogeochemical, ecological and evolutionary links. Trends Ecol Evol. 16, 287–294 (2001).il ( ) 5. Simó, R. Production of atmospheric sulfur by oceanic plankton: Biogeochemical, ecological and evolutionary links. Trends Ecol. Evol. 16, 287–294 (2001).il , ( ) 6. Lana, A. et al. An updated climatology of surface dimethlysulfide concentrations and emission fluxes in the global ocean. Globa Biogeochem. Cycles 25, GB1004 (2011).h g y 7. Klimont, Z., Smith, S. J. & Cofala, J. The last decade of global anthropogenic sulfur dioxide: 2000–2011 emissions. Environ. Res. Lett. 8, 014003 (2013). g y 7. Klimont, Z., Smith, S. J. & Cofala, J. The last decade of global anthropogenic sulfur dioxide: 2000–2011 emissions. Environ. Res. Lett 8, 014003 (2013). ( ) 8. Charlson, R. J., Lovelock, J. E., Andreae, M. O. & Warren, S. G. Oceanic phytoplankton, atmospheric sulphur, cloud albedo and climate. Nature 326, 655–661 (1987).h 8. Charlson, R. J., Lovelock, J. E., Andreae, M. O. & Warren, S. G. Oceanic phytoplankton, atmospheric sulphur, cloud albedo and climate. Nature 326, 655–661 (1987).h 9. Quinn, P. K. & Bates, T. S. The case against climate regulation via oceanic phytoplankton sulphur emissions. Nature 480, 51–56 (2011). ( ) 10. Cameron-Smith, P., Elliott, S., Maltrud, M., Erickson, D. Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 16. del Valle, D. A., Kieber, D. J., Toole, D. A., Bisgrove, J. & Kiene, R. P. Dissolved DMSO production via biological and photochem oxidation of dissolved DMS in the Ross Sea, Antarctica. Deep Sea Res. Part A. Oceanogr. Res. Pap. 56, 166–177 (2009).i p g p 17. del Valle, D. A., Kieber, D. J., Toole, D. A., Brinkley, J. & Kiene, R. P. Biological consumption of dimethylsulfide (DMS) an importance in DMS dynamics in the Ross Sea, Antarctica. Limnol. Oceanogr. 54, 785–798 (2009). ll h l h b d h l lfid d h l k h p y g ( ) 18. DiTullio, R. & Smith, W. O. Relationship between dimethylsulfide and phytoplankton pigment concentrations in the Ross Sea, Antarctica. Deep Sea Res. Part I Oceanogr. Res. Pap. 32, 873–892 (1995).ii 19. Tison, J. L., Brabant, F., Dumont, I. & Stefels, J. High-resolution dimethyl sulfide and dimethylsulfoniopropionate time series profiles in decaying summer first-year sea ice at Ice Station Polarstern, western Weddell Sea, Antarctica. J. Geophys. Res. Biogeosciences 115, G04044 (2010). 0. Stefels, J. et al. The analysis of dimethylsulfide and dimethylsulfoniopropionate in sea ice: Dry-crushing and melting using stable isotope additions. Mar. Chem. 128–129, 34–43 (2012). p 21. Kirst, G. O. et al. Dimethylsulfoniopropionate (DMSP) in icealgae and its possible biological role. Mar. Chem. 35, 381–388 (1991). 22. Levasseur, M., Gosselin, M. & Michaud, S. A new source of dimethylsulfide (DMS) for the arctic atmosphere: ice diatoms. Mar. Biol. 121 381 387 (1994) p 1. Kirst, G. O. et al. Dimethylsulfoniopropionate (DMSP) in icealgae and its possible biological role. Mar. Chem. 35, 381–388 (1991). 2 Levasseur M Gosselin M & Michaud S A new source of dimethylsulfide (DMS) for the arctic atmosphere: ice diatoms Mar Biol p 21. Kirst, G. O. et al. Dimethylsulfoniopropionate (DMSP) in icealgae and its possible biological role. Mar. Chem. 35, 381–388 (1991).i , y p p ( ) g p g , ( ) 2. Levasseur, M., Gosselin, M. & Michaud, S. A new source of dimethylsulfide (DMS) for the arctic atmosphere: ice diatoms. Mar. Biol 121, 381–387 (1994).il 3. Curran, M. A. J. & Jones, G. B. Dimethyl sulfide in the Southern Ocean: Seasonality and flux. J. Geophys. Res. 105, 20451–20459 (2000). ( ) 24. Trevena, A. J. & Jones, G. B. Dimethylsulphide and dimethylsulphoniopropionate in Antarctic sea ice and their release during sea ice melting. Mar. Chem. www.nature.com/scientificreports/ 98, 210–222 (2006).h g 25. Thomas, D. N. & Dieckmann, G. S. Antarctic Sea ice–a habitat for extremophiles. Science (80-.). 295, 641–644 (2002).i h p 26. Stefels, J. et al. Impact of sea-ice melt on dimethyl sulfide (sulfoniopropionate) inventories in surface waters of Marguerite Bay, West- Antarctic Peninsula. Philos. Trans. A. Math. Phys. Eng. Sci. 376, 20170169 (2018).i y g 7. Zemmelink, H. J., Houghton, L., Dacey, J. W. H., Worby, A. P. & Liss, P. S. Emission of dimethylsulfide from Weddell Sea leads Geophys. Res. Lett. 32, L23610 (2005).i p y 28. Zemmelink, H. J., Dacey, J. W. H., Houghton, L., Hintsa, E. J. & Liss, P. S. Dimethylsulfide emissions over the multi-year ice of the western Weddell Sea. Geophys. Res. Lett. 35, L06603 (2008).h 29. Turner, J., Maksym, T., Phillips, T., Marshall, G. J. & Meredith, M. P. The impact of changes in sea ice advance on the large winter warming on the western Antarctic Peninsula. Int. J. Climatol. 33, 852–861 (2013). 30. Stammerjohn, S. E., Martinson, D. G., Smith, R. C. & Iannuzzi, R. A. Sea ice in the western Antarctic Peninsula region: Spatio- temporal variability from ecological and climate change perspectives. Deep Sea Res. Part II Top. Stud. Oceanogr. 55, 2041–2058 (2008). 1. Steig, E. J. et al. Recent climate and ice-sheet changes in West Antarctica compared with the past 2,000 years. Nat. Geosci. 6, 372–375 (2013). 32. Montes-Hugo, M. et al. Recent changes in phytoplankton communities associated with rapid regional climate change along the Western Antarctic Peninsula. Science (80-.). 323, 1470–1473 (2009). 32. Montes-Hugo, M. et al. Recent changes in phytoplankton com 33. Orr, A. et al. A ‘low-level’ explanation for the recent large warming trend over the western Antarctic Peninsula involving i d d h i l i l ti G ph R L tt 31 L06204 (2004) 33. Orr, A. et al. A ‘low-level’ explanation for the recent large warming trend over the western Antarctic Peninsula involving blocked winds and changes in zonal circulation. Geophys. Res. Lett. 31, L06204 (2004). g p y 34. Turner, J. et al. Unprecedented springtime retreat of Antarctic sea ice in 2016. Geophys. Res. Lett. 44, 6868–6875 (2017). kl l l h l h l d l 35. Ducklow, H. W. et al. Marine pelagic ecosystems: the West Antarctic Peninsula. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 362, (2007).i 6. Berresheim, H. et al. www.nature.com/scientificreports/ Measurements of dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, and aerosol ions at Palmer Station Antarctica. J. Geophys. Res. 103, 1629–1637 (1998). p y 37. Berresheim, H. Biogenic sulfur emissions from the Subantarctic and Antarctic Oceans. J. Geophys. Res. 92, 13245–13262 (1987).h 8. Turner, S. M., Nightingale, P. D., Broadgate, W. & Liss, P. S. The distribution of dimethyl sulphide and dimethylsulfoniopropionat in Antarctic waters and sea ice. Deep. Res. II 42, 1059–1080 (1995).i p 9. Herrmann, M. et al. Diagnostic modeling of dimethylsulfide production in coastal water west of the Antarctic Peninsula. Cont. Shel Res. 32, 96–109 (2012). ( ) 0. Asher, E. C., Dacey, J. W. H., Stukel, M., Long, M. C. & Tortell, P. D. Processes driving seasonal variability in DMS, DMSP, and DMSO concentrations and turnover in coastal Antarctic waters. Limnol. Oceanogr. 62, 104–124 (2017).h 40. Asher, E. C., Dacey, J. W. H., Stukel, M., Long, M. C. & Tortell, P. D. Processes driving seasonal variability in DMS, DMSP, and DMSO concentrations and turnover in coastal Antarctic waters. Limnol. Oceanogr. 62, 104–124 (2017). 41. Webb, A. L. et al. The dynamics of DMS, DMSP and DMSO on the West Antarctic Peninsula: temporal variability over 5 years in 1. Webb, A. L. et al. The dynamics of DMS, DMSP and DMSO on the West Antarctic Peninsula: temporal variability over 5 years in Ryder Bay. Prep y y p 2. Global Wind Atlas. Available at: https://globalwindatlas.info/.i y y p 42. Global Wind Atlas. Available at: https://globalwindatlas.info/.i g 43. Bell, T. G. et al. Air – sea dimethylsulfide (DMS) gas transfer in the North Atlantic: evidence for limited interfacial gas exchange at high wind speed. Atmos. Chem. Phys. 13, 11073–11087 (2013).hl g p y 4. Pandis, S. N., Russell, L. M. & Seinfeld, J. H. The relationship between DMS flux and CCN concentration in remote marine regions J. Geophys. Res. 99, 16945–16957 (1994).i J. Geophys. Res. 99, 16945–16957 (1994). 5. Meredith, M. P. et al. Changes in the freshwater composition of the upper ocean west of the Antarctic Peninsula during the firs p y 5. Meredith, M. P. et al. Changes in the freshwater composition of the upper ocean west of the Antarctic Peninsula during the firs decade of the 21st century. Prog. Oceanogr. 87, 127–143 (2010). y g g 46. Wallace, M. I. et al. References & Wingenter, O. Changes in dimethyl sulphide oceanic distribution due to climate change. Geophys. Res. Lett. 38, L07704 (2011).hi g p y 1. Gabric, A. J., Qu, B. O., Matrai, P. & Hirst, A. C. The simulated response of dimethylsulfide production in the Arctic Ocean to globa warming. Tellus B 57, 391–403 (2005).i g 2. Kloster, S. et al. Response of dimethylsulfide (DMS) in the ocean and atmosphere to global warming. J. Geophys. Res. Biogeoscience 112, G03005 (2007).h 13. Gondwe, M., Krol, M., Gieskes, W., Klaassen, W. & de Baar, H. The contribution of ocean-leaving DMS to the global atmospheric burdens of DMS, MSA, SO2, and NSS SO4-. Global Biogeochem. Cycles 17, 1056 (2003). g y 4. Jarníková, T. & Tortell, P. D. Towards a revised climatology of summertime dimethylsulfide concentrations and sea-air fluxes in the Southern Ocean. Environ. Chem. 13, 364–378 (2016).i g y 14. Jarníková, T. & Tortell, P. D. Towards a revised climatology of summertime dimethylsulfide concentrations and sea-air fluxes i Southern Ocean. Environ. Chem. 13, 364–378 (2016).i 14. Jarníková, T. & Tortell, P. D. Towards a revised climatology of summertime dimethylsulfide concentrations and sea air fluxes in the Southern Ocean. Environ. Chem. 13, 364–378 (2016). 15. Kettle, A. J. et al. A global database of sea surface dimethylsulfide (DMS) measurements and a procedure to predict sea surface DMS Southern Ocean. Environ. Chem. 13, 364–378 (2016). 15. Kettle, A. J. et al. A global database of sea surface dimethylsulfide (DMS) measurements and a procedure to predict sea surface DMS as a function of latitude longitude and month Global Biogeochem Cycles 13 399–444 (1999) Southern Ocean. Environ. Chem. 13, 364 378 (2016). 15. Kettle, A. J. et al. A global database of sea surface dimethylsulfide (DMS) measurements and a procedure to predict sea surface DMS as a function of latitude, longitude, and month. Global Biogeochem. Cycles 13, 399–444 (1999). ( ) 5. Kettle, A. J. et al. A global database of sea surface dimethylsulfide (DMS) measurements and a procedure to predict sea surface DMS as a function of latitude, longitude, and month. Global Biogeochem. Cycles 13, 399–444 (1999). Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 9 www.nature.com/scientificreports/ Shanklin, J., Moore, C. & Colwell, S. Meteorological observing and climate in the British Antarctic Territory and South Georgia: Par 2. Weather 64, 171–177 (2009).fl 68. Loose, B. & Schlosser, P. Sea ice and its effect on CO2 flux between the atmosphere and the Southern Ocean interior. J. Geophys. Res. Ocean. 116, C11019 (2011).hl 69. Legge, O. J. et al. The seasonal cycle of ocean-atmosphere CO2 flux in Ryder Bay, west Antarctic Peninsula. Geophys. Res. Lett. 42, 2934–2942 (2015). 0. Nightingale, P. D. et al. In situ evaluation of air-sea gas exchange parameterizations using novel conservative and volatile tracers Global Biogeochem. Cycles 14, 373–387 (2000).ffii g y 71. Saltzman, E. S., King, D. B., Holmen, K. & Leck, C. Experimental determination of the diffusion coefficient of dimethylsulf water. J. Geophys. Res. 98, 16481 (1993).l p y 72. Trevena, A. & Jones, G. DMS flux over the Antarctic sea ice zone. Mar. Chem. 134–135, 47–58 (2012). l 73. McTaggart, A. R. & Burton, H. Dimethyl sulphide concentrations in the surface waters of the Australasian Antarcti Subantarctic Oceans during an Austral Summer. J. Geophys. Res. Ocean. 97, 14407–14412 (1992).i 74. Kiene, R. P. et al. Distribution and cycling of dimethylsulfide, dimethylsulfoniopropionate, and dimethylsulfoxide during spring and early summer in the Southern Ocean south of New Zealand. Aquat. Sci. 69, 305–319 (2007).i y q ang, M., Blomquist, B. W., Fairall, C. W., Archer, S. D. & Huebert, B. J. Air-sea exchange of dimethylsulfide in the Southern Ocean: Measurements from SO GasEx compared to temperate and tropical regions. J. Geophys. Res. Ocean. 116, C00F05 (2011). 75. Yang, M., Blomquist, B. W., Fairall, C. W., Archer, S. D. & Huebert, B. J. Air-sea exchange of dimethylsulfide in the Southern O Measurements from SO GasEx compared to temperate and tropical regions J Geophys Res Ocean 116 C00F05 (2011) g, , q , , , , , , J g yi Measurements from SO GasEx compared to temperate and tropical regions. J. Geophys. Res. Ocean. 116, C00F05 (2011). Acknowledgements g Funding for this study was provided by the Netherlands Organisation for Scientific Research (NWO) under the Polar Program (NPP) Project Numbers 866.10.101 and 866.14.101. RaTS is funded by the Natural Environment Research Council via National Capability support to the British Antarctic Survey. We are grateful to the scientific and technical staff at the British Antarctic Survey Rothera Station, and, in particular, the Dutch Program winter research assistants Amber Annett, Mairi Fenton and Emily Davey. Author Contributions A.W. wrote the paper with J.S and M.v.L. giving significant contributions and editing. J.S., M.v.L., D.d.O. and A.W. undertook the summer sampling at Rothera station and analysed all DMSW samples collected both winter and summer. Analysis of the 5-year dataset was undertaken by A.W. who prepared all figures. H.J.V. provided data and insights from RaTS and the localised Ryder Bay area. M.M. provided details regarding interactions of ENSO and SAM and the likely shifts in environmental parameters along the WAP during and after these events. All authors reviewed the manuscript before submission. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 57. Meredith, M. P., Murphy, E. J., Hawker, E. J., King, J. C. & Wallace, M. I. On the interannual variability of ocean temperatures around South Georgia, Southern Ocean: forcing by El Nino/Southern Oscillation and the Southern Annular Mode. Deep. Res. 55, 2007–2022 (2008). ( ) 8. Stammerjohn, S. E., Martinson, D. G., Smith, R. C., Yuan, X. & Rind, D. Trends in Antarctic annual sea ice retreat and advance and their relation to El Niño–Southern Oscillation and Southern Annular Mode variability. J. Geophys. Res. 113, C03S90 (2008). their relation to El Niño–Southern Oscillation and Southern Annular Mode variability. J. Geophys. Res. 113, C03S90 (2008). 59. Turner, J. et al. Non-annular atmospheric circulation change induced by stratospheric ozone depletion and its role in the recent 9. Turner, J. et al. Non-annular atmospheric circulation change induced by stratospheric ozone depletion and its role in the recen increase of Antarctic sea ice extent. Geophys. Res. Lett. 36, 1–5 (2009).l p y ( ) 0. McTaggart, A. R., Burton, H. R. & Nguyen, B. C. Emission and flux of DMS from the Australian Antarctic and Subantarctic Ocean during the 1988/89 summer. J. Atmos. Chem. 20, 59–69 (1995). g 1. Staubes, R. & Georgii, H.-W. Biogenic sulfur compounds in seawater and the atmosphere of the Antarctic region. Tellus B 45B 127–137 (1993).ii 62. Davis, D. et al. Dimethyl sulfide oxidation in the equatorial Pacific: Comparison for DMS, SO2, H2SO4 (g), MSA (g), MS, and NSS. J. Geophys. Res. 104, 5765–5784 (1999). p y 63. Ayers, G. P., Ivey, J. P. & Gillett, R. W. Coherence between seasonal cycles of dimethyl sulphide, methanesulphonate and sulphate in marine air. Nature 349, 404–406 (1991).h 4. Spracklen, D. V. et al. The contribution of boundary layer nucleation events to total particle concentrations on regional and globa scales. Atmos. Chem. Phys. 6, 5631–5648 (2006). y ( ) 65. Tesdal, J.-E., Christian, J. R., Monahan, A. H. & von Salzen, K. Sensitivity of modelled sulfate radiative forcing to DMS concentration and air-sea flux formulation. Atmos. Chem. Phys. 16, 10847–10864 (2016).i l y 66. Venables, H. J. & Meredith, M. P. Feedbacks between ice cov l y 66. Venables, H. J. & Meredith, M. P. Feedbacks between ice cover, ocean stratification and heat content in Ryder Bay, Western Antarctic Peninsula. J. Geophys. Res. Ocean. 119, 5323–5336 (2014). Peninsula. J. Geophys. Res. Ocean. 119, 5323–5336 (2014). p y 7. www.nature.com/scientificreports/ On the characteristics of internal tides and coastal upwelling behaviour in Marguerite Bay, west Antarctic Peninsula. Deep Sea Res. Part II Top. Stud. Oceanogr. 55, 2023–2040 (2008). . Deep Sea Res. Part II Top. Stud. Oceanogr. 55, 2023–2040 (2008). p p g 7. Van Leeuwe, M. A. et al. Microalgal community structure and primary production in Arctic and Antarctic sea ice: Asynthesis. Elem Sci. Anthr. 6, 4 (2018).i 8. Venables, H. J., Clarke, A. & Meredith, M. P. Wintertime controls on summer stratification and productivity at the western Antarctic Peninsula. Limnol. Oceanogr. 58, 1035–1047 (2013).h g 49. Clarke, A., Meredith, M. P., Wallace, M. I., Brandon, M. A. & Thomas, D. N. Seasonal and interannual variability in temperature, chlorophyll and macronutrients in northern Marguerite Bay, Antarctica. Deep. Res. Part II Top. Stud. Oceanogr. 55, 1988–2006 (2008).i 50. Simó, R. & Dachs, J. Global ocean emission of dimethylsulfide predicted from biogeophysical data. Global Biogeochem. Cycles 16, 1078 (2002). 51. Leeuwe, M. van et al. Phytoplankton species succession in the Western Antarctic Peninsula explained by photophysiological characteristics. Prep p 52. Longhurst, A. R. Ecological Geography of the Sea. https://doi.org/10.1016/B978-012455521-1/50003-6 (Academic Press, 1998) g g g p y f p g 53. Loose, B. et al. Currents and convection cause enhanced gas exchange in the ice-water boundary layer. Tellus, Ser. B Chem. Phys. Meteorol. 68, 32083 (2016).f 4. Loose, B., McGillis, W. R., Schlosser, P., Perovich, D. & Takahashi, T. Effects of freezing, growth, and ice cover on gas transpor processes in laboratory seawater experiments. Geophys. Res. Lett. 36, L05603 (2009).i 55. Frew, N. M. et al. Air-sea gas transfer: Its dependence on wind stress, small-scale roughness, and surface films. J. Geophys. Res. 109, C08S17 (2004). 56. Meredith, M. P., Renfrew, I. A., Clarke, A., King, J. C. & Brandon, M. A. Impact of the 1997/98 ENSO on upper ocean characteristics in Marguerite Bay, western Antarctic Peninsula. J. Gen. Microbiol. 109, C09013 (2004). Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4 10 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-38714-4 Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 11 Scientific Reports \| (2019) 9:2233 \| https://doi.org/10.1038/s41598-019-38714-4
https://openalex.org/W4284897379	https://www.researchsquare.com/article/rs-1765444/latest.pdf	English	null	Transformation of amides to thioamides using efficient and novel thiating reagent	Research Square (Research Square)	2,022	cc-by	4,899	Transformation of amides to thioami efficient and novel thiating reagent Walid Fathalla Port-Said University Gaber El Anany Port-Said University Ibrahim A. I. Ali Suez Canal University Samir El Rayes (  samir_elrayes@yahoo.com ) Suez Canal University Research Article Keywords: License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/11 Introduction Amides and their isosteres thioamides are very important biologically active compounds. [1] Benzothioamides are used as building blocks for the preparation of various compounds specially those containing sulfur.[2–4] Many methods have been reported for the synthesis of substituted thiobenzamides from various substrates and reagents. Typically, Beckmann rearrangement was applied to prepare thioamides using ketoxime substrates in the presence of PSCl3 [5], in situ generated Appel’s salt, Mitsunobu’s zwitterionic adduct [6] or in the presence of O,O-diethyl dithiophosphoric acid [7] as the dehydrating agents. Another interesting method for the introduction of thioamides is the modified Willgerodt–Kindler reaction using a three-component reactions involving aniline, aldehydes, and elemental sulfur powder in the presence of sulfated tungstate catalyst as acidic catalyst [8], Na2S as a basic catalyst [9], Sulfonic acid functionalized nano γ-Al2O3 [10] or in the absence of a catalyst [11, 12]. Other method was carried out using thioacyl dithiophosphates used for thioacylation of amines to afford thioamides and water soluble ammonium monothiophosphates [13, 14]. Other methods involve the N- substituted amide-thioamide transformation via thionation reagents such as Lawesson's reagent (2,4- bis(4-ethoxyphenyl)-1,3-dithia-2,4-diphosphetane 2,4-disulfide) [15] and Berzelius reagent [16] (P4S10), in dry; toluene, xylene or pyridine under reflux condition. Some of these protocols are very interesting showing high yields but still have major drawbacks harsh reaction conditions, prolonged reaction times and expensive specific reagents, ultra-dry solvents and bad smell have limited their applications. New protocol for the thioamide preparation is highly appreciable. Our group developed the heterocyclic benzamide-thioamide transformation by chlorination then reaction with N-cyclohexyl dithiocarbamate cyclohexyl ammonium salt in chloroform for 12 h. at 61oC to afford heteocyclic thioamides in excellent yields [17–20]. As a part of our continuous efforts towards the improvement of synthetic methods we now report highly efficient protocol for the open-chain amide-thioamide transformation using two steps one-pot sequential reactions: first chlorination of benzanilides, second the reaction with an efficient and novel thiating reagent; N-isopropyldithiocarbamate isopropyl ammonium salt in acetonitrile for 1h. This reagent is prepared from simple commercial substrates; isopropyl amine and carbon disulfide. Abstract Convenient protocol was developed for the transformation of N-aryl substituted benzamides to N-aryl substituted benzothioamides using N-isopropyldithiocarbamate isopropyl ammonium salt as a novel thiating reagent. The major advantages of this protocol are one-pot procedure, short reaction times, mild conditions, simple work up, high yields and pure products. Discussion N-cyclohexyl dithiocarbamate cyclohexyl ammonium salt 2 proved to be an interesting thiating reagent used in the preparation of a number of heterocyclic thioamides [17–20]. The thiating reagent N- Page 2/11 Page 2/11 cyclohexyl dithiocarbamate cyclohexyl ammonium salt 2 was simply prepared from cyclohexyl amine reaction with carbon disulfide in water at room temperature for 2h., Scheme 1. [17] Herein, we wish to examine the application of this thiating reagent to involve open chain amide-thioamide transformation. Thus, the reaction of benzoyl chloride 3 with p-toluidine 4 in benzene in the presence of triethyl amine for 6h. afforded the model precursor N-(p-tolyl)benzamide (5). The reaction of N-(p- tolyl)benzamide (5) with thionyl chloride at 70 oC for 8h. gave N-(p-tolyl)benzimidoyl chloride (6), thionyl chloride was then evaporated under reduced pressure. The imidoyl chloride 6 was kept under reduced pressure (6.7 KPa) at 120oC for 2 h. and was used in insitu without further purification [21–23]. Finally, the insitu generated acetonitrile solution of imidoyl chloride 6 was refluxed with 2 to afford a mixture of the desired compound N-(p-tolyl)benzothioamide (7) cyclohexylcarbamothioic-N-(p-tolyl)benzimidic thioanhydride (8), Scheme 2. This behavior is most probably due steric hindrance of cyclohexyl moiety, which tangle the cyclohexyl amine proton abstraction by the imine nitrogen atom. Consequently, out of necessity we should modify the structure of the thiating reagent. Recently, we reported an interesting method for the preparation of 2- arylquinazolin-4-amines by the reaction of N-(2-cyanophenyl)substituted benzimidoyl isothiocyanates with isopropyl amine through 1-isopropyl-3-(2-(4-substitutedphenyl)-quinazolin-4-yl)thiourea. [24] Due to the previous facts, we designed N-isopropyldithiocarbamate isopropyl ammonium salt 10 as our novel thiating reagent to solve the mentioned discrete problem. The reaction of three molar equivalents of isopropylamine 9 with one molar equivalent of carbon disulfide in ethyl acetate for 2 h. precipitated pure N-isopropyldithiocarbamate isopropyl ammonium salt 10 in high yield, Scheme 3. Compound 10 is hygroscopic and should be tightly packed. Indeed, our hypothesis related to applying N-isopropyldithiocarbamate isopropyl ammonium salt 10 as a novel thiating reagent gave remarkable results. Thus, N-aryl substituted benzamides 11–13(a-e) were prepared by the reaction of carboxylic acid chlorides with anilines as described earlier. N-Aryl substituted benzamides 11–13(a-e) were chlorinated with thionyl chloride at 70 oC for 8h. and the acetonitrile solution of the insitu generated benzimidoyl chloride 14–16(a-e) were treated with N- isopropyldithiocarbamate isopropyl ammonium salt (10) at room temperature for 1h. (TLC monitored). Conclusion In this work, we successfully developed a facile and convenient general method for the conversion of N- aryl substituted benzamide to N-aryl substituted benzothioamide. This protocol consists of two steps in a one-pot strategy: first, we transformed N-aryl substituted benzamide to benzimidoyl chloride derivatives by the reaction with thionyl chloride. Second, the benzimidoyl chloride reacted with N- isopropyldithiocarbamate isopropyl ammonium salt as a novel thiating reagent to finally produce N-aryl substituted benzothioamide. This method have the advantage of applying the one-pot strategy for the amide-thioamide transformations beside the operational simplicity, availability of substrates, reaction at room temperature and high yields in short reaction time. In this work, we successfully developed a facile and convenient general method for the conversion of N- aryl substituted benzamide to N-aryl substituted benzothioamide. This protocol consists of two steps in a one-pot strategy: first, we transformed N-aryl substituted benzamide to benzimidoyl chloride derivatives by the reaction with thionyl chloride. Second, the benzimidoyl chloride reacted with N- isopropyldithiocarbamate isopropyl ammonium salt as a novel thiating reagent to finally produce N-aryl isopropyldithiocarbamate isopropyl ammonium salt as a novel thiating reagent to finally produce N-aryl substituted benzothioamide. This method have the advantage of applying the one-pot strategy for the amide-thioamide transformations beside the operational simplicity, availability of substrates, reaction at room temperature and high yields in short reaction time. Discussion The reaction mixture was evaporated and ethanol was added to give bright yellow crystals as only isolated products identified as N-aryl substituted benzothioamides 17–19(a-e). Scheme 4. A total number of 15 N-aryl substituted benzothioamides 17–19(a-e) were obtained from N-aryl substituted benzamides 11–13(a-e) in a one-pot strategy and in excellent yields. This synthetic procedure using N-isopropyldithiocarbamate isopropyl ammonium salt 10 as the thiating reagent beside solving all previously mentioned problems have the advantage of applying the one-pot strategy for the amide- thioamide transformations beside the operational simplicity, availability of substrates, reaction at room temperature and high yields in short reaction time. Page 3/11 Page 3/11 A rational mechanism is described for this interesting protocol is given in Scheme 5. The reaction of N-(p- tolyl)benzamide (11b) with thionyl chloride principally afforded N-(p-tolyl)benzimidoyl chloride (14b). The insitu generated 14b solution in acetonitrile reacted with N-isopropyldithiocarbamate isopropyl ammonium salt (10) to principally afford (E)-N-(p-tolyl)benzimidic thioanhydride I as the most stable geometric isomer. Earlier results obtained by our group concerning similar structures showed that the reaction of N-(2-chloro-5-nitrophenyl)benzimidoyl isothiocyanate with tert-butyl amine afforded principally the intermediate (E)-N-(tert-butyl)carbamimidic-N-(2-chloro-5-nitrophenyl)benzimidic thioanhydride which subsequently converted to the stable bis-[N-(2-chloro-5-nitrophenyl)benzimidoyl] sulfide as Z-geometric isomer (structure assignment of this compound was corroborated by X-ray crystallographic analysis). [25] The tolyl residue directly attached to the imine nitrogen pushes electrons to it which enhances hydrogen bond acceptor character for this nitrogen atom of imine. This behavior cause the nitrogen atom of the imine group able to abstract isopropyl amine NH proton and the residual electrons will attack the thiocarbonyl group with the consequent cleavage of S-C bond and finally give our desired product 17b and isopropyl isothiocyanate, Scheme 5. A similar result was obtained by our group for the rearrangement of 2-phenylquinazolin-4-yl cyclohexylcarbamodithioate to finally produce 2- phenylquinazoline-4(3H)-thione in the presence or absence of a base. [17] Also, similar explanation was given by our group for the 2-arylquinazolin-4-amines from 1-isopropyl-3-(2-(4-substitutedphenyl)- quinazolin-4-yl)thiourea in the presence of isopropyl amine. [24] General method for the preparation of thiating reagent N-cyclohexyl dithiocarbamate cyclohexyl ammonium salt (2). To a mixture of freshly distilled cyclohexyl amine (60 mmol) and water (50 mL) was added carbon disulfide (21 mmol) dropwise. The reaction mixture was stirred at room temperature for 2h. The white solid obtained was filtered, washed with water, dried and crystalized from ethanol to provide pure product of cyclohexyl amine cyclohexyl ammonium dithiocarbamate provided the pure compound. Yield 98% (ethanol 95%) white crystals, mp 188–189°C. 1H NMR spectrum, (400 MHz, DMSO), δ, ppm (J, Hz): 8.01 (3H, bs, 3NH); 4.15–3.95 (1H, m, CH); 3.05–2.96 (1H, m, CH); 1.98–0.96 (20H, m, 10CH2). 13C NMR spectrum, (100.0 MHz, DMSO), δ, ppm: 212.4 (C = S); 55.3 (CH); 50.0 (CH); 32.3 (2CH2); 30.9 (2CH2); 25.8 (CH2); 25.5 (2CH2); 25.1 (CH2); 24.3 (2CH2). Found, %: C, 56.88; H, 9.55; N, 10.21. For C13H26N2S2 (274.2). Calculated, %: C, 56.56; H, 9.43; N, 10.09. Experimental General procedures. Solvent were purified and dried by standard procedures. The boiling range of the petroleum ether used was 40–60 oC. Thin layer chromatography (TLC): silica gel 60 F254 plastic plates (E. Merck, layer thickness 0.2 mm) detected by UV absorption. Elemental analyses were performed on a Flash EA-1112 instrument at the Microanalytical laboratory, Faculty of Science, Suez Canal University, Ismailia, Egypt. Melting points were determined on a Buchi 510 melting-point apparatus and the values are uncorrected. 1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz, respectively (Bruker AC 400) in CDCl3 and DMSO solution with tetramethylsilane as an internal standard. The NMR analyses were performed at Faculty of Science, Sohag University. Page 4/11 Page 4/11 Preparation of cyclohexylcarbamothioic- N -(p -tolyl)benzimidic thioanhydride (8). A mixture of N-(p-tolyl)benzoamide (5) (2.5 mmol) and thionyl chloride (5 mL) were heated at 70oC for 8 h. The thionyl chloride was removed under reduced pressure and was heated at 120oC under reduced pressure 50 mmHg for 2 extra hrs. to give a yellowish clear colored oil of benzimidoyl chloride 6, which was not further purified and was used directly in the next step. To a solution of benzimidoyl chloride 6 (2.5 mmol) in acetonitrile (10 mL) was added (0.69 g, 2.5 mmol) of N-cyclohexyl dithiocarbamate cyclohexyl ammonium salt (2). The reaction mixture was stirred at room temperature for 1 h. (TLC monitored), then heated for 2h.. The reaction mixture was evaporated under reduced pressure and 25 mL of ethanol was added to the solid residue. The yellowish precipitate was filtered to give 8. The crude compound was purified by crsystalization from ethyl alcohol 95%. Yield 84% yellow crystals, mp 138–139°C. 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 7.89 (2H, d, J = 8.0, ArH); 7.82 (1H, bs, NH); 7.55–7.48 (5H, m, ArH); 7.19 (2H, d, J = 8.0, ArH); 3.87–3.67 (1H, m, CH); 2.36 (3H, s, CH3); 2.06–1.59 (4H, m, 2CH2); 1.44–1.20 (6H, m, 3CH2). Found, %: C, 68.36; H, 6.48; N, 7.46. For C21H24N2S2 (368.6). Calculated, %: C, 68.44; H, 6.56; N, 7.60; S, 17.40. General method for the preparation of thiating reagent N-isopropyldithiocarbamate isopropyl ammonium salt (10). To a mixture of isopropyl amine (60 mmol) and ethyl acetate (50 mL) was added carbon disulfide (21 mmol) dropwise. The reaction mixture was stirred at room temperature for 2h. The white solid obtained was filtered, washed with ethyl acetate, dried and was packed tightly and was pure enough for further reactions. Yield 95% (acetonitrile) white crystals, mp 85–86°C. 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 8.19 (1H, bs, NH); 6.06–5.75 (3H, bs, 3NH); 3.88–3.75 (1H, m, CH); 3.72–3.51 (1H, m, CH); 1.43–1.08 Page 5/11 Page 5/11 (12H, m, 4CH3). 13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 211.8 (C = S); 52.7 (CH); 48.3 (CH); 24.5 (2CH3); 22.3 (2CH3). Found, %: C, 43.17; H, 9.23; N, 14.38. For C7H18N2S2 (194.4). Calculated, %: C, 43.26; H, 9.34; N, 14.41; S, 32.99. N -Phenylbenzothioamide (17a). Yield 89% (ethanol 95%) yellow crystals, mp 100–101°C (lit. [26] 102°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 9.06 (1H, bs, NH); 8.20–7.80 (4H, m, ArH); 7.64–7.29 (6H, m, ArH).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 197.5 (C = S), 143.5, 139.6, 130.2, 129.3, 128.9, 127.6, 125.8, 124.7. Found, %: C, 73.15; H, 5.17; N, 6.53. For C13H11NS (213.3). Calculated, %: C, 73.20; H, 5.20; N, 6.57; S, 15.03. General method for the preparation of N-aryl substituted benzthioamide 17–19(a-e) A mixture of N-aryl substituted benzamide 11–13(a-e) (2.5 mmol) and thionyl chloride (5 mL) were heated at 70oC for 8 h. The thionyl chloride was removed under reduced pressure and was heated at 120oC under reduced pressure 50 mmHg for 2 extra hrs. to give a yellowish clear colored oil of benzimidoyl chloride 14–16(a-e), which was not further purified and was used directly in the next step. To a solution of benzimidoyl chloride 14–16(a-e) (2.5 mmol) in acetonitrile (10 mL) was added (0.49 g, 2.5 mmol) of N-isopropyldithiocarbamate isopropyl ammonium salt (10). The reaction mixture was stirred at room temperature for 1 h. (TLC monitored). The reaction mixture was evaporated under reduced pressure and 25 mL of ethanol was added to the solid residue. The yellowish precipitate was filtered to give the desired N-aryl substituted benzothioamides 17–19(a-e). The crude compounds were pure enough for analytical purposes. Purification of products for analysis was achieved by crystallization from the appropriate solvent. N -(p -Tolyl)benzothioamide (17b). Yield 93% (ethanol 95%) yellow crystals, mp 126–127°C (lit. [8] 130°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 8.99 (1H, bs, NH); 7.88–7.77 (2H, m, ArH); 7.66 (2H, d, J = 8.0, ArH); 7.51 (2H, d, J = 8.0, ArH); 7.42–7.39 (3H, m, ArH); 2.37 (3H, s, CH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 198.8 (C = S), 143.7, 136.7, 135.1, 130.6, 129.9, 128.4, 127.6, 125.9, 124.3, 21.7 (CH3). Found, %: C, 73.93; H, 5.74; N, 6.09. For C14H13NS (227.3). Calculated, %: C, 73.97; H, 5.76; N, 6.16; S, 14.10. 4-Methoxy- N -(p -tolyl)benzothioamide (18b). Yield 93% (ethanol 95%) yellow crystals, mp 172–173°C (lit. [28] 174°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 7.85 (2H, d, J = 6.0, ArH); 7.73 (1H, bs, NH); 7.52 (2H, d, J = 6.0, ArH); 7.19 (2H, d, J = 6.0, ArH); 6.99 (2H, d, J = 6.0, ArH); 3.73 (3H, s, OCH3); 2.36 (3H, s, CH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 200.5 (C = S), 158.7, 136.6, 135.8, 134.1, 129.9, 127.4 124.7, 113.8, 55.7 (OCH3), 21.5 (CH3). Found, %: C, 69.94; H, 5.76; N, 5.38. For C15H15NOS (257.4). Calculated, %: C, 70.01; H, 5.88; N, 5.44; S, 12.46. N -(4-Methoxyphenyl)benzothioamide (17e). Yield 96% (ethanol 95%) yellow crystals, mp 130–131°C (lit. [26] 127°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 8.89 (1H, bs, NH); 7.87–7.76 (2H, m, ArH); 7.54 (2H, d, J = 8.0, ArH); 7.53–7.42 (3H, m, ArH); 6.88 (2H, d, J = 8.0, ArH); 3.73 (3H, s, OCH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 199.6 (C = S), 158.6, 142.8, 131.4, 130.2, 128.3, 127.5, 125.3, 113.8, 55.3 (OCH3). Found, %: C, 69.03; H, 5.36; N, 5.72. For C14H13NOS (243.3). Calculated, %: C, 69.11; H, 5.39; N, 5.76; S, 13.18. N -(o -Tolyl)benzothioamide (17d). Yield 84% (ethanol 95%) yellow crystals, mp 148–150°C (lit. [26] 151°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 7.86 (1H, d, J = 6.0, ArH); 7.81 (2H, d, J = 6.0, ArH); 7.59 (1H, bs, NH); 7.59–7.41 (3H, m, ArH); 7.22–7.15 (2H, m, ArH); 7.13 (1H, t, J = 6.0, ArH) 2.27 (3H, s, CH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 198.2 (C = S), 142.9, 139.1, 133.6, 131.8, 131.3, 128.9,126.0, 125.6,125.1, 18.4 (CH3). Found, %: C, 73.85; H, 5.70; N, 6.12. For C14H13NS (227.3). Calculated, %: C, 73.97; H, 5.76; N, 6.16; S, 14.10. 4-Methoxy- N -phenylbenzothioamide (18a). Yield 82% (ethanol 95%) yellow crystals, mp 145–146°C (lit. [8] 150°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 7.86 (2H, d, J = 6.0, ArH); 7.71 (1H, bs, NH); 7.45–7.36 (2H, m, ArH); 7.34–7.29 (3H, m, ArH); 6.93 (2H, d, J = 6.0, ArH); 3.82 (3H, s, OCH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 199.4 (C = S), 158.4, 139.1, 134.5, 129.0, 127.2, 126.9, 123.4, 114.1, 55.5 (OCH3). Found, %: C, 69.09; H, 5.38; N, 5.72. For C14H13NOS (243.3). Calculated, %: C, 69.11; H, 5.39; N, 5.76; S, 13.18. N -(m -Tolyl)benzothioamide (17c). Yield 76% (ethanol 95%) yellow crystals, mp 87–88°C (lit. [27] 81°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 9.00 (1H, bs, NH); 7.88–7.72 (2H, m, ArH); 7.67–7.54 (2H, m, ArH); 7.46–7.36 (3H, m, ArH); 7.31–7.18 (2H, m, ArH); 2.42 (3H, s, CH3). Found, %: C, 73.89; H, 5.72; N, 6.11. For C14H13NS (227.3). Calculated, %: C, 73.97; H, 5.76; N, 6.16; S, 14.10. Page 6/11 Page 6/11 4-Methoxy- N -(o -tolyl)benzothioamide (18d). Yield 94% (ethanol 95%) yellow crystals, mp 125–126°C (lit. [29] 119°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 7.94 (1H, d, J = 6.0, ArH); 7.88 (2H, d, J = 6.0, ArH); 7.64 (1H, bs, NH); 7.29–7.24 (2H, m, ArH); 7.13 (1H, t, J = 6.0, ArH); 7.00 (2H, d, J = 6.0, ArH); 3.98 (3H, s, OCH3); 2.36 (3H, s, CH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 199.1 (C = S), 159.3, 139.2, 136.5, 134.3, 133.6, 130.1, 127.4, 126.4, 125.6, 113.9, 55.5 (OCH3), 18.9 (CH3). Found, %: C, 69.92; H, 5.79; N, 5.41. For C15H15NOS (257.4). Calculated, %: C, 70.01; H, 5.88; N, 5.44; S, 12.46. 4-Chloro- N -phenylbenzothioamide (19a). Yield 92% (ethanol 95%) yellow crystals, mp 157–158°C (lit. [30] 153°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 9.03 (1H, bs, NH); 7.67 (2H, d, J = 6.0, ArH); 7.45–7.36 (4H, m, ArH); 7.34–7.29 (3H, m, ArH).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 198.6 (C = S), 140.3, 139.8, 135.3, 129.0, 128.5, 127.9, 127.3, 123.6. Found, %: C, 62.89; H, 4.05; N, 5.61. For C13H10ClNS (247.7). Calculated, %: C, 63.03; H, 4.07; Cl, 14.31; N, 5.65; S, 12.94. 4-Methoxy- N -(4-methoxyphenyl)benzothioamide (18e). Yield 90% (ethanol 95%) yellow crystals, mp 149–150°C (lit. [8] 152°C). 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 7.75 (2H, d, J = 6.0, ArH); 7.61 (1H, bs, NH); 7.44 (2H, d, J = 6.0, ArH); 6.89 (2H, d, J = 6.0, ArH); 6.82 (2H, d, J = 6.0, ArH); 3.82 (3H, s, OCH3); 3.74 (3H, s, OCH3). 13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 202.1 (C = S), 159.7, 157.6, 134.1, 131.3 127.8, 125.6, 114.4, 113.7, 55.6 (OCH3), 55.2 (OCH3). Found, %: C, 65.88; H, 5.51; N, 5.04. For C15H15NO2S (273.4). Calculated, %: C, 65.91; H, 5.53; N, 5.12; S, 11.73. 4-Methoxy N-(m-tolyl)benzothioamide (18c). Yield 86% (ethanol 95%) yellow crystals, mp 132–133°C. 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 7.82 (2H, d, J = 6.0, ArH); 7.67 (1H, bs, NH); 7.62–7.54 (2H, m, ArH); 7.31–7.11 (2H, m, ArH); 6.93 (2H, d, J = 6.0, ArH); 3.86 (3H, s, OCH3); 2.39 (3H, s, CH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 200.2 Page 7/11 Page 7/11 (C = S), 158.5, 138.9, 136.3, 135.4, 134.4, 129.8, 127.6, 125.9, 124.5, 114.6, 21.2 (CH3). Found, %: C, 69.87; H, 5.74; N, 5.36. For C15H15NOS (257.4). Calculated, %: C, 70.01; H, 5.88; N, 5.44; S, 12.46. (C = S), 158.5, 138.9, 136.3, 135.4, 134.4, 129.8, 127.6, 125.9, 124.5, 114.6, 21.2 (CH3). Found, %: C, 69.87; H, 5.74; N, 5.36. For C15H15NOS (257.4). Calculated, %: C, 70.01; H, 5.88; N, 5.44; S, 12.46. 4-Chloro- N -(p -tolyl)benzothioamide (19b). Yield 96% (ethanol 95%) yellow crystals, mp 150–151°C. 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 9.03 (1H, bs, NH); 7.71 (2H, d, J = 6.0, ArH); 7.52 (2H, d, J = 6.0, ArH); 7.42 (2H, d, J = 6.0, ArH); 7.19 (2H, d, J = 6.0, ArH); 2.37 (3H, s, CH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 199.5 (C = S), 141.2, 136.3, 135.6, 134.7, 129.2, 128.9, 127.6, 124.5, 21.3 (CH3). Found, %: C, 64.14; H, 4.59; N, 5.28. For C14H12ClNS (261.8). Calculated, %: C, 64.24; H, 4.62; Cl, 13.54; N, 5.35; S, 12.25. 4-Chloro- N -(o -tolyl)benzothioamide (19d). Yield 73% (ethanol 95%) yellow crystals, mp 129–130°C. 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 9.03 (1H, bs, NH); 7.94 (1H, d, J = 6.0, ArH); 7.67 (2H, d, J = 6.0, ArH); 7.39 (2H, d, J = 6.0, ArH); 7.31– 7.24 (2H, m, ArH); 7.16 (1H, t, J = 6.0, ArH); 2.36 (3H, s, CH3). 13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 199.3 (C = S), 140.5, 139.4, 136.5, 134.5, 133.8, 130.5, 128.8, 127.9, 127.3, 125.6, 18.5 (CH3). Found, %: C, 64.12; H, 4.48; N, 5.31. For C14H12ClNS (261.8). Calculated, %: C, 64.24; H, 4.62; Cl, 13.54; N, 5.35; S, 12.25. 4-Chloro- N -(m -tolyl)benzothioamide (19c). Yield 98% (ethanol 95%) yellow crystals, mp 136–137°C. 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 9.01 (1H, bs, NH); 7.67–7.54 (4H, m, ArH); 7.39 (2H, d, J = 6.0, ArH); 5.31–7.13 (2H, m, ArH); 2.36 (3H, Page 8/11 Page 8/11 s, CH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 199.6 (C = S), 141.5, 138.4, 136.7, 135.6, 134.9, 129.6, 129.0, 127.2, 125.8, 123.4. 21.5 (CH3). Found, %: C, 64.18; H, 4.54; N, 5.29. For C14H12ClNS (261.8). Calculated, %: C, 64.24; H, 4.62; Cl, 13.54; N, 5.35; S, 12.25. s, CH3).13C NMR spectrum, (100.0 MHz, CDCl3), δ, ppm: 199.6 (C = S), 141.5, 138.4, 136.7, 135.6, 134.9, 129.6, 129.0, 127.2, 125.8, 123.4. 21.5 (CH3). Found, %: C, 64.18; H, 4.54; N, 5.29. For C14H12ClNS (261.8). Calculated, %: C, 64.24; H, 4.62; Cl, 13.54; N, 5.35; S, 12.25. 4-Chloro- N -(4-methoxyphenyl)benzothioamide (19e). Yield 85% (ethanol 95%) yellow crystals, mp 166–167°C (lit. [31] 172°C).. 1H NMR spectrum, (400 MHz, CDCl3), δ, ppm (J, Hz): 8.89 (1H, bs, NH); 7.82 (2H, d, J = 6.0, ArH); 7.64 (2H, d, J = 6.0, ArH); 7.36 (2H, d, J = 6.0, ArH); 6.91 (2H, d, J = 6.0, ArH); 3.75 (3H, s, OCH3).13C NMR spectrum, (100.0 MHz, DMSO), δ, ppm: 199.8 (C = S), 158.9, 140.4, 134.2, 131.9, 128.6, 127.6, 126.3, 114.5, 55.4 (OCH3). Found, %: C, 60.49; H, 4.32; N, 4.87. For C14H12NClNOS (277.8). Calculated, %: C, 60.54; H, 4.35; Cl, 12.76; N, 5.04; S, 11.54. Ethics approval and consent to participate Not applicable Availability of data and materials All data are already presented in details along the manuscript in each section and title has its corresponding data Competing interests The authors declare that they have no competing interests Page 9/11 Acknowledgements Not applicable Authors' contributions aber, Ibrahim and Samir substantial contribution to conception and design Walid, Gaber, Ibrahim and Samir substantial contribution to conception and design Walid, Ibrahim and Samir substantial contribution to acquisition of data Walid, Gaber, Ibrahim and Samir substantial contribution to analysis and interpretation of data Walid, Gaber, Ibrahim and Samir drafting the article Walid, Gaber, Ibrahim and Samir substantial contribution to analysis and interpretation of data Walid, Gaber, Ibrahim and Samir drafting the article Walid, Gaber, Ibrahim and Samir substantial contribution to analysis and interpretation of data Walid, Gaber, Ibrahim and Samir drafting the article Walid, Gaber and Samir critically revising the article for important intellectual content Walid, Gaber, Ibrahim and Samir final approval of the version to be published Walid, Gaber and Samir critically revising the article for important intellectual content Walid, Gaber, Ibrahim and Samir final approval of the version to be published References 1. N. Mahanta, M. Szantai-Kis, E. J. Petersson, D. A. Mitchell ACS Chem Biol. 2019, 14(2): 142–163. 2. T. S. Jagodzin´ ski, Chem. Rev. 2003, 103, 197–228. 3. T. Murai, H. Aso, Y. Tatematsu, Y. Itoh, H. Niwa, S. Kato, J. Org. Chem. 2003, 68, 8514–8519. 4. R. N. Hurd and G. DeLaMater, Chem. Rev., 1961, 61, 45. 3. T. Murai, H. Aso, Y. Tatematsu, Y. Itoh, H. Niwa, S. Kato, J. Org. Chem. 2003, 68, 8514–8519. 4. R. N. Hurd and G. DeLaMater, Chem. Rev., 1961, 61, 45. 5. U. Pathak, L. K. Pandey, S. Mathur, M. V. S. Suryanarayana. Chem. Commun., 2009, 5409–5411. 6 Y Wei Y Liu L-G Xie Chinese Chemical Letters 2022 33(5) 2407-2410 5. U. Pathak, L. K. Pandey, S. Mathur, M. V. S. Suryanarayana. Chem. Commun., 2009, 5409–5411. 5. U. Pathak, L. K. Pandey, S. Mathur, M. V. S. Suryanarayana. Chem. Commun., 2009, 5409 5411. 6. Y. Wei, Y. Liu, L-G. Xie. Chinese Chemical Letters, 2022, 33(5), 2407-2410. 6. Y. Wei, Y. Liu, L-G. Xie. Chinese Chemical Letters, 2022, 33(5), 2407-2410. 7. A. K. Yadav, V. P. Srivastava, L. D. S. Yadav. Tetrahedron Letters 2012, 53 7. A. K. Yadav, V. P. Srivastava, L. D. S. Yadav. Tetrahedron Letters 2012, 53, 7113–7116 8. S. P. Pathare, P. S. Chaudhari, K. G. Akamanchi. Applied Catalysis A: General 2012, 425(426), 125– 129. 9. K. Okamoto, T. Yamamoto, T. Kanbara. SYNLETT 2007, 17, 2687–2690 10 Z Yi B Zh F Ai Ph h S lf d Sili 2013 188 1412 1420 9. K. Okamoto, T. Yamamoto, T. Kanbara. SYNLETT 2007, 17, 2687–2690 9. K. Okamoto, T. Yamamoto, T. Kanbara. SYNLETT 2007, 17, 2687–2690 10. Z. Yin, B. Zheng, F. Ai. Phosphorus, Sulfur, and Silicon, 2013, 188, 1412–1420, 11. H. Xu, H. Deng, Z. Li, H. Xiang, X. Zhou. Eur. J. Org. Chem. 2013, 7054–7057 , , , , 10. Z. Yin, B. Zheng, F. Ai. Phosphorus, Sulfur, and Silicon, 2013, 188, 1412–1420, 11. H. Xu, H. Deng, Z. Li, H. Xiang, X. Zhou. Eur. J. Org. Chem. 2013, 7054–7057 12. N. T. Do, K. M. Tran, H. T. Phan, T. A. To, T. T. Nguyen,N. T. S. Phan. Org. Biomol. Chem., 2019, 17, 8987 13. L. Doszczak, J. Rachon. J. Chem. Soc., Perkin Trans. 1, 2002, 1271–1279. 13. L. Doszczak, J. Rachon. J. Chem. Soc., Perkin Trans. 1, 2002, 1271–1279 14. L. Doszczak, J. Rachon. Chem. Schemes Schemes 1 to 5 are available in the Supplemental Files section. Schemes 1 to 5 are available in the Supplemental Files section. References Varun, A. Sood, K. R. Prabhu. RSC Advances, 2014, 4, 60798. 29. F. G. Stevens, C. J. McCall, P. Lelievald, P. Alexander, A. Richter, D. E. Davies. Journal of Medicinal Chemistry 1994, 37, 1689. 30. H. Xu, H. Deng,Z. Li, H. Xiang, X. Zhou European Journal of Organic Chemistry, 2013, 31, 7054. 31. K. Kobayashi, D. Fujiwara, and M. Tanmatsu. Heterocycles, 2018, 96, 902. K. Kobayashi, D. Fujiwara, and M. Tanmatsu. Heterocycles, 2018, 96, 902. References Commun., 2000, 2093–2094 15. R. S. Varma, D. Kumar. Org. Lett., 1999, 1(5), 697. 15. R. S. Varma, D. Kumar. Org. Lett., 1999, 1(5), 697. 15. R. S. Varma, D. Kumar. Org. Lett., 1999, 1(5), 697. 16. D. Cho, J. Ahn, K. A. De Castro, H. Ahn, H. Rheeb. Tetrahedron 2010, 66, 5583-5588. 16. D. Cho, J. Ahn, K. A. De Castro, H. Ahn, H. Rheeb. Tetrahedron 2010, 66, 5583-5588. 17. Walid Fathalla, Ibrahim A. I. Ali and Pavel Pazdera. Beilstein J. Org. Chem. 2017, 13, 174–181. 18. Mohamed Megahed, Walid Fathalla, Amer Elsheikh J. Heterocyclic Chem., 2018, 55, 2799. 17. Walid Fathalla, Ibrahim A. I. Ali and Pavel Pazdera. Beilstein J. Org. Chem. 2017, 13, 174–181. 17. Walid Fathalla, Ibrahim A. I. Ali and Pavel Pazdera. Beilstein J. Org. Chem. 2017, 13, 174–181. 18 Mohamed Megahed Walid Fathalla Amer Elsheikh J Heterocyclic Chem 2018 55 2799 17. Walid Fathalla, Ibrahim A. I. Ali and Pavel Pazdera. Beilstein J. Org. Chem 17. Walid Fathalla, Ibrahim A. I. Ali and Pavel Pazdera. Beilstein J. Org. Chem. 2017, 13, 174 181. 18. Mohamed Megahed, Walid Fathalla, Amer Elsheikh J. Heterocyclic Chem., 2018, 55, 2799. 18. Mohamed Megahed, Walid Fathalla, Amer Elsheikh J. Heterocyclic Chem., 2018, 55, 2799. Page 10/11 Page 10/11 19. El Rayes, Samir; Abou Elmagd, Ahmed; Gomaa, Mohammed S.; Ahmed I. Ali, Ibrahim; Fathalla, Walid; Pottoo, Faheem; Khan, Firdos. ACS Omega, 2019, 4, 20, 18555-18566. 20. Soliman, M.H.A., Ali, I.A.I., El-Sakka, S.S.A., Mohamed, O.E.-S.A.-B. Journal of Molecular Structure, 2022, 1254, 132325. 21. Fathalla, W.; Pazdera, P. Tetrahedron 2017, 73, 4573-4583. 21. Fathalla, W.; Pazdera, P. Tetrahedron 2017, 73, 4573-4583. 22. Fathalla, W.; Pazdera, P. Chem. Pap. 2018, 72, 209–219 22. Fathalla, W.; Pazdera, P. Chem. Pap. 2018, 72, 209–219 23. Fathalla, W.; Pazdera, P. Organic Preparation and procedures international 2018, 50, 385–407. 24. Walid Fathalla, Pavel Pazdera, Mohamed E. Khalifa, Ibrahim A. I. Ali, Samir. M. El Rayes. J. Heterocyclic Chem. 2022, 59, 933–942. 25. Walid Fathalla, Ibrahim. A.I. Ali, Jaromir Marek, Pavel Pazdera. Journal of Sulfur Chemistry 2012, 33, 49–63. 26. L. Nahakpam, F. A.S. Chipem, B. S. Chingakham, W. S. Laitonjam. New J. Chem., 2015, 39, 2240- 2247. 27. N. K. Downer-Riley, Y. A. Jackson. Tetrahedron, 2008, 64, 7741. 27. N. K. Downer-Riley, Y. A. Jackson. Tetrahedron, 2008, 64, 7741. 28. B. V. Varun, A. Sood, K. R. Prabhu. RSC Advances, 2014, 4, 60798. 28. B. V. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Scheme1.jpg Scheme2.jpg Scheme3.jpg Scheme4.png Scheme5.jpg Scheme1.jpg Page 11/11
https://openalex.org/W2523798614	https://europepmc.org/articles/pmc5968893?pdf=render	English	null	Muscle- and Mode-Specific Responses of the Forearm Flexors to Fatiguing, Concentric Muscle Actions	Sports	2,016	cc-by	7,340	Muscle- and Mode-Speciﬁc Responses of the Forearm Flexors to Fatiguing, Concentric Muscle Actions Ethan Hill , Terry Housh, Cory Smith, Richard Schmidt and Glen Johnson Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68505, USA; thoush1@unl.edu (T.H.); csmith@unl.edu (C.S.); rschmidt1@unl.edu (R.S.); gojohnson22@gmail.com (G.J.) Correspondence: ethan.hill@unl.edu; Tel.: +1-402-472-2690 Ethan Hill , Terry Housh, Cory Smith, Richard Schmidt and Glen Johnson Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68505, USA; thoush1@unl.edu (T.H.); csmith@unl.edu (C.S.); rschmidt1@unl.edu (R.S.); gojohnson22@gmail.com (G.J.) Correspondence: ethan.hill@unl.edu; Tel.: +1-402-472-2690 Received: 26 July 2016; Accepted: 22 September 2016; Published: 30 September 2016 Abstract: Background: Electromyographic (EMG) and mechanomyographic (MMG) studies of fatigue have generally utilized maximal isometric or dynamic muscle actions, but sport- and work-related activities involve predominately submaximal movements. Therefore, the purpose of the present investigation was to examine the torque, EMG, and MMG responses as a result of submaximal, concentric, isokinetic, forearm ﬂexion muscle actions. Methods: Twelve men performed concentric peak torque (PT) and isometric PT trials before (pretest) and after (posttest) performing 50 submaximal (65% of concentric PT), concentric, isokinetic (60◦·s−1), forearm ﬂexion muscle actions. Surface EMG and MMG signals were simultaneously recorded from the biceps brachii and brachioradialis muscles. Results: The results of the present study indicated similar decreases during both the concentric PT and isometric PT measurements for torque, EMG mean power frequency (MPF), and MMG MPF following the fatiguing workbout, but no changes in EMG amplitude (AMP) or MMG AMP. Conclusions: These ﬁndings suggest that decreases in torque as a result of fatiguing, dynamic muscle actions may have been due to the effects of metabolic byproducts on excitation–contraction coupling as indicated by the decreases in EMG MPF and MMG MPF, but lack of changes in EMG AMP and MMG AMP from both the biceps brachii and brachioradialis muscles. Keywords: muscle fatigue; isokinetic; EMG; MMG Keywords: muscle fatigue; isokinetic; EMG; MMG Sports 2016, 4, 47; doi:10.3390/sports4040047 www.mdpi.com/journal/sports sports Article Muscle- and Mode-Speciﬁc Responses of the Forearm Flexors to Fatiguing, Concentric Muscle Actions Ethan Hill , Terry Housh, Cory Smith, Richard Schmidt and Glen Johnson sports 1. Introduction Surface electromyography (EMG) records and quantiﬁes the action potentials that activate skeletal muscle ﬁbers [1]. The amplitude (AMP) of the EMG signal is generated by the summation of the action potential trains from the active motor units and is inﬂuenced by the number of active motor units, their ﬁring rates, and synchronization [1,2]. The power spectrum of the EMG signal is, in part, determined by average muscle ﬁber action potential conduction velocity [3] and the shape of the action potential waveforms [4]. Mechanomyography (MMG) is a non-invasive technique that provides information related to muscle function, which is unique compared with EMG. Gordon and Holbourn (1948) [5] described the MMG signal as the mechanical counterpart of motor unit activity as measured by EMG. MMG quantiﬁes the lateral oscillations of activated muscle ﬁbers that are generated by (a) gross lateral movement of the muscle at the initiation of a contraction generated by the non-simultaneous activation of muscle ﬁbers; (b) smaller subsequent lateral oscillations generated at the resonance frequency of the muscle; and (c) dimensional changes of the active ﬁbers [6]. It has been suggested that the amplitude of the MMG signal is related to motor unit recruitment and that the frequency content of the MMG signal is qualitatively related to the motor unit ﬁring rate [6–8]. A number of anatomical, physiological, and non-physiological factors can affect the time and frequency domain parameters of the EMG signal including subcutaneous tissue layer, environmental noise, wave cancellation, and electrode placement [1,9–11]. In addition, the MMG signal can be inﬂuenced by Sports 2016, 4, 47; doi:10.3390/sports4040047 2 of 10 Sports 2016, 4, 47 muscle temperature, muscle stiffness, mass, the viscosity of the intracellular and extracellular ﬂuids, and intramuscular ﬂuid pressure [6,12,13]. A unique application of EMG and MMG involves the assessment of muscle fatigue. Muscle fatigue has been described as “an acute impairment of performance that includes both an increase in perceived effort necessary to exert a desired force and an eventual inability to produce this force” [14] (p. 1631) and “can occur despite continued and successful performance of a submaximal task” [15] (p. 133). Previous investigations have examined muscle fatigue as assessed by changes in torque, EMG, and MMG during maximal and submaximal, isometric muscle actions [6,16–21]. For example, following maximal, intermittent, isometric, leg extension muscle actions, Camic et al. 1. Introduction (2013) [16] reported decreases in isometric peak torque (PT), EMG AMP, EMG mean power frequency (MPF), and MMG MPF, but no change in MMG AMP from the vastus lateralis. During fatiguing, submaximal, intermittent isometric muscle actions, Orizio (1993) [6] reported intensity-speciﬁc responses for MMG AMP during sustained, isometric, forearm ﬂexion muscle actions at submaximal intensities of 20%–80% of isometric PT. Furthermore, Seghers and Spaepen (2004) [20] reported a decrease and no change in EMG median frequency from the biceps brachii (BB) during intermittent, isometric, forearm ﬂexion muscle actions at submaximal intensities of 25% and 50% of isometric PT, respectively. A number of investigations [18,22–27] have also examined the responses as a result of maximal, dynamic movements, but a limited number of investigations [28] have examined the effects of fatiguing, submaximal, dynamic workbouts on EMG or MMG responses. For example, Camic et al. (2013) [16] examined the effects of 30 repeated, maximal, concentric muscle actions at 30◦·s−1 and reported decreases in concentric PT, EMG AMP, EMG MPF, MMG AMP, and MMG MPF from the vastus lateralis. During fatiguing, maximal, eccentric workbouts at velocities of 60◦·s−1, 120◦·s−1, and 180◦·s−1, Hill et al. (2015) [27] reported no changes in eccentric torque, but isometric PT decreased 17.9%, 16.1%, and 8.9%, respectively. In addition, Perry-Rana et al. (2003) [24] reported muscle-speciﬁc EMG and MMG responses for the vastus lateralis, vastus medialis, and rectus femoris as a result of fatiguing, maximal, eccentric muscle actions at 120◦·s−1. Together, these investigations have shown that fatigue-related changes in the time and frequency domain parameters of EMG and MMG signals can be affected by the mode, intensity, and velocity of muscle action employed (isometric vs. concentric vs. eccentric), as well as the muscles groups involved. Most sport- and work-related activities involve predominately submaximal, dynamic movements. Thus, understanding the fatigue-related neuromuscular responses associated with submaximal, dynamic muscle actions may have implications regarding work- and sport-related interventions to improve athletic performance, reduce sport- and work-related musculoskeletal injuries, or both [29–33]. Therefore, the purpose of the present investigation was to examine the torque, EMG, and MMG responses as a result of submaximal, concentric, isokinetic, forearm ﬂexion muscle actions. Based on previous investigations [20,27], we hypothesized that concentric PT, isometric PT, EMG MPF, MMG AMP, and MMG MPF would decrease as a result of the 50 submaximal, concentric muscle actions, while EMG AMP would remain unchanged. 2.2. Concentric PT and Isometric PT Neuromuscular Responses 2.2. Concentric PT and Isometric PT Neuromuscular Responses EMG AMP: There were no significant 3- (Muscle × Mode × Time, p = 0.325, η2p = 0.088) or 2-way interactions (Time × Mode, p = 0.382, η2p = 0.070; Muscle × Mode, p = 0.165, η2p = 0.167; Muscle × Time, p = 0.065, η2p = 0.276) for normalized EMG AMP. In addition, there were no significant main effects for Mode (p = 0.435, η2p = 0.056), Time (p = 0.701, η2p = 0.014), or Muscle (p = 0.179, η2p = 0.158). Thus, there was no change in EMG AMP as a result of the fatiguing workbout (Figure 2). EMG AMP: There were no signiﬁcant 3- (Muscle × Mode × Time, p = 0.325, η2 p = 0.088) or 2-way interactions (Time × Mode, p = 0.382, η2 p = 0.070; Muscle × Mode, p = 0.165, η2 p = 0.167; Muscle × Time, p = 0.065, η2 p = 0.276) for normalized EMG AMP. In addition, there were no signiﬁcant main effects for Mode (p = 0.435, η2 p = 0.056), Time (p = 0.701, η2 p = 0.014), or Muscle (p = 0.179, η2 p = 0.158). Thus, there was no change in EMG AMP as a result of the fatiguing workbout (Figure 2). there was no change in EMG AMP as a result of the fatiguing workbout (Figure 2). EMG MPF: There were no significant 3- (Muscle × Mode × Time, p = 0.185, η2p = 0.154) or 2-way interactions (Time × Mode, p = 0.115, η2p = 0.210; Muscle × Mode, p = 0.567, η2p = 0.031; Muscle × Time, p = 0.891, η2p = 0.002) for normalized EMG MPF. There was, however, a significant main effect for Time (pretest > posttest; p = 0.045, η2p = 0.317), collapsed across Muscle and Mode, but no significant main effects for Mode (p = 0.745, η2p = 0.010) or Muscle (p = 0.810, η2p = 0.005). Thus, EMG MPF d d l f h f i i kb (Fi 2) EMG MPF: There were no signiﬁcant 3- (Muscle × Mode × Time, p = 0.185, η2 p = 0.154) or 2-way interactions (Time × Mode, p = 0.115, η2 p = 0.210; Muscle × Mode, p = 0.567, η2 p = 0.031; Muscle × Time, p = 0.891, η2 p = 0.002) for normalized EMG MPF. 2.2. Concentric PT and Isometric PT Neuromuscular Responses 2.2. Concentric PT and Isometric PT Neuromuscular Responses There was, however, a signiﬁcant main effect for Time (pretest > posttest; p = 0.045, η2 p = 0.317), collapsed across Muscle and Mode, but no signiﬁcant main effects for Mode (p = 0.745, η2 p = 0.010) or Muscle (p = 0.810, η2 p = 0.005). Thus, EMG MPF decreased as a result of the fatiguing workbout (Figure 2). decreased as a result of the fatiguing workbout (Figure 2). MMG AMP: There was a significant 3-way (p = 0.042, η2p = 0.324) interaction for normalized MMG AMP that was decomposed into separate 2-way ANOVAs (Mode × Time) for each muscle. There were no significant Mode × Time interactions for the BB (p = 0.172, η2p = 0.686) or the BR (p = 0.053, η2p = 0.299). There were, however, significant main effects for Mode from the BB (concentric PT > isometric PT; p = 0.004, η2p = 0.578) and BR (concentric PT > isometric PT; p = 0.001, η2p = 0.755), but no main effects for Time from the BB (p = 0.112, η2p = 0.538) or BR (p = 0.827, η2p = 0.005). Thus, MMG MMG AMP: There was a signiﬁcant 3-way (p = 0.042, η2 p = 0.324) interaction for normalized MMG AMP that was decomposed into separate 2-way ANOVAs (Mode × Time) for each muscle. There were no signiﬁcant Mode × Time interactions for the BB (p = 0.172, η2 p = 0.686) or the BR (p = 0.053, η2 p = 0.299). There were, however, signiﬁcant main effects for Mode from the BB (concentric PT > isometric PT; p = 0.004, η2 p = 0.578) and BR (concentric PT > isometric PT; p = 0.001, η2 p = 0.755), but no main effects for Time from the BB (p = 0.112, η2 p = 0.538) or BR (p = 0.827, η2 p = 0.005). Thus, MMG AMP was greater during the concentric PT than the isometric PT measurements. p p AMP was greater during the concentric PT than the isometric PT measurements. MMG MPF: There were no significant 3- (Muscle × Mode × Time, p = 0.466, η2p = 0.049) or 2-way interactions (Time × Mode, p = 0.925, η2p = 0.001; Muscle × Mode, p = 0.402, η2p = 0.065; Muscle × Time, p = 0.951, η2p < 0.001) for normalized MMG MPF. 2.1. Concentric PT and Isometric PT Responses 2.1. Concentric PT and Isometric PT Responses Figure 1 shows the pretest and posttest concentric PT and isometric PT responses. There was no signiﬁcant Mode × Time interaction for torque (p = 0.347, η2 p = 0.081). There were, however, signiﬁcant main effects for Mode (isometric PT > concentric PT; p < 0.001, η2 p = 0.872) and Time (pretest > posttest; p < 0.001, η2 p = 0.841). Thus, torque decreased during both the concentric PT (23.3%) and isometric PT (17.8%) muscle actions as a result of the fatiguing workbout, and concentric PT was less than isometric PT at pretest and posttest. Sports 2016, 4, 47 3 of Sports 2016, 4, 47 3 of 3 of 10 3 of 10 Sports 2016, 4, 47 Figure 1. Pretest and posttest values (±SEM) for concentric peak torque (PT) and isometric PT. Significant at p ≤ 0.05 for pretest > posttest. † Significant at p ≤ 0.05 for concentric PT < isometric PT. Figure 1. Pretest and posttest values (±SEM) for concentric peak torque (PT) and isometric PT. * Signiﬁcant at p ≤0.05 for pretest > posttest. † Signiﬁcant at p ≤0.05 for concentric PT < isometric PT. 3.1. The Effects of the Submaximal, Dynamic Workbout The purpose of the present investigation was to 3.1. The Effects of the Submaximal, Dynamic Workbout p p p g q p as a result of submaximal, concentric, isokinetic, forearm flexion muscle actions. The findings of the present study indicated that there were no mode-specific effects for torque, EMG AMP, EMG MPF, or MMG MPF. There was, however, a mode-specific effect for MMG AMP which was greater during the concentric PT than the isometric PT measurements. Furthermore, consistent with our hypothesis and previous investigations [20,27], concentric PT, isometric PT, EMG MPF, and MMG MPF decreased, while EMG AMP remained unchanged. Unlike our hypothesis, however, there was no change in MMG AMP. 3.2. Torque The purpose of the present investigation was to examine the torque, EMG, and MMG responses as a result of submaximal, concentric, isokinetic, forearm ﬂexion muscle actions. The ﬁndings of the present study indicated that there were no mode-speciﬁc effects for torque, EMG AMP, EMG MPF, or MMG MPF. There was, however, a mode-speciﬁc effect for MMG AMP which was greater during the concentric PT than the isometric PT measurements. Furthermore, consistent with our hypothesis and previous investigations [20,27], concentric PT, isometric PT, EMG MPF, and MMG MPF decreased, while EMG AMP remained unchanged. Unlike our hypothesis, however, there was no change in MMG AMP. 2.2. Concentric PT and Isometric PT Neuromuscular Responses 2.2. Concentric PT and Isometric PT Neuromuscular Responses Pretest and posttest values (±SEM) expressed as percentage changes from pretest (collapsed across Muscle (biceps brachii and brachioradialis) and Mode (concentric peak torque and isometric peak torque)) for electromyographic amplitude (EMG AMP), EMG mean power frequency (EMG MPF), mechanomyographic amplitude (MMG AMP), and MMG MPF. * Signiﬁcant at p ≤0.05 for pretest > posttest. Figure 2. Pretest and posttest values (±SEM) expressed as percentage changes from pretest (collapsed across Muscle (biceps brachii and brachioradialis) and Mode (concentric peak torque and isometric peak torque)) for electromyographic amplitude (EMG AMP), EMG mean power frequency (EMG MPF), mechanomyographic amplitude (MMG AMP), and MMG MPF. * Significant at p ≤ 0.05 for pretest > posttest. Figure 2. Pretest and posttest values (±SEM) expressed as percentage changes from pretest (collapsed across Muscle (biceps brachii and brachioradialis) and Mode (concentric peak torque and isometric peak torque)) for electromyographic amplitude (EMG AMP), EMG mean power frequency (EMG MPF), mechanomyographic amplitude (MMG AMP), and MMG MPF. * Signiﬁcant at p ≤0.05 for pretest > posttest. The i i 3.2. Torque in maximal torque following the submaximal, fatiguing workbout. That is, even though the fatiguing workbout involved only concentric, isokinetic muscle actions, there was no difference in the percent declines in concentric PT (23.3%) and isometric PT (17.8%). These findings were consistent with those of Camic (2011) [34] who reported a 20.3% decline in concentric PT and a 16.5% decline in isometric PT following 30 maximal, concentric, isokinetic, leg extension muscle actions in women. Thus, the results of the present study, in conjunction with those of Camic (2011) [34], indicated that, for both genders, there were no mode-specific declines in maximal torque and similar decreases in concentric PT and isometric PT following submaximal or maximal, fatiguing, concentric workbouts. 3.3. Pretest Versus Posttest EMG and MMG Responses Th l d ifi f diff i The results of the present study indicated that there was no mode-speciﬁc effect for the decline in maximal torque following the submaximal, fatiguing workbout. That is, even though the fatiguing workbout involved only concentric, isokinetic muscle actions, there was no difference in the percent declines in concentric PT (23.3%) and isometric PT (17.8%). These ﬁndings were consistent with those of Camic (2011) [34] who reported a 20.3% decline in concentric PT and a 16.5% decline in isometric PT following 30 maximal, concentric, isokinetic, leg extension muscle actions in women. Thus, the results of the present study, in conjunction with those of Camic (2011) [34], indicated that, for both genders, there were no mode-speciﬁc declines in maximal torque and similar decreases in concentric PT and isometric PT following submaximal or maximal, fatiguing, concentric workbouts. 2.2. Concentric PT and Isometric PT Neuromuscular Responses 2.2. Concentric PT and Isometric PT Neuromuscular Responses There was, however, a significant main effect for Time (pretest > posttest; p < 0.001, η2p = 0.715), collapsed across Muscle and Mode, but no significant main effects for Mode (p = 0 674 η2 = 0 017) or Muscle (p = 0 877 η2 = 0 002) Thus MMG MPF MMG MPF: There were no signiﬁcant 3- (Muscle × Mode × Time, p = 0.466, η2 p = 0.049) or 2-way interactions (Time × Mode, p = 0.925, η2 p = 0.001; Muscle × Mode, p = 0.402, η2 p = 0.065; Muscle × Time, p = 0.951, η2 p < 0.001) for normalized MMG MPF. There was, however, a signiﬁcant main effect for Time (pretest > posttest; p < 0.001, η2 p = 0.715), collapsed across Muscle and Mode, but no signiﬁcant main effects for Mode (p = 0.674, η2 p = 0.017) or Muscle (p = 0.877, η2 p = 0.002). Thus, MMG MPF decreased as a result of the fatiguing workbout (Figure 2). orts 2016, 4, 47 4 of Sports 2016, 4, 47 4 of 10 Figure 2. Pretest and posttest values (±SEM) expressed as percentage changes from pretest (collapsed across Muscle (biceps brachii and brachioradialis) and Mode (concentric peak torque and isometric peak torque)) for electromyographic amplitude (EMG AMP), EMG mean power frequency (EMG MPF), mechanomyographic amplitude (MMG AMP), and MMG MPF. * Significant at p ≤ 0.05 for pretest > posttest. Figure 2. Pretest and posttest values (±SEM) expressed as percentage changes from pretest (collapsed across Muscle (biceps brachii and brachioradialis) and Mode (concentric peak torque and isometric peak torque)) for electromyographic amplitude (EMG AMP), EMG mean power frequency (EMG MPF), mechanomyographic amplitude (MMG AMP), and MMG MPF. * Signiﬁcant at p ≤0.05 for pretest > posttest. 4 of 10 f 10 Sports 2016, 4, 47 orts 2016, 4, 47 Sports 2016, 4, 47 Figure 2. Pretest and posttest values (±SEM) expressed as percentage changes from pretest (collapsed across Muscle (biceps brachii and brachioradialis) and Mode (concentric peak torque and isometric peak torque)) for electromyographic amplitude (EMG AMP), EMG mean power frequency (EMG MPF), mechanomyographic amplitude (MMG AMP), and MMG MPF. * Significant at p ≤ 0.05 for pretest > posttest. Figure 2. p p neuromuscular responses as a result of the submaxim 3.3. Pretest Versus Posttest EMG and MMG Responses collapsed across Time and Muscle MMG AMP was greater during the concentric PT than the isometric PT measurements. The greater MMG AMP values during the concentric PT than isometric There were no muscle- or mode-speciﬁc patterns of differences in pretest versus posttest neuromuscular responses as a result of the submaximal, fatiguing workbout in the present study, but collapsed across Time and Muscle MMG AMP was greater during the concentric PT than the isometric PT measurements. The greater MMG AMP values during the concentric PT than isometric PT 5 of 10 Sports 2016, 4, 47 measurements may have been due to the dynamic nature of the muscle action [8,35]. It is also possible that the lower concentric PT values (84.0 and 64.4 Nm at pretest and posttest, respectively), compared with the isometric PT (98.6 and 81.1 Nm at pretest and posttest, respectively) values, were associated with less muscle stiffness, greater muscle compliance, or both, which resulted in less restriction of the lateral oscillations of the activated muscle ﬁbers and, therefore, greater MMG AMP values [8,35]. g Fatigue is typically associated with decreases in EMG MPF which reﬂect the effects of metabolic byproducts on muscle ﬁber action potential conduction velocity [1,2]. Thus, the pretest versus posttest decrease in EMG MPF in the present study, along with the decreases in concentric PT and isometric PT, supported the fatiguing nature of the workbout. Furthermore, the pretest versus posttest decreases in concentric PT and isometric PT, without changes in EMG AMP and MMG AMP suggested excitation–contraction coupling failure. That is, the buildup of metabolic byproducts such as lactate, inorganic phosphate, and ammonia interfere with contractile properties of the activated muscle ﬁbers [36–40]. Although there is some disagreement [41,42], the effect of lactate and inorganic phosphate accumulation on force production may be due to the effects of calcium release and reuptake by the sarcoplasmic reticulum, actin-myosin binding afﬁnity, troponin-calcium binding afﬁnity, ATP breakdown via ATPase, and ATP production in the metabolic pathways [39]. In addition, ammonia accumulation during exercise can adversely affect action potential propagation [37,40]. Thus, it is possible that the fatigue-induced buildup of metabolic byproducts caused excitation–contraction coupling failure and contributed to the decreases in concentric PT and isometric PT. In the present study, the decrease in global motor unit ﬁring rate (MMG MPF) may have been related to the effects of muscle wisdom [43,44]. p p neuromuscular responses as a result of the submaxim 3.3. Pretest Versus Posttest EMG and MMG Responses Muscle wisdom is a motor unit activation strategy characterized by decreased muscle relaxation times and motor neuron discharge rates as well as greater fusion of motor unit twitches to optimize force production [43–47]. The applicability of muscle wisdom during dynamic muscle actions, however, has been questioned [44]. In addition, the decrease in global motor unit ﬁring rate (MMG MPF) without changes in EMG AMP or MMG AMP did not support the suggestion of Orizio (1993) [6] regarding fatigue-related de-recruitment of motor units [6], but may have been due to synchronization. Speciﬁcally, EMG AMP is a function of motor unit recruitment, ﬁring rate, and synchronization [1,2,48]. Although there is some disagreement regarding the occurrence of synchronization [49,50], the lack of change in EMG AMP despite decreases in motor unit ﬁring rate (MMG MPF) and no change in motor unit recruitment (MMG AMP) suggested that motor unit synchronization, which results in a more efﬁcient and synchronous activation of motor units to optimize force production [1,51–55], compensated for the decrease in ﬁring rate. 4.1. Subjects Thirteen men volunteered to participate in this investigation, but 1 subject was unable to complete all testing procedures (n = 12; mean age ± SD = 22.6 ± 2.2 years; body weight = 84.0 ± 8.3 kg; height = 178.6 ± 8.3 cm). The subjects regularly participated in resistance training (8.1 ± 2.2 h per week) and had no known cardiovascular, pulmonary, metabolic, muscular, or coronary heart disease, or regularly used prescription medication. The subjects visited the laboratory on 2 occasions separated by at least 48-h and were instructed not to perform upper body exercise 48-h prior to each visit. The study was approved by the University Institutional Review Board for Human Subjects, and all subjects completed a health history questionnaire and signed written informed consent prior to testing. 4.2. Procedures Familiarization (Visit 1): The ﬁrst laboratory visit consisted of an orientation session to familiarize the subjects with the testing protocols. During the orientation, the subjects performed submaximal and maximal concentric, isokinetic (60◦·s−1) and isometric muscle actions of the forearm ﬂexors. The subjects visually tracked torque production using real-time torque displayed on a computer 6 of 10 Sports 2016, 4, 47 monitor programmed using LabVIEW 13.0 software (National Instruments, Austin, TX, USA) and practiced performing concentric, isokinetic muscle actions at 65% of concentric PT. Determination of Concentric PT and Isometric PT: During Visit 2, the subjects performed a warm-up consisting of 10–15 submaximal (approximately 50%–75% of PT), concentric, isokinetic muscle actions of the dominant (based on throwing preference) forearm ﬂexors at 60◦·s−1 on a calibrated Cybex II dynamometer. After 2 min of rest, the subjects randomly performed 5 concentric PT and 5 isometric PT trials [56]. The concentric muscle actions were performed at 60◦·s−1 through a 90◦range of motion (from 170◦to 80◦), and the isometric muscle actions were performed for 4 s at an elbow joint angle of 115◦where 180◦corresponded to full extension [57–60]. The highest values were selected as the pretest concentric PT and isometric PT, respectively. Submaximal, Concentric, Isokinetic Muscle Actions: Following the determination of the pretest concentric PT and pretest isometric PT, the subjects performed 50 submaximal (65% of their pretest concentric PT), concentric, isokinetic muscle actions at 60◦·s−1 followed by passive forearm extension at 60◦·s−1 that were assisted by the investigator. Real-time torque was displayed on a computer monitor. In addition, a light bulb indicated the start and end of each repetition, which was displayed on the same computer monitor as the real-time torque. For analyses, only subjects that maintained 65% (±5%) of PT during all 50 submaximal, concentric muscle actions were used. Thus, only the data from 12 of the 13 subjects were analyzed. After completing the 50 submaximal, concentric muscle actions, the subjects randomly performed 5 posttest concentric PT and 5 posttest isometric PT trials using the same procedures as the pretest. Electrode and Accelerometer Placements: During Visit 2, bipolar (30 mm center-to-center) surface EMG electrode (circular 4-mm diameter silver–silver chloride, BIOPAC Systems, Inc., Santa Barbara, CA, USA) arrangements were placed on the dominate arm over the BB and BR muscles according to the recommendations of Barbero et al. (2012) [61]. The reference electrode was placed over the acromion process. 4.2. Procedures Prior to each electrode placement, the skin was shaved, carefully abraded, and cleaned with alcohol. The MMG signals from the BB and BR were detected using accelerometers (Entran EGAS FT 10, dimensions: 1.0 × 1.0 × 0.5 cm3, mass: 1.0 g, sensitivity: 651.6 and 624.3 mV/g) that were placed between the proximal and distal EMG electrodes of each of the bipolar arrangement using double-sided adhesive tape. Signal Processing: The raw EMG and MMG signals were digitized at 1000 Hz with a 32-bit analog-to-digital converter (Model MP100, Biopac Systems, Inc., Santa Barbara, CA, USA) and stored in a personal computer (ATIV Book 9 Intel Core i7 Samsung Inc., Dallas, TX, USA) for subsequent analyses. The EMG signals were ampliﬁed (gain: ×1000) using differential ampliﬁers (EMG 100, Biopac Systems, Inc., Santa Barbara, CA, USA). The EMG and MMG signals were digitally bandpass ﬁltered (fourth-order Butterworth, zero-phase shift) at 10–500 Hz and 5–100 Hz, respectively. All signal processing was performed using custom programs written with the LabVIEW programming software. The EMG (µV root-mean-square, µVrms) and MMG (m·s−2) AMP and MPF (Hz) values for the concentric and isometric muscle actions were calculated for the middle third of each contraction. Thus, during the concentric and isometric muscle actions, signal epochs of 0.50 s and 1.33 s were used, respectively, to calculate the AMP and MPF values of the EMG and MMG signals. These portions of the signals were selected to avoid the acceleration and deceleration phases that are typical of isokinetic dynamometers [62] and to avoid the initial gross lateral movement of the muscle at the onset of muscle contraction [6]. For the MPF analyses, each data segment was processed with a Hamming window and the Discrete Fourier transform (DFT) algorithm [63,64]. The MPF was selected to represent the power spectrum in accordance with the recommendations of Hermens et al. (2000) [65]. All EMG and MMG analyses were performed using normalized (to pretest isometric PT) values (Table 1). 7 of 10 Sports 2016, 4, 47 Table 1. Descriptive data (means ± SD) for the normalized pretest and posttest neuromuscular responses from the biceps brachii (BB) and brachioradialis (BR) during the concentric peak torque (PT) and isometric PT muscle actions. 4.3. Statistical Analyses A 2 (Mode (concentric PT, isometric PT)) × 2 (Time (pretest, posttest)) repeated measures ANOVA was used to analyze the absolute concentric PT and isometric PT. In addition, separate 2 (Muscle (BB, BR)) × 2 (Mode (concentric PT, isometric PT)) × 2 (Time (pretest, posttest)) repeated measures ANOVAs were used to analyze the normalized (to values during the pretest isometric PT) EMG AMP, EMG MPF, MMG AMP, and MMG MPF values. Partial eta squared effect sizes (η2 p) were calculated for each ANOVA. Signiﬁcant 3-way interactions were decomposed with follow-up repeated measures ANOVAs, and signiﬁcant 2-way interactions were decomposed with follow-up, Bonferroni-corrected dependent samples t-tests. In addition, Greenhouse–Geisser corrections were applied when the assumption of sphericity was not met according to Maulchy’s test of sphericity. All statistical analyses were performed using IBM SPSS v. 21 (Armonk, NY, USA), and an alpha of p ≤0.05 was considered statistically signiﬁcant. 5. Conclusions In summary, the results of the present study indicated that there were no mode-speciﬁc declines in maximal torque and similar decreases in concentric PT and isometric PT following the submaximal fatiguing, concentric workbout. In addition, there were no muscle- or mode-speciﬁc patterns of differences in neuromuscular responses as a result of the fatiguing workbout. MMG AMP, however, was greater during the concentric PT than the isometric PT measurements, which may have been due to the dynamic nature of the muscle action, muscle stiffness, muscle compliance, or a combination thereof. As a result of the fatiguing workbout, there were decreases from pretest to posttest for EMG MPF and MMG MPF, but no changes in EMG AMP or MMG AMP (Figure 2). The pretest versus posttest decreases in concentric PT and isometric PT, without changes in EMG AMP and MMG AMP suggested excitation–contraction coupling failure. The decrease in global motor unit ﬁring rate (MMG MPF) without changes in EMG AMP or MMG AMP may have been related to the effects of muscle wisdom, motor unit synchronization, or both. Author Contributions: All authors have contributed substantially to the manuscript including research design, data collection and analysis, and the editing and writing of the ﬁnal manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. 4.2. Procedures Neuromuscular Parameters Concentric PT Isometric PT BB BR BB BR EMG AMP (µV) Pretest 1.08 ± 0.20 1.04 ± 0.16 1.00 ± 0.00 1.00 ± 0.00 Posttest 1.12 ± 0.40 0.90 ± 0.16 1.05 ± 0.37 0.97 ± 0.17 EMG MPF (Hz) Pretest 1.05 ± 0.27 1.01 ± 0.12 1.00 ± 0.00 1.00 ± 0.00 Posttest 0.93 ± 0.24 0.97 ± 0.13 1.03 ± 0.27 0.96 ± 0.15 MMG AMP (m·s−2) Pretest 1.23 ± 0.42 1.21 ± 0.32 1.00 ± 0.00 1.00 ± 0.00 Posttest 1.42 ± 0.46 1.43 ± 0.38 1.12 ± 0.27 0.81 ± 0.22 MMG MPF (Hz) Pretest 1.23 ± 0.49 1.13 ± 0.38 1.00 ± 0.00 1.00 ± 0.00 Posttest 1.12 ± 0.35 1.19 ± 0.27 0.98 ± 0.33 0.95 ± 0.31 1. Basmajian, J.V. Muscles Alive, Their Functions Revealed by Electromyography; Williams & Wilkins: Philadelphia, PA, USA, 1978. References 1. Basmajian, J.V. Muscles Alive, Their Functions Revealed by Electromyography; Williams & Wilkins: Philadelphia, PA, USA, 1978. Sports 2016, 4, 47 8 of 10 2. De Luca, C.J. Physiology and mathematics of myoelectric signals. IEEE Trans. Biomed. Eng. 1979, 26, 313–325. [CrossRef] [PubMed] 2. De Luca, C.J. Physiology and mathematics of myoelectric signals. IEEE Trans. Biomed. Eng. 1979, 26, 313–325. [CrossRef] [PubMed] 3. Lindstrom, L.; Magnusson, R.; Petersen, I. Muscular fatigue and action potential conduction velocity changes studied with frequency analysis of EMG signals. Electromyography 1970, 10, 341–356. [PubMed] 4. Mills, K.R. Power spectral analysis of electromyogram and compound muscle action potential during muscle fatigue and recovery. J. Physiol. 1982, 326, 401–409. [CrossRef] [PubMed] 5. Gordon, G.; Holbourn, A.H.S. The sounds from single motor units in a contracting muscle. J. Physiol. Lond. 1948, 107, 456–464. [CrossRef] [PubMed] 6. Orizio, C. Muscle sound—Bases for the introduction of a mechanomyographic signal in muscle studies. Crit. Rev. Biomed. Eng. 1993, 21, 201–243. [PubMed] 7. Orizio, C.; Perini, R.; Veicsteinas, A. Muscular sound and force relationship during isometric contraction in man. Eur. J. Appl. Physiol. Occup. Physiol. 1989, 58, 528–533. [CrossRef] [PubMed] 8. Beck, T.W.; Housh, T.J.; Johnson, G.O.; Weir, J.P.; Cramer, J.T.; Coburn, J.W.; Malek, M.H. Mechanomyographic amplitude and mean power frequency versus torque relationships during isokinetic and isometric muscle actions of the biceps brachii. J. Electromyogr. Kinesiol. 2004, 14, 555–564. [CrossRef] [PubMed] 9. Farina, D.; Merletti, R.; Enoka, R.M. The extraction of neural strategies from the surface EMG. J. Appl. Physiol. 2004, 96, 1486–1495. [CrossRef] [PubMed] 10. Farina, D.; Merletti, R.; Enoka, R.M. The extraction of neural strategies from the surface EMG: An update. J. Appl. Physiol. 2014, 96, 1486–1495. [CrossRef] [PubMed] 11. Keenan, K.G.; Valero-Cuevas, F.J. Epoch length to accurately estimate the amplitude of interference EMG is likely the result of unavoidable amplitude cancellation. Biomed. Signal Process. Control 2008, 3, 154–162. [CrossRef] [PubMed] 12. Beck, T.W.; Housh, T.J.; Cramer, J.T.; Weir, J.P.; Johnson, G.O.; Coburn, J.W.; Malek, M.H.; Mielke, M. Mechanomyographic amplitude and frequency responses during dynamic muscle actions: A comprehensive review. Biomed. Eng. Online 2005, 4. [CrossRef] [PubMed] 13. Stokes, M.J.; Dalton, P.A. Acoustic myography for investigating human skeletal muscle fatigue. J. Appl. Physiol. 1991, 71, 1422–1426. [PubMed] Enoka, R.M.; Stuart, D.G. Neurobiology of muscle fatigue. J. Appl. Physiol. 1992, 72, 1631–1648. [PubMed] 14. Enoka, R.M.; Stuart, D.G. Neurobiology of muscle fatigue. J. Appl. Physiol. 1992, 72, 1631–1648. [PubMed] 15. References Hunter, S.K. Sex Differences and Mechanisms of Task-Speciﬁc Muscle Fatigue. Exerc. Sport Sci. Rev. 2009, 37, 113–122. [CrossRef] [PubMed] gy g pp y 15. Hunter, S.K. Sex Differences and Mechanisms of Task-Speciﬁc Muscle Fatigue. Exerc. Sport Sci. Rev. 2009, 37, 113–122. [CrossRef] [PubMed] 16. Camic, C.L.; Housh, T.J.; Zuniga, J.M.; Hendrix, C.R.; Bergstrom, H.C.; Traylor, D.A.; Schmidt, R.J.; Johnson, G.O. Electromyographic and mechanomyographic responses across repeated maximal isometric and concentric muscle actions of the leg extensors. J. Electromyogr. Kinesiol. 2013, 23, 342–348. [CrossRef] [PubMed] 17. Yeung, S.S.; Au, A.L.; Chow, C.C. Effects of fatigue on the temporal neuromuscular control of vastus medialis muscle in humans. Eur. J. Appl. Physiol. Occup. Physiol. 1999, 80, 379–385. [CrossRef] [PubMed] 18. Babault, N.; Desbrosses, K.; Fabre, M.-S.; Michaut, A.; Pousson, M. Neuromuscular fatigue development during maximal concentric and isometric knee extensions. J. Appl. Physiol. 2006, 100, 780–785. [CrossRef] [PubMed] 19. Crenshaw, A.G.; Karlsson, S.; Styf, J.; Backlund, T.; Friden, J. Knee extension torque and intramuscular pressure of the vastus lateralis muscle during eccentric and concentric activities. Eur. J. Appl. Physiol. Occup. Physiol. 1995, 70, 13–19. [CrossRef] [PubMed] 20. Seghers, J.; Spaepen, A. Muscle fatigue of the elbow ﬂexor muscles during two intermittent exercise protocols with equal mean muscle loading. Clin. Biomech. 2004, 19, 24–30. [CrossRef] 21. Mathiassen, S.E. The inﬂuence of exercise/rest schedule on the physiology and psychophysiological response to isometric shoulder-neck exercise. Eur. J. Appl. Physiol. Occup. Physiol. 1993, 67, 528–539. [CrossRef] [PubMed] 22. Perry-Rana, S.R.; Housh, T.J.; Johnson, G.O.; Bull, A.J.; Berning, J.M.; Cramer, J.T. MMG and EMG responses during fatiguing isokinetic muscle contractions at different velocities. Muscle Nerve 2002, 26, 367–373. [CrossRef] [PubMed] 23. Camic, C.L.; Housh, T.J.; Zuniga, J.M.; Bergstrom, H.C.; Schmidt, R.J.; Johnson, G.O. Mechanomyographic and electromyographic responses during fatiguing eccentric muscle actions of the leg extensors. J. Appl. Biomech. 2014, 30, 255–261. [CrossRef] [PubMed] 9 of 10 Sports 2016, 4, 47 24. Perry-Rana, S.R.; Housh, T.J.; Johnson, G.O.; Bull, A.J.; Cramer, J.T. MMG and EMG responses during 25 maximal, eccentric, isokinetic muscle actions. Med. Sci. Sports Exerc. 2003, 35, 2048–2054. [CrossRef] [PubMed] 25. Tesch, P.A.; Dudley, G.A.; Duvoisin, M.R.; Hather, B.M.; Harris, R.T. Force and EMG signal patterns during repeated bouts of concentric or eccentric muscle actions. Acta Physiol. Scand. 1990, 138, 263–271. [CrossRef] [PubMed] 26. Gray, J.C.; Chandler, J.M. Percent Decline in Peak Torque Production during Repeated Concentric and Eccentric Contractions of the Quadriceps Femoris Muscle. J. References Orthop. Sports Phys. Ther. 1989, 10, 309–314. [CrossRef] [PubMed] 27. Hill, E.C.; Housh, T.J.; Camic, C.L.; Smith, C.M.; Cochrane, K.C.; Jenkins, N.D.; Cramer, J.T.; Schmidt, R.J.; Johnson, G.O. The Effects of Velocity on Electromyographic, Mechanomyographic, and Torque Responses to Repeated Eccentric Muscle Actions. J. Strength Cond. Res. 2015, 30, 1743–1751. [CrossRef] [PubMed] 28. Hunter, S.K. Sex differences in human fatigability: Mechanisms and insight to physiological responses. Acta Physiol. Oxf. 2014, 210, 768–789. [CrossRef] [PubMed] 29. Barr, A.E.; Barbe, M.F. Pathophysiological Tissue Changes Associated With Repetitive Movement: A Review of the Evidence. Phys. Ther. 2002, 82, 173–187. [PubMed] 30. Sjøgaard, G.; Søgaard, K. Muscle Injury in Repetitive Motion Disorders. Clin. Orthop. Relat. Res. 19 21–31. [PubMed] 31. Järvinen, T.A.H.; Kannus, P.; Maffulli, N.; Khan, K.M. Achilles Tendon Disorders: Etiology and Epidemiology. Foot Ankle Clin. 2005, 10, 255–266. [CrossRef] [PubMed] 32. Rohmert, W. Problems in determining rest allowances Part 1: Use of modern methods to evaluate stress and strain in static muscular work. Appl. Ergon. 1973, 4, 91–95. [CrossRef] 33. Noakes, T.D. Implications of exercise testing for prediction of athletic performance: A contemporary perspective. Med. Sci. Sports Exerc. 1988, 20, 319–330. [CrossRef] [PubMed] 34. Camic, C.L. An Assessment of the Motor Control Strategies and Effects of Fatigue Speciﬁc to Isometric, Concentric, and Eccentric Muscle Actions. Available online: http://digitalcommons.unl.edu/dissertations/ AAI3449431/ (accessed on 28 September 2016). 5. Ettema, G.J.; Huijing, P.A. Skeletal muscle stiffness in static and dynamic contractions. J. Biomech. 1994 1361–1368. [CrossRef] 36. Edwards, R.H. Human muscle function and fatigue. Ciba Found. Symp. 1981, 82, 1–18. [PubMed] 37. Maclaren, D.P.; Gibson, H.; Parry-Billings, M.; Edwards, R.H. A review of metabolic and physiological factors in fatigue. Exerc. Sport Sci. Rev. 1989, 17, 29–66. [CrossRef] [PubMed] 38. Fortune, E.; Lowery, M.M. Effect of Extracellular Potassium Accumulation on Muscle Fiber Conduction Velocity: A Simulation Study. Ann. Biomed. Eng. 2009, 37, 2105–2117. [CrossRef] [PubMed] 39. Westerblad, H.; Allen, D.G.; Lannergren, J. Muscle fatigue: Lactic acid or inorganic phosphate the major cause? News Physiol. Sci. 2002, 17, 17–21. [PubMed] 40. Malek, M.H.; Housh, T.J.; Crouch, L.D.; Johnson, G.O.; Hendrix, C.R.; Beck, T.W.; Mielke, M.; Schmidt, R.J.; Housh, D.J. Plasma ammonia concentrations and the slow component of oxygen uptake kinetics during cycle ergometry. J. Strength Cond. Res. 2008, 22, 2018–2026. [CrossRef] [PubMed] y g y g 41. Gladden, L.B. Lactate metabolism: A new paradigm for the third millennium. J. Physiol. 2004, 558, 5–30. [CrossRef] [PubMed] 42. References Robergs, R.A.; Ghiasvand, F.; Parker, D. Biochemistry of exercise-induced metabolic acidosis. A Regul. Integr. Comp. Physiol. 2004, 287, R502–R516. [CrossRef] [PubMed] 42. Robergs, R.A.; Ghiasvand, F.; Parker, D. Biochemistry of exercise-induced metabolic acidosis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2004, 287, R502–R516. [CrossRef] [PubMed] 43. Marsden, C.D.; Meadows, J.C.; Merton, P.A. “Muscular wisdom” that minimizes fatigue during prolonged effort in man: Peak rates of motoneuron discharge and slowing of discharge during fatigue. Adv. Neurol. 1983, 39, 169–211. [PubMed] 43. Marsden, C.D.; Meadows, J.C.; Merton, P.A. “Muscular wisdom” that minimizes fatigue during prolonged effort in man: Peak rates of motoneuron discharge and slowing of discharge during fatigue. Adv. Neurol. 1983, 39, 169–211. [PubMed] 44. Garland, S.J.; Gossen, E.R. The muscular wisdom hypothesis in human muscle fatigue. Exerc. Sport Sci. Rev. 2002, 30, 45–49. [CrossRef] [PubMed] 44. Garland, S.J.; Gossen, E.R. The muscular wisdom hypothesis in human muscle fatigue. Exerc. Sport Sci. Rev. 2002, 30, 45–49. [CrossRef] [PubMed] 45. Fuglevand, A.J. Neural Aspects of Fatigue. Neuroscientist 1996, 2, 203–206. [CrossRef] 46. Fuglevand, A.J.; Keen, D.A. Re-evaluation of muscle wisdom in the human adductor pollicis using physiological rates of stimulation. J. Physiol. 2003, 549, 865–875. [CrossRef] [PubMed] 10 of 10 10 of 10 Sports 2016, 4, 47 47. Fuglevand, A.J.; Zackowski, K.M.; Huey, K.A.; Enoka, R.M. Impairment of neuromuscular propagation during human fatiguing contractions at submaximal forces. J. Physiol. 1993, 460, 549–572. [CrossRef] [PubMed] 8. De Luca, C.J.; Lefever, R.S.; McCue, M.P.; Xenakis, A.P. Control scheme governing concurrently active hu motor units during voluntary contractions. J. Physiol. (Lond.) 1982, 329, 129–142. [CrossRef] 49. De Luca, C.J.; Erim, Z. Common drive of motor units in regulation of muscle force. Trends Neurosci. 1994, 17, 299–305. [CrossRef] 50. Kline, J.C.; de Luca, C.J. Synchronization of motor unit ﬁrings: An epiphenomenon of ﬁring rate characteristics not common inputs. J. Neurophysiol. 2016, 115, 178–192. [CrossRef] [PubMed] 51. Nigg, B.M.; MacIntosh, B.R.; Mester, J. Biomechanics and Biology of Movement; Human Kinetics: Champaign, IL, USA, 2000. 52. Defreitas, J.M.; Beck, T.W.; Ye, X.; Stock, M.S. Synchronization of low- and high-threshold motor units. Muscle Nerve 2014, 49, 575–583. [CrossRef] [PubMed] 53. Kleine, B.U.; Stegeman, D.F.; Mund, D.; Anders, C. Inﬂuence of motoneuron ﬁring synchronization on SEMG characteristics in dependence of electrode position. J. Appl. Physiol. 2001, 91, 1588–1599. [PubMed] 54. Yao, W.X.; Fuglevand, A.J.; Enoka, R.M. Motor-unit synchronization increases EMG amplitude and decreases force steadiness of simulated contractions. J. Neurophysiol. References 2000, 83, 441–452. [PubMed] 55. Semmler, J.G. Motor unit synchronization and neuromuscular performance. Exerci. Sport Sci. Rev. 2002, 30, 8–14. [CrossRef] 56. Brown, L.E. Isokinetics in Human Performance; Human Kinetics: Champaign, IL, USA, 2000. 57. Orizio, C.; Perini, R.; Diemont, B.; Figini, M.M.; Veicsteinas, A. Spectral analysis of muscular sound during isometric contraction of biceps brachii. J. Appl. Physiol. 1990, 68, 508–512. [PubMed] 58. Orizio, C.; Gobbo, M.; Diemont, B.; Esposito, F.; Veicsteinas, A. The surface mechanomyogram as a tool to describe the inﬂuence of fatigue on biceps brachii motor unit activation strategy. Historical basis and novel evidence. Eur. J. Appl. Physiol. 2003, 90, 326–336. [CrossRef] [PubMed] 59. Kang, T.; Seo, Y.; Park, J.; Dong, E.; Seo, B.; Han, D. The effects of elbow joint angle change on the elbow ﬂexor muscle activation in pulley with weight exercise. J. Phys. Ther. Sci. 2013, 25, 1133–1136. [CrossRef] [PubMed] 60. Yang, J.; Lee, J.; Lee, B.; Kim, S.; Shin, D.; Lee, Y.; Lee, J.; Han, D.; Choi, S. The Effects of Elbow Joint Angle Changes on Elbow Flexor and Extensor Muscle Strength and Activation. J. Phys. Ther. Sci. 2014, 26, 1079–1082. [CrossRef] [PubMed] 61. Barbero, M.; Merletti, R.; Rainoldi, A.; Jull, G.A. Atlas of Muscle Innervation Zones: Understanding Surface Electromyography and Its Applications; Springer: Milan, Italy, 2012. 62. Brown, L.E.; Whitehurst, M.; Gilbert, R.; Buchalter, D.N. The effect of velocity and gender on load range during knee extension and ﬂexion exercise on an isokinetic device. J. Orthop. Sports Phys. Ther. 1995, 21, 107–112. [CrossRef] [PubMed] 63. Diemont, B.; Figini, M.M.; Orizio, C.; Perini, R.; Veicsteinas, A. Spectral analysis of muscular sound at low and high contraction level. Int. J. Biomed. Comput. 1988, 23, 161–175. [CrossRef] 63. Diemont, B.; Figini, M.M.; Orizio, C.; Perini, R.; Veicsteinas, A. Spectral analysis of muscular sound at low and high contraction level. Int. J. Biomed. Comput. 1988, 23, 161–175. [CrossRef] 64. Kwatny, E.; Thomas, D.H.; Kwatny, H.G. An application of signal processing techniques to the study of myoelectric signals. IEEE Trans. Biomed. Eng. 1970, 17, 303–313. [CrossRef] [PubMed] 65. Hermens, H.J.; Freriks, B.; Disselhorst-Klug, C.; Rau, G. Development of recommendations for SEMG sensors 64. Kwatny, E.; Thomas, D.H.; Kwatny, H.G. An application of signal processing techniques to the study of myoelectric signals. IEEE Trans. Biomed. Eng. 1970, 17, 303–313. [CrossRef] [PubMed] 64. Kwatny, E.; Thomas, D.H.; Kwatny, H.G. References An application of signal processing techniques to the study of myoelectric signals. IEEE Trans. Biomed. Eng. 1970, 17, 303–313. [CrossRef] [PubMed] 65. Hermens, H.J.; Freriks, B.; Disselhorst-Klug, C.; Rau, G. Development of recommendations for SEMG sensors and sensor placement procedures. J. Electromyogr. Kinesiol. 2000, 10, 361–374. [CrossRef] 65. Hermens, H.J.; Freriks, B.; Disselhorst-Klug, C.; Rau, G. Development of recommendations for SEMG sensors and sensor placement procedures. J. Electromyogr. Kinesiol. 2000, 10, 361–374. [CrossRef] © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4213162651	https://www.zora.uzh.ch/id/eprint/218061/1/PhysRevD.105.L031904.pdf	English	null	Møller scattering at NNLO	Physical review. D/Physical review. D.	2,022	cc-by	5,442	Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2022 Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. Møller scattering at NNLO Pulak Banerjee ,1 Tim Engel ,1,2 Nicolas Schalch,3 Adrian Signer ,1,2 and Yannick Ulrich 4 1Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland 2Physik-Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland 3Albert Einstein Center for Fundamental Physics, Institut für Theoretische Physik, Universität Bern, Sidlerstrasse 5, CH-3012 Bern, Switzerland 4Institute for Particle Physics Phenomenology, University of Durham, South Road, Durham DH1 3LE, United Kingdom Pulak Banerjee ,1 Tim Engel ,1,2 Nicolas Schalch,3 Adrian Signer ,1,2 and Yannick Ulrich 4 1Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland 2Physik-Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland 3Albert Einstein Center for Fundamental Physics, Institut für Theoretische Physik, Universität Bern, Sidlerstrasse 5, CH-3012 Bern, Switzerland 4Institute for Particle Physics Phenomenology, University of Durham, South Road, Durham DH1 3LE, United Kingdom Pulak Banerjee ,1 Tim Engel ,1,2 Nicolas Schalch,3 Adrian Signer ,1,2 and Yannick Ulrich 4 1Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland 2Physik-Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland 3Albert Einstein Center for Fundamental Physics, Institut für Theoretische Physik, Universität Bern, Sidlerstrasse 5, CH-3012 Bern, Switzerland 4Institute for Particle Physics Phenomenology, University of Durham, South Road, Durham DH1 3LE, United Kingdom (Received 2 August 2021; accepted 12 January 2022; published 23 February 2022) We present a calculation of the full set of next-to-next-to-leading-order QED corrections to unpolarized Møller scattering. This encompasses photonic, leptonic, and nonperturbative hadronic corrections and includes electron mass effects as well as hard photon radiation. The corresponding matrix elements are implemented in the Monte Carlo framework MCMULE allowing for the computation of fully differential observables. As a first application we show results tailored to the kinematics and detector design of the PRad II experiment where a high-precision theory prediction for Møller scattering is required to achieve the targeted precision. We observe that the corrections become essential to reliably calculate the corresponding differential distributions especially in regions where the leading-order contribution is absent. Møller scattering at NNLO DOI: https://doi.org/10.1103/physrevd.105.l031904 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-218061 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Banerjee, Pulak; Engel, Tim; Schalch, Nicolas; Signer, Adrian; Ulrich, Yannick (2022). Møller scattering at NNLO. Physical review D, 105(3):L031904. DOI: https://doi.org/10.1103/physrevd.105.l031904 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-218061 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4 0 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-218061 Journal Article Published Version Originally published at: Banerjee, Pulak; Engel, Tim; Schalch, Nicolas; Signer, Adrian; Ulrich, Yannick (2022). Møller scattering at NNLO. Physical review D, 105(3):L031904. DOI: https://doi.org/10.1103/physrevd.105.l031904 PHYSICAL REVIEW D 105, L031904 (2022) Letter L031904-1 Published by the American Physical Society DOI: 10.1103/PhysRevD.105.L031904 I. INTRODUCTION experiments rely on Monte Carlo event generators that combine matrix elements to physical observables. At NLO accuracy in QED such Monte Carlo codes have been developed [8–10]. While for most experiments this level of precision is sufficient, the situation is different for the planned PRad II experiment [7], the upgraded version of its predecessor PRad [6]. Recent developments at the low-energy precision fron- tier have renewed interest in high-precision calculations in QED. In order to meet the experimental accuracy when using state-of-the-art lepton beams, next-to-leading-order (NLO) or even next-to-next-to-leading-order (NNLO) cor- rections in QED have become highly desirable. Both experiments measure the elastic scattering of electrons and protons to extract the proton charge radius. These efforts were triggered after a recent high-precision spectroscopy experiment using muonic hydrogen at the Paul Scherrer Institute [11,12] had measured a significantly smaller value for the proton charge radius compared to earlier results [13]. The PRad and PRad II experiments therefore play an important role in the resolution of this “proton radius puzzle.” Electron-electron or Møller scattering is a prime example where a high-precision theory prediction has become important. As it is a ubiquitous process at electron beam lines, it has been investigated in connection with luminosity measurements [1,2] and background studies [3], including a recent dedicated measurement at very low energies to study electron mass effects [4]. Precise knowledge of QED effects might also be useful for the MOLLER experiment [5] that searches for parity violation as an indication toward new physics. The main motivation, however, is given by PRad [6,7] to which we turn below. A key feature of the PRad experiments is the suppression of systematic uncertainties by normalizing to a simulta- neous measurement of Møller scattering. To be precise, the differential cross section for electron-proton scattering ðdσ=dθÞexp ep is extracted from the measured events Nexp and the theoretical prediction for the Møller cross section ðdσ=dθÞth ee via [14] All of these experiments rely on Standard Model theory predictions for Møller scattering. Since at low energies the corresponding radiative corrections are QED-dominated, a high-precision theory calculation is feasible. To account for nontrivial detector geometries and acceptances these dσ dθ exp ep ¼ Nexpðe−p →e−pÞ Nexpðe−e−→e−e−Þ dσ dθ th ee : ð1Þ ð1Þ It is therefore essential that the theory prediction for Møller scattering matches the experimental precision. Published by the American Physical Society BANERJEE, ENGEL, SCHALCH, SIGNER, and ULRICH integrating the tree-level matrix element (squared) with two additional photons in the final sate over the photon phase space. Second, real-virtual corrections require the interfer- ence of the one-loop amplitude and tree-level amplitude with one additional photon. Finally, the double-virtual corrections involve the interference of the two-loop ampli- tude for ee →ee with its tree-level amplitude as well as the corresponding one-loop amplitude squared. These three individual parts are physically indistinguishable for soft photons and are infrared divergent. Only the combination of all contributions leads to a finite, physical result. As alluded to before, the existing NLO Monte Carlo event generators were sufficient for PRad. This, however, is not the case for PRad II that aims at improving the experimental precision significantly. It is therefore expected that missing higher-order QED corrections become the dominant source of systematic uncertainty [7]. We have therefore calculated the full set of NNLO QED corrections to Møller scattering. This includes not only photonic and leptonic corrections but also nonperturbative hadronic contributions. The corresponding matrix elements wereimplementedinthe MCMULEframework, a MonteCarlo for muons and other leptons [15]. This allows for the computation of arbitrary fully differential observables. As alluded to before, the existing NLO Monte Carlo event generators were sufficient for PRad. This, however, is not the case for PRad II that aims at improving the experimental precision significantly. It is therefore expected that missing higher-order QED corrections become the dominant source of systematic uncertainty [7]. We have therefore calculated the full set of NNLO QED corrections to Møller scattering. This includes not only photonic and leptonic corrections but also nonperturbative hadronic contributions. The corresponding matrix elements wereimplementedinthe MCMULEframework, a MonteCarlo for muons and other leptons [15]. This allows for the computation of arbitrary fully differential observables. The corrections can be split into purely photonic con- tributions and fermionic ones that are due to vacuum polarization (VP). The fermionic part takes into account all three lepton flavors as well as nonperturbative hadronic effects, indicated by the grey blob in Fig. 1(b). The corresponding method will be presented in the second part of this section after discussing our approach toward photonic corrections first. The double-real contribution of the fermionic part, shown top left in Fig. 1(b) corre- sponds to the process This paper is organized as follows: we begin by briefly summarizing our calculational methods in Sec. II. BANERJEE, ENGEL, SCHALCH, SIGNER, and ULRICH Next, we present results tailored to the kinematics and detector design of the PRad II experiment in Sec. III and conclude in Sec. IV. II. CALCULATION We have calculated the full set of NNLO QED correc- tions to the process e−ðp1Þe−ðp2Þ →e−ðp3Þe−ðp4Þlþðp5Þl−ðp6Þ ð3Þ ð3Þ e−ðp1Þe−ðp2Þ →e−ðp3Þe−ðp4Þfγðp5Þγðp6Þg ð2Þ with additional leptons (or hadrons) in the final state. Since this is a final state with a measurable difference it can be disentangled from Møller scattering. It is separately finite if fermion masses are not neglected. Hence, we have chosen not to include the double-real fermionic part in our results presented below. including effects due to the nonvanishing electron mass. These corrections are composed of three parts, as illustrated in Fig. 1. First, double-real corrections are obtained by (a) (b) FIG. 1. Representative contributions to the squared amplitude, double-real (top left), real-virtual (top right), and double-virtual involving the two-loop amplitude (bottom left) and one-loop amplitude squared (bottom right). (a) Examples of photonic correc- tions at NNLO and (b) examples of fermionic corrections at NNLO. (a) (a) The matrix elements are regularized in d ¼ 4 −2ϵ dimensions and renormalized in the on-shell scheme. They are implemented in the parton-level integrator MCMULE that is based on FKSl [16], a QED extension of the FKS subtraction scheme [17,18] beyond NLO. MCMULE allows to consistently perform the phase-space integration in the presence of soft singularities and to calculate physical observables in a fully differential way. (a) I. INTRODUCTION 04(7) L031904-1 Published by the American Physical Society 2470-0010=2022=105(3)=L031904(7) L031904-1 Published by the American Physical Society 2470-0010=2022=105(3)=L031904(7) L031904-1 PHYS. REV. D 105, L031904 (2022) BANERJEE, ENGEL, SCHALCH, SIGNER, and ULRICH A. Photonic corrections (b) All tree-level and one-loop photonic matrix elements were calculated analytically with the full dependence on the electron mass. The diagrams were generated with QGraf [19] and calculated with the Mathematica code PACKAGE-X [20]. While for most of the obtained expressions a sufficiently stable and efficient implementation was pos- sible, a different approach had to be taken in the case of the numerically delicate real-virtual matrix element. In this case next-to-soft stabilization, previously developed in the context of Bhabha scattering [21], was used. This method is based on the observation that the numerical instabilities are strongly enhanced when the emitted photon becomes soft. We have therefore expanded the real-virtual matrix element for small photon energies including the nonuni- versal next-to-soft contribution. This allows us to rely on (b) FIG. 1. Representative contributions to the squared amplitude, double-real (top left), real-virtual (top right), and double-virtual involving the two-loop amplitude (bottom left) and one-loop amplitude squared (bottom right). (a) Examples of photonic correc- tions at NNLO and (b) examples of fermionic corrections at NNLO. L031904-2 PHYS. REV. D 105, L031904 (2022) MØLLER SCATTERING AT NNLO OpenLoops [22,23] in its standard mode for the bulk of the phase space and otherwise to switch to the next-to-soft approximation. This approach therefore yields a stable and fast implementation of the problematic real-virtual contribution. OpenLoops [22,23] in its standard mode for the bulk of the phase space and otherwise to switch to the next-to-soft approximation. This approach therefore yields a stable and fast implementation of the problematic real-virtual contribution. parton shower algorithm matched to the exact NLO result [33]. As presented in [21], we have found exact agreement at NLO and a deviation at the level of 17% for the NNLO correction, consistent with the expected precision of the logarithmic approximation from the parton shower. The two-loop matrix element for Møller scattering can be derived from corresponding Bhabha results via the crossing relation p2 ↔−p4. However, even though Bhabha scattering is one of the best studied processes in particle physics, the exact two-loop contribution is cur- rently not known. The approximation where the electron mass m is set to zero was calculated some time ago [24]. Building upon this result it was later possible to determine the leading mass effects based on the universal factorizing structure of collinear divergences [25–27]. B. Fermionic corrections As mentioned above it is not sensible to apply the methods used for photonic corrections to the fermionic contributions because of the occurrence of the factorization anomaly in the massification procedure. Moreover, at low energies nonperturbative hadronic contributions become relevant. Fortunately, all leptonic and hadronic contribu- tions to Møller scattering are due to VP insertions. We can therefore calculate all of them simultaneously by including all contributions in the VP Π ¼ Πe þ Πμ þ Πτ þ Πhad: ð4Þ ð4Þ The one-loop contribution to the leptonic VP can be calculated trivially and the two-loop result can be extracted from [34]. For the nonperturbative hadronic VP, on the other hand, we rely on the Fortran library alphaQED [35–37] that calculates Πhad based on experimental data. The full photon propagator −igμν q2ð1 −Πðq2ÞÞ ¼ −igμν q2 ð1 þ Πðq2Þ þ Π2ðq2Þ þ Þ ð5Þ ð5Þ automatically resums VP insertions to all orders in pertur- bation theory. In this paper, however, we take a strict fixed- order approach for all contributions. In what follows we therefore use the expanded version of the photon propagator given on the right-hand side of (5). The impact of missing higher-order effects—together with resummation of soft and collinear photon emission—is left for future studies. The analytic continuation that has to be performed after crossing is nontrivial. Fortunately, this step was de facto already performed in the case of the massless two-loop matrix element [24] as it was calculated for all three possible kinematic channels for 2 →2 processes. It was therefore straightforward to construct the massless two- loop matrix element for Møller scattering based on this. We then massified the corresponding expression ourselves arriving at the desired result. For a large class of diagrams, such as the two diagrams on the right of Fig. 1(b), the VP contributions factorize and can thus be calculated straightforwardly based on photonic NLO matrix elements. The verification of the photonic corrections described in Sec. II A therefore also presents a strong check in this case. The calculation of the non- factorizable vertex and box diagrams, however, is more involved. Traditionally these contributions are calculated dispersively, see e.g., [38]. In our case we have instead used the hyperspherical formalism [39,40] where it was possible to reuse many of the results of the analogous calculation in muon-electron scattering [41]. A. Photonic corrections In this approxi- mation power suppressed terms of Oðm2=q2Þ are neglected with q2 corresponding to any of the Mandelstam invariants s ¼ ðp1 þ p2Þ2, t ¼ ðp1 −p3Þ2, and u ¼ ðp1 −p4Þ2. Even for low-energy observables this approximation is well justified due to the small electron mass. This massi- fication therefore allows us to straightforwardly calculate both the logarithmically enhanced and the constant mass terms based on the massless result. Recently, this procedure was extended to processes that include a heavy mass [28]. In this more general approach a factorization anomaly [29,30] was discovered in fermionic contributions resulting in the breaking of naive factorization. This in turn explains the occurrence of additional powers of large logarithms that result in a larger massification error. We have therefore only used the massification procedure for the photonic two-loop contribution and followed a different approach in the fermionic case. III. RESULTS As expected, the results for the fiducial and the simple cuts agree for the elastic corrections σð0Þ and σð1Þ VP. We further observe large NLO contributions compared to the naive counting of the expansion parameter α=π. The NNLO K factors, on the other hand, are much smaller and in agreement with the naive prediction. For the total cross section we can thus conclude that the NNLO result yields a robust prediction for the considered observable with miss- ing higher-order corrections well under control. Comparing the fiducial and simple results reveals that the inelasticity and coplanarity cut have a noticeable impact on the higher- order corrections. If hard photon emission was even more severely restricted, the NNLO corrections, too, would become large and the resummation of corresponding large logarithms would be required. As expected, the results for the fiducial and the simple cuts agree for the elastic corrections σð0Þ and σð1Þ VP. We further observe large NLO contributions compared to the naive counting of the expansion parameter α=π. The NNLO K factors, on the other hand, are much smaller and in agreement with the naive prediction. For the total cross section we can thus conclude that the NNLO result yields a robust prediction for the considered observable with miss- ing higher-order corrections well under control. Comparing the fiducial and simple results reveals that the inelasticity and coplanarity cut have a noticeable impact on the higher- order corrections. If hard photon emission was even more severely restricted, the NNLO corrections, too, would become large and the resummation of corresponding large logarithms would be required. With the contributions discussed in the previous section implemented in MCMULE we can compute any infrared safe observable of Møller scattering at NNLO in QED. g To illustrate this, as a first application we use a kinematic set up tailored to the PRad II experiment. We consider an electron beam with energy Eb ¼ 1.4 GeV incident on a target electron at rest. We refer to the outgoing electron with the smaller (larger) scattering angle as the “narrow” (“wide”) electron. The corresponding scattering angles are denoted by θn and θw, respectively. We further define the inelasticity η ¼ Eb þ m −En −Ew and the coplanarity ζ ¼ j180° −jϕn −ϕwjj with Ei and ϕi the energy and azimuth of the narrow and wide electrons. B. Fermionic corrections In this approach the non- factorizable two-loop contribution is written as an integral over the radial part of the Wick-rotated loop momentum We have verified the nonradiative part of our calculation at NLO by comparing to corresponding results in [31] and found exact agreement. We have further compared to full NLO results provided by [32] using the Monte Carlo code from [10]. On account of this code being an event generator and not a full dedicated integrator, we found agreement, up to Monte Carlo errors, at the level of precision achieved. To still allow for a robust check of our calculation we have implemented all photonic matrix elements in MCMULE such that they can directly be crossed to Bhabha scattering. This then allows us to verify our calculation indirectly by comparing to the much richer literature of Bhabha scatter- ing. In particular, we have compared to the state-of-the-art Monte Carlo generator BABAYAGA that is based on a Mnon−fac VP ¼ Z ∞ 0 dQ2Πð−Q2ÞKðQ2; s; t; uÞ ð6Þ ð6Þ which can be performed numerically. The kernel function K results from the analytic integration over the solid angle L031904-3 BANERJEE, ENGEL, SCHALCH, SIGNER, and ULRICH PHYS. REV. D 105, L031904 (2022) TABLE I. The integrated cross section for the fiducial and the simplified cuts at LO, NLO, and NNLO. All digits are significant compared to the numerical integration error. of the loop momentum. In the case of triangles this integration can be elegantly performed after writing the propagators in terms of Gegenbauer polynomials and making use of the corresponding orthogonality relation. For box integrals, however, this method does not work and the corresponding calculation is therefore more compli- cated. In this case the general result can be found in [42]. σ=μb δKðiÞ=% Fiducial Simple Fiducial Simple σð0Þ 2291.02 2291.02 σð1Þ γ −148.36 78.23 −6.476 3.415 σð1Þ VP 19.33 19.33 0.844 0.844 σð2Þ γ 2.82 −0.17 0.131 −0.007 σð2Þ VP −1.31 −0.06 −0.061 −0.002 σ2 2163.50 2388.35 The main advantage of the hyperspherical approach compared to the dispersive calculation is that the VP is integrated over spacelike momenta avoiding hadronic resonances. Furthermore, the threshold singularities are transformed into logarithmic divergences in the kernel functions through the angular integration. Nevertheless, the numerical integration over Q2 has to be performed with care. Since large cancellations can occur in the kernel functions, they have to be expanded in these problematic regions. B. Fermionic corrections In particular, this is the case for Q2 →0, Q2 →∞, and around threshold singularities. corresponding restrictions for the t- and u-channel momen- tum transfers using the kinematic relations for elastic scattering. The corresponding cuts for this simplified scenario then read We have verified our calculation of the nonfactorizable fermionic contributions by comparing to corresponding crossed results for Bhabha scattering. First of all, we have obtained complete agreement in the case of closed electron loops with the exact analytic calculation in [43]. Furthermore, we have compared the muon and the tau contributions to approximate results from [44] that neglect terms of Oðm2 f=q2Þ with mf the mass of any of the fermions and q2 ∈fs; t; ug. As expected we do not find exact agreement but instead observe the correct converging behavior when the energy scale q2 is increased. −1295 MeV2 ≲t; u ≲−135 MeV2: ð8Þ ð8Þ The order-by-order contributions, σðiÞ, to the integrated cross section, σ2 ¼ σð0Þ þ σð1Þ þ σð2Þ, for both the fiducial and the simplified cuts are presented in Table I. The photonic and fermionic corrections are given separately and are denoted by σðiÞ γ and σðiÞ VP, respectively. Additionally, we show the corresponding K factors defined as KðiÞ ¼ 1 þ δKðiÞ ¼ σi σi−1 : ð9Þ ð9Þ III. RESULTS Both quantities are zero for elastic events and can therefore be used to restrict hard photon emission. We approximate the experimental design with the cuts For the more realistic fiducial observable we present differential distributions in Fig. 2. Figure 2(a) shows the corresponding results with respect to the energy of the “narrow” electron. The angular distributions for both the narrow and wide electron are given in Fig. 2(b). For both figures the differential cross section is displayed in the upper panel. In addition, the middle panel shows the differential version of the K factor defined in (9). In regions where the LO cross section is zero the NNLO 0.5° < θn; θw < 6.5°; η < 3.5σE; ζ < 3.5σϕ; ð7Þ ð7Þ with σE ¼ 37.7 MeV and σϕ ¼ 2.1° the expected detector resolutions for the considered beam energy [45]. In addition to this fiducial observable we also consider a simpler version that does not restrict hard photon radiation. To this end we convert the angular cuts in (7) to L031904-4 MØLLER SCATTERING AT NNLO PHYS. REV. D 105, L031904 (2022) (a) (b) FIG. 2. Differential cross section for the fiducial observable defined in (7). (a) Energy distribution of the “narrow” electron and (b) angular distribution of the “narrow” and the “wide” electron. (a) nonperturbative hadronic corrections. The fermionic part was calculated with a seminumerical approach using the hyperspherical formalism without any approximation. The photonic two-loop matrix element, on the other hand, was computed based on the crossed massless Bhabha result via massification resulting in a parametrically small error of Oðα2m2=q2Þ relative to LO. Apart from the calculation of the two-loop matrix element, the main challenge in the computation of the photonic corrections is the numerical stability of the real-virtual matrix element. We have obtained a fast and stable implementation of this delicate contribution using a combination of OpenLoops and the next- to-soft stabilization method recently developed in the context of Bhabha scattering. All matrix elements were implemented in the parton- level Monte Carlo integrator MCMULE. While not an event generator, this framework allows us to compute arbitrary fully differential observables by consistently removing soft divergences originating in the phase-space integration. As a first application of our NNLO calculation we have gen- erated integrated and differential cross sections relevant for the planned PRad II experiment. ACKNOWLEDGMENTS We are grateful to A. Antognini for calling to our attention the necessity of the calculation and assuring us that anyone can do it. We thank the PRad collaboration for providing the kinematical configuration relevant for the PRad II experiment. We are indebted to C. Carloni Calame for engaging in a detailed comparison with BABAYAGA. We also thank C. Epstein for the comparison with his NLO event generator. It is further a pleasure to thank M. Fael for his advice regarding the hyperspherical formalism and for helping us verify the corresponding results. We are also grateful to M. Zoller for his help in using OpenLoops. Our thanks are further extended to P. Mastrolia and R. Bonciani for providing the analytic results for the massive fermionic two-loop contributions in electronic form. Last but not least, we thank L. Dixon for a digital version of the massless two-loop matrix element. P. B. and T. E. acknowl- edge support by the Swiss National Science Foundation (SNF) under Contracts No. 200021_178967. P. B. further acknowledges support by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 701647. Y. U. acknowledges support by the UK Science and Technology Facilities Council (STFC) under Grant No. ST/T001011/1. contribution effectively corresponds to a NLO correction resulting in a large K factor. For this reason the lower panel zooms into the other region where the NNLO K factor is small. In this case we observe a similar behavior as for the total cross section with large NLO and small NNLO corrections. Contrary to the integrated case, however, the NNLO calculation is indispensable for distributions in order to obtain a reliable prediction also in the region where the LO contribution is zero. We have presented integrated and differential cross sections for only one possible scenario for PRad II. Other configurations of interest to PRad and PRad II will be studied in the future and made available on https://mule- tools.gitlab.io/user-library. The data that enter the results presented in this section can also be found there. III. RESULTS For the total cross section we obtain large NLO and small NNLO corrections ensuring a high-precision prediction. In the case of differential distributions the situation is more subtle. In the absence of a LO contribution the NNLO correction becomes essential to achieve a moderate precision of about 10%. To improve this theory accuracy further, missing higher- order corrections could be approximated with a parton shower. (b) FIG. 2. Differential cross section for the fiducial observable defined in (7). (a) Energy distribution of the “narrow” electron and (b) angular distribution of the “narrow” and the “wide” electron. IV. CONCLUSION We have calculated the full set of NNLO QED correc- tions to Møller scattering including photonic, leptonic, and L031904-5 PHYS. REV. D 105, L031904 (2022) BANERJEE, ENGEL, SCHALCH, SIGNER, and ULRICH [1] A. Schmidt, C. O’Connor, J. C. Bernauer, and R. Milner, A novel technique for determining luminosity in electron-scattering/positron-scattering experiments from multi-interaction events, Nucl. Instrum. Methods Phys. Res., Sect. A 877, 112 (2018). [20] H. H. Patel, PACKAGE-X: A mathematica package for the analytic calculation of one-loop integrals, Comput. Phys. Commun. 197, 276 (2015). [21] P. Banerjee, T. Engel, N. Schalch, A. Signer, and Y. Ulrich, Bhabha scattering at NNLO with next-to-soft stabilisation, Phys. Lett. B 820, 136547 (2021). [2] T. Benisch, S. Bernreuther, E. Devitsin, V. Kozlov, S. Potashov, K. Rith, A. Terkulov, and Ch. Weiskopf, The luminosity monitor of the HERMES experiment at DESY, Nucl. Instrum. Methods Phys. Res., Sect. A 471, 314 (2001). [22] F. Buccioni, S. Pozzorini, and M. Zoller, On-the-fly reduc- tion of open loops, Eur. Phys. J. C 78, 70 (2018). [23] F. Buccioni, J.-N. Lang, J. M. Lindert, P. Maierhöfer, S. Pozzorini, H. Zhang, and M. F. Zoller, OpenLoops 2, Eur. Phys. J. C 79, 866 (2019). [3] R. Alarcon et al. (The DarkLight Collaboration), Search for New Physics in eþe−Final States Near an Invariant Mass of 17 MeV Using the CEBAF Injector. [24] Z. Bern, L. J. Dixon, and A. Ghinculov, Two loop correction to Bhabha scattering, Phys. Rev. D 63, 053007 (2001). g j [4] C. S. Epstein et al., Measurement of Møller scattering at 2.5 MeV, Phys. Rev. D 102, 012006 (2020). [25] A. A. Penin, Two-loop photonic corrections to massive Bhabha scattering, Nucl. Phys. B734, 185 (2006). [26] A. Mitov and S. Moch, The singular behavior of massive QCD amplitudes, J. High Energy Phys. 05 (2007) 001. [5] J. Benesch et al. (MOLLER Collaboration), The MOLLER experiment: An ultra-precise measurement of the weak mixing angle using Møller scattering, arXiv:1411.4088. [27] T. Becher and K. Melnikov, Two-loop QED corrections to Bhabha scattering, J. High Energy Phys. 06 (2007) 084. [6] W. Xiong et al., A small proton charge radius from an electron–proton scattering experiment, Nature (London) 575, 147 (2019). [28] T. Engel, C. Gnendiger, A. Signer, and Y. Ulrich, Small- mass effects in heavy-to-light form factors, J. High Energy Phys. 02 (2019) 118. [7] A. Gasparian et al. IV. CONCLUSION (PRad Collaboration), PRad-II: A new upgraded high precision measurement of the proton charge radius, arXiv:2009.10510. [29] T. Becher and M. Neubert, Drell-Yan production at small qT, transverse parton distributions and the collinear anomaly, Eur. Phys. J. C 71, 1665 (2011). [8] A. Afanasev, E. Chudakov, A. Ilyichev, and V. Zykunov, MERADGEN 1.0: Monte Carlo generator for the simulation of radiative events in polarized Moller scattering, Comput. Phys. Commun. 176, 218 (2007). [30] T. Becher, G. Bell, and M. Neubert, Factorization and resummation for jet broadening, Phys. Lett. B 704, 276 (2011). y [9] I. Akushevich, H. Gao, A. Ilyichev, and M. Meziane, Radiative corrections beyond the ultra relativistic limit in unpolarized ep elastic and Møller scatterings for the PRad Experiment at Jefferson Laboratory, Eur. Phys. J. A 51, 1 (2015). [31] N. Kaiser, Radiative corrections to lepton-lepton scattering revisited, J. Phys. G 37, 115005 (2010). [32] C. S. Epstein (private communication). [33] G. Balossini, C. M. Carloni Calame, G. Montagna, O. Nicrosini, and F. Piccinini, Matching perturbative and parton shower corrections to Bhabha process at flavour factories, Nucl. Phys. B758, 227 (2006). [10] C. S. Epstein and R. G. Milner, QED radiative corrections to low-energy Møller and Bhabha scattering, Phys. Rev. D 94, 033004 (2016). [34] A. Djouadi and P. Gambino, Electroweak gauge bosons self- energies: Complete QCD corrections, Phys. Rev. D 49, 3499 (1994). [11] R. Pohl et al., The size of the proton, Nature (London) 466, 213 (2010). [35] F. Jegerlehner, The effective fine structure constant at TESLA energies, arXiv:hep-ph/0105283. [12] A. Antognini et al., Proton structure from the measurement of 2S −2P transition frequencies of muonic hydrogen, Science 339, 417 (2013). [36] F. Jegerlehner, Precision measurements of σhadronic for αeffðEÞ at ILC energies and ðg −2Þμ, Nucl. Phys. B, Proc. Suppl. 162, 22 (2006). [13] P. J. Mohr, B. N. Taylor, and D. B. Newell, Codata recom- mended values of the fundamental physical constants: 2006, Rev. Mod. Phys. 80, 633 (2008). [37] F. Jegerlehner, Electroweak effective couplings for future precision experiments, Nuovo Cimento C 034S1, 31 (2011). [14] A. Gasparian et al., Jlab experiment proposal, Jefferson Lab Report No. C12-11-106, 2011, https://www.jlab.org/ exp_prog/proposals/12/C12-11-106.pdf. [38] M. Fael and M. Passera, Muon-Electron Scattering at Next- To-Next-To-Leading Order: The Hadronic Corrections, Phys. Rev. Lett. 122, 192001 (2019). p p g p p p [15] P. Banerjee, T. Engel, A. Signer, and Y. Ulrich, QED at NNLO with McMule, SciPost Phys. IV. CONCLUSION 9, 027 (2020). [39] M. J. Levine and R. Roskies, Hyperspherical approach to quantum electrodynamics—sixth-order magnetic moment, Phys. Rev. D 9, 421 (1974). [16] T. Engel, A. Signer, and Y. Ulrich, A subtraction scheme for massive QED, J. High Energy Phys. 01 (2020) 085. [40] M. J. Levine, R. C. Perisho, and R. Roskies, Analytic contributions to the G factor of the electron, Phys. Rev. D 13, 997 (1976). [17] S. Frixione, Z. Kunszt, and A. Signer, Three-jet cross sections to next-to-leading order, Nucl. Phys. B467, 399 (1996). [18] R. Frederix, S. Frixione, F. Maltoni, and T. Stelzer, Automation of next-to-leading order computations in QCD: The FKS subtraction, J. High Energy Phys. 10 (2009) 003. [41] M. Fael, Hadronic corrections to μ-e scattering at NNLO with space-like data, J. High Energy Phys. 02 (2019) 027. [42] S. Laporta, Hyperspherical integration and the triple cross vertex graphs, Nuovo Cimento Soc. Ital. Fis. 107A, 1729 (1994). [19] P. Nogueira, Automatic Feynman graph generation, J. Comput. Phys. 105, 279 (1993). L031904-6 [43] R. Bonciani, A. Ferroglia, P. Mastrolia, E. Remiddi, and J. J. van der Bij, Two-loop NF ¼ 1 QED Bhabha scattering differential cross section, Nucl. Phys. B701, 121 (2004). [44] S. Actis, M. Czakon, J. Gluza, and T. Riemann, Two-loop fermionic corrections to massive Bhabha scattering, Nucl. Phys. B786, 26 (2007). [45] PRad Collaboration (private communication). [44] S. Actis, M. Czakon, J. Gluza, and T. Riemann, Two-loop fermionic corrections to massive Bhabha scattering, Nucl. Phys. B786, 26 (2007). [45] PRad Collaboration (private communication). PHYS. REV. D 105, L031904 (2022) PHYS. REV. D 105, L031904 (2022) [43] R. Bonciani, A. Ferroglia, P. Mastrolia, E. Remiddi, and J. J. van der Bij, Two-loop NF ¼ 1 QED Bhabha scattering differential cross section, Nucl. Phys. B701, 121 (2004). L031904-7
https://openalex.org/W4210672800	https://www.nature.com/articles/s41598-022-05325-5.pdf	English	null	COVID-19 reinfections among naturally infected and vaccinated individuals	Scientific reports	2,022	cc-by	8,860	COVID‑19 reinfections among naturally infected and vaccinated individuals OPEN Sezanur Rahman1, M. Mahfuzur Rahman1, Mojnu Miah1, Mst Noorjahan Begum1 Monira Sarmin2, Mustafa Mahfuz2, Mohammad Enayet Hossain1, Mohammed Ziaur Rahman 1, Mohammod Jobayer Chisti2, Tahmeed Ahmed2, Shams El Arifeen3 & Mustafizur Rahman1* Sezanur Rahman1, M. Mahfuzur Rahman1, Mojnu Miah1, Mst Noorjahan Begum1, Monira Sarmin2, Mustafa Mahfuz2, Mohammad Enayet Hossain1, Mohammed Ziaur Rahman 1, Mohammod Jobayer Chisti2, Tahmeed Ahmed2, Shams El Arifeen3 & Mustafizur Rahman1* The protection against emerging SARS-CoV-2 variants by pre-existing antibodies elicited due to the current vaccination or natural infection is a global concern. We aimed to investigate the rate of SARS-CoV-2 infection and its clinical features among infection-naïve, infected, vaccinated, and post- infection-vaccinated individuals. A cohort was designed among icddr,b staff registered for COVID-19 testing by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). Reinfection cases were confirmed by whole-genome sequencing. From 19 March 2020 to 31 March 2021, 1644 (mean age, 38.4 years and 57% male) participants were enrolled; where 1080 (65.7%) were tested negative and added to the negative cohort. The positive cohort included 750 positive patients (564 from baseline and 186 from negative cohort follow-up), of whom 27.6% were hospitalized and 2.5% died. Among hospitalized patients, 45.9% had severe to critical disease and 42.5% required oxygen support. Hypertension and diabetes mellitus were found significantly higher among the hospitalised patients compared to out-patients; risk ratio 1.3 and 1.6 respectively. The risk of infection among positive cohort was 80.2% lower than negative cohort (95% CI 72.6–85.7%; p < 0.001). Genome sequences showed that genetically distinct SARS-CoV-2 strains were responsible for reinfections. Naturally infected populations were less likely to be reinfected by SARS-CoV-2 than the infection-naïve and vaccinated individuals. Although, reinfected individuals did not suffer severe disease, a remarkable proportion of naturally infected or vaccinated individuals were (re)-infected by the emerging variants. Bangladesh observed the third wave of COVID-19 pandemic and faced a record upsurge during June–Septem- ber 2021, fueled by the highly contagious Delta variant1. Many of these COVID-19 positive cases had reported previous experience with natural infection or vaccination. In Bangladesh, the COVID-19 vaccination started on 27 January 2021 with COVISHIELD™ (ChAdOx1 nCoV-19 Corona Virus Vaccines manufactured by Serum Institute of India Pvt Ltd). Until July 2021, six additional COVID-19 vaccines (mRNA-1273, BNT162b2, Sputnik V, Ad26.COV2.S, BBIBP-CorV/Vero Cells, and CoronaVac) have been approved by the Govt. of Bangladesh2. www.nature.com/scientificreports www.nature.com/scientificreports COVID‑19 reinfections among naturally infected and vaccinated individuals OPEN Recent studies suggest that natural infections are protective against reinfection at least for 8–12 months3 and vaccination confers strong resistance against variants of concern, including the Delta variant. However, even with high vaccine coverage, many countries face multiple waves with faster high altitude spread than the previous1,4–7. Therefore, the protection against the new variants with pre-existing antibodies due to natural infection or vac- cination becomes a global concern8–10. g More than 95% of symptomatic cases develop antibodies within 14 days, and by day 30, 100% symptomatic and 45% asymptomatic cases become fully seroconverted11. But, the concentration of neutralising antibodies is another factor to confer protection against SARS-CoV-2 reinfection12. Recently, the number of reinfection cases have been increasing globally13. Although, CDC considered symptomatic infection < 35 Ct-value with ≥ 45 days interval between two rRT-PCR tests as reinfection cases14; Tang et al. identified reinfection cases within 19 days by different PANGO Lineage SARS-CoV-215. Therefore, how long an individual is protected from further 1Virology Laboratory, Infectious Diseases Division, icddr,b: International Centre for Diarrhoeal Disease Research, Bangladesh, 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh. 2Nutrition and Clinical Services Division, icddr,b: International Centre for Diarrhoeal Disease Research, Bangladesh, Mohakhali, Dhaka 1212, Bangladesh. 3Maternal and Child Health Division, icddr,b: International Centre for Diarrhoeal Disease Research, Bangladesh, Mohakhali, Dhaka 1212, Bangladesh. *email: mustafizur@icddrb.org Scientific Reports \| (2022) 12:1438 \| https://doi.org/10.1038/s41598-022-05325-5 www.nature.com/scientificreports/ Figure 1. Study profile (participants were enrolled until 31 March 2021, and all participants were followed up until 10 July 2021). igure 1. Study profile (participants were enrolled until 31 March 2021, and all participants were followed up ntil 10 July 2021). Figure 1. Study profile (participants were enrolled until 31 March 2021, and all participants were followed up until 10 July 2021). tudy profile (participants were enrolled until 31 March 2021, and all participants were followed up 2021) SARS-CoV-2 infection after recovering from COVID-19, becomes an important research question during this prolonged pandemic.f Recent studies showed that complete vaccination was effective against SARS-CoV-2 even for emerging vari- ants; and infection was significantly lower among vaccinated individuals than non-vaccinated16–19. In contrast, several studies showed low vaccine efficacy against Delta (B.1.617.2) variant compared to Wuhan (B.1) or Alpha (B.1.1.7) variants20–22. Another study from a town in Massachusetts showed that among 469 cases, mostly infected by the Delta variant, 74% were fully vaccinated23. COVID‑19 reinfections among naturally infected and vaccinated individuals OPEN While, another study identified a small fraction of vaccine breakthrough infections in USA24; in contrast, data from densely populated and low vaccine coverage regions is limited25. Therefore, it is important to evaluate the vaccine’s effectiveness against emerging variants. hf g g g Although host immunity26,27 and chance of exposure28 are the main factors, genomic evidence shows that recurrent cases were infected with phylogenetically distinct SARS-CoV-2 strains29–36. Antibodies against the spike protein were effective in inhibiting SARS-CoV entry into the host cell; however, mutations in the receptor-binding domain of S-protein helps them to escape host immunity and lead to the emergence of new variants37–39. Emerg- ing new variants are not only capable of escaping immunity and causing reinfection but also show increased transmissibility, severity and mortality40–42. Therefore, molecular surveillance for variant monitoring is crucial, and several countries and research organisations have already started surveillance programs1,43. g y p g icddr,b (International Centre for Diarrhoeal Disease Research, Bangladesh) is an international health research institution with approximately 4000 national (~ 95%) and international clinicians, health workers, scientists and non-scientific staff. This organisation has been providing an extensive staff-clinic facility for staff and their fam- ily members. Since March 2020, icddr,b staff-clinic started COVID-19 testing and treatment support. Taking advantage of this ongoing COVID-19 testing and treatment facility, we designed a cohort study and investigated the frequency of SARS-CoV-2 infection among infection naïve, previously infected, vaccinated, vaccinated with previous infection and non-vaccinated staff. Here we provide comparative data analysis of (1) SARS-CoV-2 infections between COVID-19 negative and positive cohort; (2) clinical features and genomic variations between first episode and reinfection; and finally (3) data on SARS-CoV-2 infection among vaccinated and unvaccinated persons. Results From 19 March 2020 to 31 March 2021, a total of 1644 staff of icddr,b were enrolled for the COVID-19 testing cohort (Fig. 1). Males were predominant (n = 939; 57.2%), and the mean age was 38.4 years (median 37 years) (Table 1). 1080 (65.7%) participants were tested negative using rRT-PCR for SARS-CoV-2 and enrolled as a https://doi.org/10.1038/s41598-022-05325-5 Scientific Reports \| (2022) 12:1438 \| www.nature.com/scientificreports/ Table 1. Characteristics of study participants by baseline cohort allocation and follow-up. a First infections. b Reinfections. c Symptomatic case. d During follow-up. All participant (N = 1644) Baseline Follow-up Negative cohort (N = 1080) Positive cohort (N = 750) Negative cohorta (N = 277) Positive cohortb (N = 38) Gender Male 940 (57.2%) 609 (56.4%) 446 (59.4%) 167 (60.3%) 27 (71.1%) Female 704 (42.8%) 471 (43.6%) 304 (40.6%) 110 (39.7%) 11 (28.9%) Age (year) Mean 38.4 38.3 39.1 40.5 40.2 Median (Std. devia- tion) 37.0 (10.2) 37.0 (10.4) 38.0 (9.9) 40.0 (9.9) 38.0 (10.7) IQR 30.0–45.8 30.0–45.0 31.0–46.7 32.0–48.0 30.7–50.3 Cause for testing Influenza like illness 587 (54.4%) 607 (80.8%) 203 (73.3%) 32 (84.2%) Contact with COVID-19 case 112 (10.4%) 79 (10.7%) 38 (13.7%) 6 (15.8%) Asymptomatic peri- odic test 381 (35.2%) 64 (8.5%) 36 (13.0%) – Symptomsc Fever 358 (33.1%) 466 (62.1%) 180 (64.9%) 29 (76.3%) Cough 383 (35.4%) 328 (43.7%) 130 (46.9%) 21 (55.3%) Short of breath 44 (4.1%) 25 (3.3%) 13 (4.7%) 2 (5.3%) Sore throat 77 (7.1%) 39 (5.2%) 11 (4.0%) 3 (7.9%) Other symptom 44 (4.1%) 49 (6.5%) 22 (7.9%) 5 (13.2%) Day intervalsd Mean (Std. error of mean) 173.6 (7.7) 195.6 (19.4) Median (Std. devia- tion) 138.0 (128.0) 156.5 (119.5) IQR 68–257 93.5–287.8 Minimum 1 47 Maximum 566 460 Table 1. Characteristics of study participants by baseline cohort allocation and follow-up. a First infections. b Reinfections. c Symptomatic case. d During follow-up. Table 1. Characteristics of study participants by baseline cohort allocation and follow-up. a First infections. b Reinfections. c Symptomatic case. d During follow-up. negative cohort. The positive cohort includes 750 positive cases, 564 from baseline and 186 from the negative cohort (Fig. 1). negative cohort. The positive cohort includes 750 positive cases, 564 from baseline and 186 from the negative cohort (Fig. 1). Negative cohort. In the negative cohort baseline, 609 (56.4%) participants were male, and the mean age of the participants was 38.3 years (Table 1). Results For the rest https://doi.org/10.1038/s41598-022-05325-5 Scientific Reports \| (2022) 12:1438 www.nature.com/scientificreports/ 1-Mar-20 1-Apr-20 1-May-20 1-Jun-20 1-Jul-20 1-Aug-20 1-Sep-20 1-Oct-20 1-Nov-20 1-Dec-20 1-Jan-21 1-Feb-21 1-Mar-21 1-Apr-21 1-May-21 1-Jun-21 1-Jul-21 Negative Infections Re-infections (A) Negative Cohort (B) Positive Cohort 1080 277 15 750 38 1-Mar-20 1-Apr-20 1-May-20 1-Jun-20 1-Jul-20 1-Aug-20 1-Sep-20 1-Oct-20 1-Nov-20 1-Dec-20 1-Jan-21 1-Feb-21 1-Mar-21 1-Apr-21 1-May-21 1-Jun-21 1-Jul-21 0 200 400 600 800 1000 Figure 2. Cumulative number of cases in Y-axis from (A) negative cohort and (B) positive cohort and X-axis indicate timeline (date). The green line indicates a cumulative number of negative cohort enrolment; the blue line indicates a cumulative incidence of infection (first infection in negative cohort and enrollment in the positive cohort), and the red line indicates a cumulative number of reinfection. (A) Negative Cohort 38 Figure 2. Cumulative number of cases in Y-axis from (A) negative cohort and (B) positive cohort and X-axis indicate timeline (date). The green line indicates a cumulative number of negative cohort enrolment; the blue line indicates a cumulative incidence of infection (first infection in negative cohort and enrollment in the positive cohort), and the red line indicates a cumulative number of reinfection. f the cases (n = 312, 41.6%), no medical attention was required due to low-mild symptoms, and they were ecovered after homestay. t Disease severity, complication/outcome was recorded only for the in-patient group (n = 207) (Fig. 3A); where 95 (45.9%) cases had severe disease based on national guideline44. Among them, Acute Respiratory Distress Syn- drome (ARDS) had developed in 43 (20.8%) and septic shock in 15 (7.2%) participants. 88 (42.5%) participants required oxygen support (Fig. 3B); through a nasal cannula (n = 57, 64.8%), mask (n = 12, 13.6%), through High Flow Nasal Cannula (n = 10, 11.4%) and mechanical ventilation (n = 9, 10.1%). Comorbidities (hypertension, diabetes mellitus, and asthma/COPD) were recorded for both in-patient and out-patient groups (Fig. 3C). Hypertension (RR 1.4; 95% CI 1.1–1.7) and diabetes mellitus (RR 1.5; 95% CI 1.3–1.8) were found significantly (p ≤ 0.001) higher among the in-patient group than out-patient. Asthma/COPD was also found in a higher number in the in-patient group but was not statistically significant. Reinfection. From the positive cohort, 731 (excluding death cases) COVID-19 positive participants (epi- sode-1) were followed up for 0.91-person-years, and 38 (5.7 reinfections per 100 person-year) were identified as reinfection (episode-2) with SARS-CoV-2. Results As far as causes of testing were concerned, 112 (10.4%) participants were tested for having confirmed contact with other COVID-19 patients, while 586 (54.3%) participants were tested for having influenza-like illness and 382 (35.4%) for asymptomatic routine check-up. Cough (n = 383, 35.4%), fever 358 (33.1%), sore throat 77 (7.1%) and shortness of breath 44 (4.1%) were common symptoms. Other symptoms including headache, vomiting, diarrhoea, and asthma were also recorded. y p g g During follow-up till 31 July 2021 (0.99-person year), 277 cases (25.8 infections per 100 person-year) were infected with SARS-CoV-2 (Fig. 2A), where the male (n = 167; 60.3%) were predominant. Both mean (40.5 yeas) and median (40.0 years) age for infected cases were slightly higher than baseline participants. Also, the symptomatic follow-up cases were more likely to be positive (n = 203, 73.3%) than asymptomatic cases (n = 36). Among infected participants, 180 (64.9%) came with fever, while 130 (46.9%) had a cough. The mean interval between negative cohort enrolment and infection was 173.6 days (median, 138.0). Positive cohort. A total of 750 SARS-CoV-2 positive cases were followed up under the positive cohort for reinfection identification (Fig. 2B). Gender (n = 446, 59.5% male) and age distribution (mean 39.1 years and median 38.0 years) for positive cohort participants were similar to the negative cohort (Table 1). The sympto- matic cases were much highly represented (n = 607, 80.9%) in the positive cohort as opposed to the negative cohort (n = 586, 54.3%). The symptomatic cases in the positive cohort were more likely to present with fever (n = 466, 62.1%) and cough (n = 328, 43.7%) than the other symptoms. The mean Ct-value of the RdRp gene for symptomatic cases 22.9 (median 20.4, IQR 17.0–27.6) was significantly (p = 0.01) lower than asymptomatic (mean 25.3, median 22.8, IQR 16.8–35.1). Among all positive cases, 207 (27.6%) were admitted to the icddr,b COVID-19 management hospital and received treatment (in-patient group) according to the national guideline on clinical management of COVID- 1944 and 19 of them died. Thus the overall case fatality rate was 2.5% (19/750). Also, 232 (30.9%) positive cases received medication and support from the staff-clinic but were not admitted (out-patient group). Results 112 95 0 20 40 60 80 100 120 Mild/ moderate Severe/ critical ARDS Septic shock Death Diseases severity Complication/ outcome 112 (54.1%) 95 (45.9%) 43 (20.8%) 15 (7.2%) 19 (9.2%) (A) N = 207 0 50 100 150 200 250 Hypertension Diabetes Mellitus In-Patient Out-Patient In-Patient Out-Patient In-Patient Out-Patient Asthma/ COPD 94 (45.4%) 71 (30.6%) 73 (35.3%) 43 (18.5%) 33 (15.9%) 27 (11.6%) (C) g O2 Not required O2 required Nasal Cannula (upto 6 L) Face mask (upto 10 L) Non rebreather mask (10-15 L) HFNC Mechanical ventilation 119 (57.5%) 88 (42.5%) 57 (64.8%) 10 (11.4%) 10 (11.4%) 9 (10.1%) 2 (2.3%) (B) N = 207 N = 88 (B) O2 Not required Figure 3. Clinical features of the positive cohort. (A) Diseases severity, complication, and outcome of in-patient group. (B) Oxygen requirement of in-patient group. (C) Comorbidity of COVID-19 positive in-patient (hospitalised, n = 200) and out-patient (not hospitalised but seek treatment in staff clinic, n = 207) group. and did not require oxygen support, while one hospitalisation case (case 16) during reinfection was critical and required oxygen support through High-Flow Nasal Cannula (HFNC) having oxygen flow up to 20 L/min in Intentive Care Unit45. Nearly complete genome sequences from both episode-1 and episode-2 infections were retrieved from 18 reinfections out of 38 cases. The amino acid sequence comparison among the strains from episode-1 and epi- sode-2 with reference Wuhan strain (GenBank Accession# NC_045512.2) is shown in Fig. 4. The sequencing results revealed that the reinfection was caused by a different (PANGO Lineage) strain than the first infection, except one (case-08). For case numbers 09, 10 and 12, both episodes were caused by the same Wuhan-like variant while others were caused by different variants than first infections. The case-16 was infected by the Wuhan-like variant in episode-1 and by the Alpha variant in episode-2, which was the only severe case during reinfection. Case numbers 17, 20, 21, 24, 26 were reinfected by Beta variant, and case numbers 27, 30, 32, 36, 37 were rein- fected by Delta variant, while all these cases were first infected by Wuhan-like variant. Case numbers 28, 31 and 34 were infected by Beta in episode-1 and reinfected by Delta variant. Case-08 was the only case infected and reinfected by the same PANGO Lineage (B.1.1.25); however, three amino acids were different between episode-1 and episode-2. Results The risk of reinfection in the positive cohort was significantly lower (RR 0.3; 95% CI 0.19–0.35; p < 0.001) than primary infection in the negative cohort. The mean Ct-value of RdRp gene in episode-1 of reinfected cases (29.6, median 34.5, IQR 20.4–36.3) was significantly (p < 0.001) higher than non-reinfected cases (22.9, median 20.4, IQR 16.8–28.2). During reinfection, the mean Ct-value of RdRp gene was 26.3 (median 26.2, IQR 18.9–33.3), which was lower than episode-1 but not statistically significant (p = 0.1). Mean intervals for reinfection (episode-1 to episode-2) was 195.6 days, and the median was 156.5. The earliest day of reinfection was 47 days after the primary infection, and the longest was 460 days (Appendix 1E). y yt y g y Among reinfected cases, 9 (23.7%) received one dose of the COVISHIELD™ vaccine at least 34 days (mean 68.6) before reinfection, while three received full vaccination before reinfection. At least one comorbidity among obesity, diabetes, asthma, heart disease, lung disease, and high blood pressure (BP) was present in 19 (50%) reinfection cases. During episode-2, confirm contact with another COVID-19 patient was higher than episode-1 (14 vs. 8). Although several mild symptoms were recorded for primary infection and reinfection (Appendix 1E), no one required hospitalisation except one (case-25) during primary infection and one (case-16) during reinfection. The hospitalisation case during primary infection (case 25) and reinfection (case 7) was not severe https://doi.org/10.1038/s41598-022-05325-5 Scientific Reports \| (2022) 12:1438 \| www.nature.com/scientificreports/ 112 95 0 20 40 60 80 100 120 Mild/ moderate Severe/ critical ARDS Septic shock Death Diseases severity Complication/ outcome 112 (54.1%) 95 (45.9%) 43 (20.8%) 15 (7.2%) 19 (9.2%) (A) N = 207 0 50 100 150 200 250 Yes No Missing Hypertension Diabetes Mellitus In-Patient Out-Patient In-Patient Out-Patient In-Patient Out-Patient Asthma/ COPD 94 (45.4%) 71 (30.6%) 73 (35.3%) 43 (18.5%) 33 (15.9%) 27 (11.6%) (C) O2 Not required O2 required Nasal Cannula (upto 6 L) Face mask (upto 10 L) Non rebreather mask (10-15 L) HFNC Mechanical ventilation 119 (57.5%) 88 (42.5%) 57 (64.8%) 10 (11.4%) 10 (11.4%) 9 (10.1%) 2 (2.3%) (B) N = 207 N = 88 Figure 3. Clinical features of the positive cohort. (A) Diseases severity, complication, and outcome of in-patient group. (B) Oxygen requirement of in-patient group. (C) Comorbidity of COVID-19 positive in-patient (hospitalised, n = 200) and out-patient (not hospitalised but seek treatment in staff clinic, n = 207) group. Results The case was asymptomatic and tested positive on 6 September 2020 as confirmed contact with another COVID-19 patient. After 20 days, on 26 September, the follow-up rRT-PCR test was negative. On 23 October 2020, after 47 days from episode-1, the case was found positive with high fever, cough, diarrhoea, and muscle aches. Thus, the case was confirmed as reinfection by the definition provided in Appendix 1B. SARS‑CoV‑2 infection among vaccinated individuals. A subset of patients were analyzed to calculate the association between vaccination and (re)-infection. From 1 May 2021 to 31 July 2021, 381 symptomatic staff provided specimens for SARS-CoV-2 rRT-PCR testing; among them, 115 (30.2%) received at least one dose, and 96 (25.2%) received full doses of COVISHIELD™ vaccine before the test date. Among full dose vaccinated individuals, 37 (38.5%) were infected and among non-vaccanated 96 (36.1%) were infected; while the risk ratio between vaccinated and non-vaccinated individuals was statistically insignificant (RR = 1.08, 95% CI 0.76–1.534 and p = 0.66). Also risk ratio between full dose vaccinated and single dose vaccinated individuals was statistically insignificant (Appendix-1). Mean intervals from vaccination date (full dose) to SARS-CoV-2 positive test date was 65.1 (median 66.0, IQR: 51.3–82.0) days. Among the positive cases, no one required medical attention except three who had co-morbidites. Two vaccinated individuals had high blood pressure, attended the hospital and were discharged with conventional COVID-19 medication after a few hours of observation. Another case was non-vaccinated and had asthma with thyroiditis, which required oxygen support with a nasal cannula. https://doi.org/10.1038/s41598-022-05325-5 Scientific Reports \| (2022) 12:1438 \| www.nature.com/scientificreports/ B.1.617.2 Delta A S L L S L I F A R ^ G R K G R T N L L A I I ^ G M C Y Non-polar Amino acid Polar Amino acid Polar Amino acid (Acidic) Polar Amino acid (Basic) Deleted Stop codon Figure 4. Amino acid substitution map of reinfected cases (for both infection and reinfection events) confirming reinfection by genetically distinct SARS-CoV-2. Results 261 265 300 309 621 677 983 1001 1230 1260 1306 1500 1640 1655 1708 1785 1862 1994 1997 2000 2018 2046 2087 2092 2224 2287 2350 2389 2596 2930 3209 3255 3310 3353 3395 3510 3593 3606 3618 3620 3646 3718 4311 4398 18 19 67 80 97 145 158 173 215 417 446 452 478 484 490 501 570 614 681 701 716 879 929 950 982 1118 1141 1247 26 28 53 54 57 100 116 170 213 238 275 71 82 82 116 120 40 27 52 54 68 73 87 89 3 63 194 203 204 205 215 235 243 327 362 377 380 385 Wuhan-Hu-1 B K T I P Y T D T K N A H P K A I P V A K P P T H S P S S N V A T E K P V V L I A T V T S L T A D K Y R Q D K G L T E F N A D P A T A S D S D L C S F L A Q G Q S Q D L P I V L T T Q R S K Y T N D D S R G T G S G S T D Q R B.1.1.25 F G I K R B.1.1.25 F V G R K R B.1.540 I G D B.1 N G L B.1 G L B.1.36.16 L G F H L H B.1.540 G B.1.1.25 F F G R K R B.1.1.25 F N V G R C K R B.1.1.7 Alpha I D T K ^ ^ ^ Y D G H I A H # I # C L K R F B.1.540 G B.1.351.3 Beta I N L S R V ^ F A G ^ N K Y G V H L F L I B.1 F G B.1.351.3 Beta I N L S R ^ F A G ^ N K Y G V F H L L I I B.1 G B.1.351.3 Beta I N R L S R ^ F V A N G ^ N K Y G V H L L I B.1 I N G H L I B.1.351.3 Beta I N L S R S ^ F A G ^ N K Y G V H L L I B.1 F I H G B.1.351.3 Beta I N L L R S R ^ F A G ^ N K Y G V H L L I B.1.1.25 F A G K R B.1.617.2 Delta N S L S L I R A ^ G R K G R S N L K Y T A I I ^ G M C Y B.1.351.3 Beta I N L S R ^ F A G ^ N K Y G V H L L I B.1.617.2 Delta L V V R K G N W L T ^ G M Y B G H B.1.617.2 Delta S L S L I A R ^ G R K G R T N L T A I I ^ G M C Y B.1.351.3 Beta I N L L S R ^ F V A G ^ N K Y G V H L L I B.1.617.2 Delta L Y L Y V A R ^ G R K G R N Y L V T A F I ^ G M I Y K B.1.1 S L L G K R B.1.617.2 Delta S S L S L I A R ^ G R K G R N L T A I I ^ G M C Y B.1.351.3 Beta I N L S R ^ F V A G ^ N K Y G V H L L I B.1.617.2 Delta S L L I A V R K G R N L H T I ^ G M C L Y B.1.1 G R I K R B.1.617.2 Delta L C I L Y V A R ^ G H R K G R N L C T A F I ^ G M C Y K B.1.1.25 F G L K R B.1.617.2 Delta A S L L S L I F A R ^ G R K G R T N L L A I I ^ G M C Y Case-34 Case-36 Case-37 Pango-liniage/ Variant Case-27 Case-28 Case-30 Case-31 Case-32 Case-17 Case-20 Case-21 Case-24 Case-26 Case-08 Case-09 Case-10 Case-12 Case-16 SGF3675 HV69 EF156 LAL242 DF119 Non-polar Amino acid Polar Amino acid Polar Amino acid (Acidic) Polar Amino acid (Basic) Deleted Stop codon Figure 4. Results Amino acid substitution map of reinfected cases (for both infection and reinfection events) confirming reinfection by genetically distinct SARS-CoV-2. Results 6 Scientific Reports \| (2022) 12:1438 \| https://doi.org/10.1038/s41598-022-05325-5 261 265 300 309 621 677 983 1001 1230 1260 1306 1500 1640 1655 1708 1785 1862 1994 1997 2000 2018 2046 2087 2092 2224 2287 2350 2389 2596 2930 3209 3255 3310 3353 3395 3510 3593 3606 3618 3620 3646 3718 4311 4398 18 19 67 80 97 145 158 173 215 417 446 452 478 484 490 501 570 Wuhan-Hu-1 B K T I P Y T D T K N A H P K A I P V A K P P T H S P S S N V A T E K P V V L I A T V T S L T A D K Y R Q D K G L T E F N A B.1.1.25 F B.1.1.25 F V B.1.540 I B.1 N B.1 B.1.36.16 L B.1.540 B.1.1.25 F F B.1.1.25 F N V B.1.1.7 Alpha I D T K ^ ^ ^ Y D B.1.540 B.1.351.3 Beta I N L S R V ^ F A G ^ N K Y B.1 F B.1.351.3 Beta I N L S R ^ F A G ^ N K Y B.1 B.1.351.3 Beta I N R L S R ^ F V A N G ^ N K Y B.1 I N B.1.351.3 Beta I N L S R S ^ F A G ^ N K Y B.1 F I H B.1.351.3 Beta I N L L R S R ^ F A G ^ N K Y B.1.1.25 F A B.1.617.2 Delta N S L S L I R A ^ G R K B.1.351.3 Beta I N L S R ^ F A G ^ N K Y B.1.617.2 Delta L V V R K B B.1.617.2 Delta S L S L I A R ^ G R K B.1.351.3 Beta I N L L S R ^ F V A G ^ N K Y B.1.617.2 Delta L Y L Y V A R ^ G R K B.1.1 S L L B.1.617.2 Delta S S L S L I A R ^ G R K B.1.351.3 Beta I N L S R ^ F V A G ^ N K Y B.1.617.2 Delta S L L I A V R K B.1.1 B.1.617.2 Delta L C I L Y V A R ^ G H R K B.1.1.25 F B.1.617.2 Delta A S L L S L I F A R ^ G R K Case-34 Case-36 Case-37 Pango-liniage/ Variant Case-27 Case-28 Case-30 Case-31 Case-32 Case-17 Case-20 Case-21 Case-24 Case-26 Case-08 Case-09 Case-10 Case-12 Case-16 SGF3675 HV69 EF156 LAL242 Non-polar Amino acid Polar Amino acid Polar Amino acid (Acidic) Polar Amin Figure 4. Discussion Th d This study represents infection and reinfection events of SARS-CoV-2 by a robust prospective cohort study from a densely populated country, Bangladesh. We did a systematic search to identify studies that described clinical characteristics and/or potential factors and/or rates for recurrence of positive SARS-CoV-2 (Appendix 1A). A total of 22 cohorts were identified, and no cohort confirmed reinfection by whole-genome sequencing. In addi- tion, among 118 published case reports, only 12 confirmed reinfections by whole-genome sequencing. Therefore, our cohort is unique in identifying reinfection cases with genomic evidence, which is essential to confirm true reinfections. Here we have provided evidence that: (1) being naturally infected confers better protection against the SARS-CoV-2; (2) SARS-CoV-2 infection was associated with an 80% lower risk for concurrent infection, with more than six-month protective effect after primary infection; (3) COVISHIELD™ vaccine showed reduced effectiveness against new variants of SARS-CoV-2. f In this cohort, both symptomatic and asymptomatic cases were enrolled for rRT-PCR test; however, symp- tomatic cases were 1.3 times more likely to be positive than asymptomatic cases. Also, the high representation of symptomatic cases in the positive cohort compared to the negative cohort suggests that in a population and setting that this sample is representative of, SARS-CoV-2 is a more likely etiology for influenza-like illness. The significantly higher infection rate among infection naïve participants than patients previously infected with SARS-CoV-2 indicates possible protection by natural infections. Mean rRT-PCR Ct-value during episode-1 of reinfected cases was significantly higher than others. Also, the fact that the mean Ct-value of the symptomatic cases was found to be significantly lower than asymptomatic ones, similar to another study46, indicates that a relatively low Ct-value or high viral load was required to develop COVID-19-like symptoms47–49 as well as for seroconversion11,50. Although seroconversion and concurrent protection against any pathogen depend on several host factors, a median protective effect of over 5-months protective effect was observed in this study, confirming previous studies51–53. Additionally, previous infection history was associated with an 80% lower risk of further infection; a similar result was observed in previous studies45,54,55. Discussion Th d However, seroconversion is not the only protective measure because, after a few months, the amount of neutralising antibodies decline56,57 while cellular immunity induced by natural infection plays a significant role in preventing subsequent infections58.t i On the other hand, vaccination after SARS-CoV-2 infection increased T-cell immunity, antibody-secreting memory B-cell response to the spike protein, and neutralising antibodies effectivity even after the first dose of mRNA-based vaccines (Pfizer-BioNTech or Moderna)59–61. While our data showed vaccination with COV- ISHIELD™, a vector based recombinant DNA vaccine was unable to prevent infection or reinfection62. Reynolds et al. and Stamatatos et al., provided evidence of cross-variant neutralisation of Alpha (B.1.1.7) and Beta (B.1.351) variants by the Pfizer-BioNTech or Moderna vaccination after natural infection. However, molecular evidence of reinfection from this study, along with others63 indicating that genetically distinct strains or new variants can escape immunity whether it was achieved by natural infection or vaccination. Although existing data showed that vaccinated COVID-19 patients are lower at risk for hospitalization64, our data failed to correlate vaccination status with hospitalisation and variants due to a low number of vaccinated individuals infected with different variants. Therefore, a large study with vaccinated, non-vaccinated, hospitalised, and non-hospitalised COVID-19 patent infected with different SARS-CoV-2 variants is needed.ii pf In Bangladesh, the first COVID-19 case was identified on 8 March 2020, and since then icddr,b started SARS- CoV-2 rRT-PCR test for staff with influenza-like illnesses. Upon the first case identification among icddr,b staff on 19 March 2020, the cohort was started. Therefore, an antibody test against SARS-CoV-2 was not required to confirm infection naïve participants. On the other hand, rRT-PCR test is considered to be the gold standard for its high sensitivity and specificity, which was used to define the cohort (negative or positive). Hence there was an inconsiderable chance for selection bias in this study. But there might be some Hawthorne effect in the patients’ self reporting of symptoms and cause of testing due to the limitations imposed by the phone-call registration system for COVID-19 testing. Also, reinfected cases were extensively interviewed after the second infection, allowing the possibility of recall bias. Despite the minimal Hawthorne effect and recall bias, this was a robust cohort study from a densely populated country like Bangladesh in order to understand SARS-CoV-2 infection dynamics. y In summary, our data indicate that prior infection ensures some degree of protection against SARS-CoV-2 reinfections. Results Amino acid substitution map of reinfected cases (for both infection and reinfection events) con Scientific Reports \| (2022) 12:1438 \| https://doi.org/10.1038/s41598-022-05325-5 www.nature.com/scientificreports/ Discussion Th d However, emerging variants could (re)-infect naturally infected or vaccinated individuals. Therefore, along with vaccination, other non-pharmaceutical interventions and protective measures need to be implemented for infection control. Our data also warrant evaluation of the vaccine effectiveness against emerging variants. Methods S i Specimen and data collection. This prospective cohort study was designed among icddr,b staff regis- tered for COVID-19 testing. During registration over phone call, the staff-clinic received clinical and behav- ioural data from the participants. These data include cause of testing, age, sex, symptom, date of symptom onset, travel history, and possible contact history with confirmed COVID-19 patients. In terms of the cause of testing all participants were classified into; (1) contact cases (having symptoms or not), (2) symptomatic/influenza-like illness (do not have contact history), and (3) asymptomatic (for routine check-up). Since the national COVID- 19 vaccination started, the staff-clinic included and updated the vaccination statuses of the staff in the database. After registration, all suspected cases provided nasopharyngeal swab samples in viral transport media at the icddr,b COVID-19 sample collection booth. All specimens and data collected by the staff-clinic were then sent to the Virology Laboratory of icddr,b for the SARS-CoV-2 rRT-PCR test. The Virology Laboratory also shared test results with the staff-clinic for further patient management. https://doi.org/10.1038/s41598-022-05325-5 Scientific Reports \| (2022) 12:1438 \| www.nature.com/scientificreports/ Ethical statements. The test and treatment of staff for COVID-19 was conducted under the icddr,b activity number ACT-01108 and ACT-01207; and this cohort study was conducted under protocol number PR-21065 and approved by the institutional review board of icddr,b. All participants provided informed consent during enrolment. In addition, all methods were performed in accordance with the relevant guidelines and regulations. Ethical statements. The test and treatment of staff for COVID-19 was conducted under the icddr,b activity number ACT-01108 and ACT-01207; and this cohort study was conducted under protocol number PR-21065 and approved by the institutional review board of icddr,b. All participants provided informed consent during enrolment. In addition, all methods were performed in accordance with the relevant guidelines and regulations. COVID‑19 detection. One aliquot of the collected nasopharyngeal swab was used for SARS-CoV-2 screen- ing, and one was stored for future references. rRT-PCR was used to detect SARS-CoV-2 using RdRp (ORF1ab) and N gene-specific primers and probes65. The iTaq universal probes one-step kit (Bio-Rad Laboratories, CA, USA) was used in the Bio-Rad CFX96 Touch real-time PCR system. Threshold cycle (Ct) values of ≤ 37 were considered positive. Data management and analysis. Participants enrolled from 19 March 2020 to 31 March 2021 were included for the current study and were followed up until 31 July 2021. The baseline COVID-19 test data defined the cohort (Appendix 1B). Methods S i Participants with negative rRT-PCR results during registration/baseline were fol- lowed up as a negative cohort. Participants with positive rRT-PCR results during registration or follow-up were considered a positive cohort and further followed-up for reinfection according to CDC guidelines for investigat- ing the SARS-CoV-2 reinfection cases14. Participants who had two positive rRT-PCR tests 90 days apart were considered reinfection cases, including symptomatic cases testing positive ≥ 45 days after the first infection with paired respiratory specimens14. After each reinfection identification, an extended interview was conducted with a structured questionnaire in a Case Record Form. All data were recorded and analysed using SPSS (version 20). The proportion of each variable was analysed through chi-square or Fisher’s exact test, where appropriate. SARS‑CoV‑2 genome sequencing and analysis. Besides our regular genomic surveillances1, rein- fection cases were subjected to comparative genome characterisation; therefore, cases with Ct-values of ≤ 33 for both episodes (first infection and reinfection) were selected for whole-genome sequencing using the NGS method (Appendix 1C). All SARS-CoV-2 sequences under this study were submitted to publicly available online databases; GISAID (www.gisaid.org) and/or GenBank (www.ncbi.nlm.nih.gov/genbank/). Accession numbers are in Appendix 1D. Data availabilityh y The metadata and supplementary data will be available with the manuscript as a supplementary documen Appendix-1. Received: 22 November 2021; Accepted: 3 January 2022 References 1. Rahman, M. et al. The emergence of SARS-CoV-2 variants in Dhaka City, Bangladesh. Transbound. Emerg. Diseases 00, 1–2. https:// doi.org/10.1111/tbed.14203 (2021).h g 2. DGHS. Coronavirus (COVID-19) Update. Directorate General of Health Services (DGHS), The People’s Republic of Bangladesh (2021). 3. Radbruch, A. & Chang, H.-D. A long-term perspective on immunity to COVID. 595, 359-360. https://doi.org/10.1038/d41586- 021-01557-z (2021).h 4. Soriano, V., de Mendoza, C., Gómez-Gallego, F., Corral, O. & Barreiro, P. Third wave of COVID-19 in Madrid, Spain. Int. J. Infect. Dis. 107, 212–214. https://doi.org/10.1016/j.ijid.2021.04.074 (2021). p g j j 5. Seong, H. et al. Comparison of the second and third waves of the COVID-19 pandemic in South Korea: Importance of early public health intervention. Int. J. Infect. Dis. 104, 742–745. https://doi.org/10.1016/j.ijid.2021.02.004 (2021).f f p g j j ( ) 6. Ishimaru, T. et al. Workplace measures against COVID-19 during the winter third wave in Japan: Company size-based differences J. Occup. Health 63, e12224. https://doi.org/10.1002/1348-9585.12224 (2021). Health 63, e12224. https://doi.org/10.1002/1348-9585.12224 (202 p p g 7. Moona, A. A., Daria, S., Asaduzzaman, M. & Islam, M. R. Bangladesh reported delta variant of coronavirus among its citizen: Actionable items to tackle the potential massive third wave. Infect. Prevent. Practice 3, 100159. https://doi.org/10.1016/j.infpip. 2021.100159 (2021). ( ) 8. Xie, X. et al. Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera Nat. Med. 27, 620–621. https://doi.org/10.1038/s41591-021-01270-4 (2021).f 8. Xie, X. et al. Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y vari Nat. Med. 27, 620–621. https://doi.org/10.1038/s41591-021-01270-4 (2021). p g 9. Greaney, A. J. et al. Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition b l l l h l b d C ll b h //d / / h ( ) p g 9. Greaney, A. J. et al. Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies. Cell Host Microbe 29, 463-476.e466. https://doi.org/10.1016/j.chom.2021.02.003 (2021). 10. Weisblum, Y. et al. Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants. Elife 9, e61312. https://doi.org/10. 7554/eLife 61312 sa2 (2020) 9. Greaney, A. J. et al. Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies. Cell Host Microbe 29, 463-476.e466. https://doi.org/10.1016/j.chom.2021.02.003 (2021). y p y p p g j 0. Weisblum, Y. et al. Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants. Elife 9, e61312. https://doi.org/10 7554/eLife.61312.sa2 (2020).t 11. Received: 22 November 2021; Accepted: 3 January 2022 www.nature.com/scientificreports/ 28.1: Clinical, diagnostic therapeutic and public health implications. Viruses 13, 439. https://doi.org/10.3390/v13030439 (2021). p p p p g 39. Wang, R. et al. Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use A receptors from additional species. Immunity 54, 1611-1621.e1615. https://doi.org/10.1016/j.immuni.2021.06.003 (2021). p p y p g j 0. Haim-Boukobza, S. et al. Detecting rapid spread of SARS-CoV-2 variants, France, January 26–February 16, 2021. Emerg. Infect Dis. 27, 1496. https://doi.org/10.3201/eid2705.210397 (2021). g 1. Nyberg, T. et al. Risk of hospital admission for patients with SARS-CoV-2 variant B. 1.1. 7: Cohort analysis. BMJ 373, 1. https:// doi.org/10.1136/bmj.n1412 (2021). doi.org/10.1136/bmj.n1412 (2021). 42. Davies, N. G. et al. Increased mortality in community-tested cases of SARS-CoV-2 lineage B. 1.1. 7. Nature 593, 270–274. https:// d i /10 1038/ 41586 021 03426 1 (2021) g j 2. Davies, N. G. et al. Increased mortality in community-tested cases of SARS-CoV-2 lineage B. 1.1. 7. Nature 593, 270–274. https:// doi.org/10.1038/s41586-021-03426-1 (2021). g ( ) 43. Hossain, M. E. et al. Genome sequence of a SARS-CoV-2 strain from Bangladesh that is nearly identical to United Kingdom SARS-CoV-2 variant B. 1.1. 7. Microbiol. Resource Announcements. https://doi.org/10.1128/MRA.00100-21 (2021). l l d l l l f ( l f l h l d h p g 4. CDC-DGHS. Vol. National Guidelines on Clinical Management of COVID-19 (Directorate General of Health Services, Bangladesh Communicable Disease Control, 2021). 5. Hossain, M. E. et al. A case report: Genetically distinct severe acute respiratory syndrome coronavirus-2 variant causing reinfec tion. Front. Microbiol. 12, 792514. https://doi.org/10.3389/fmicb.2021.792514 (2021). p g 46. Hall, V. J. et al. SARS-CoV-2 infection rates of antibody-positive compared with antibody-negative health-care workers in England: A large, multicentre, prospective cohort study (SIREN). Lancet 397, 1459–1469. https://doi.org/10.1016/S0140-6736(21)00675-9 (2021). 7. Singanayagam, A. et al. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19 England, January to May 2020. Eurosurveillance 25, 2001483. https://doi.org/10.2807/1560-7917.ES.2020.25.32.2001483 (2020). 8. Zhou, R. et al. Viral dynamics in asymptomatic patients with COVID-19. Int. J. Infect. Dis. 96, 288–290. https://doi.org/10.1016/j ijid.2020.05.030 (2020). j ( ) 49. Rao, S. N., Manissero, D., Steele, V. R. & Pareja, J. A narrative systematic review of the clinical utility of cycle threshold values in the context of COVID-19. Infect. Diseases Therapy 9, 573–586. https://doi.org/10.1007/s40121-020-00324-3 (2020). fh py p g 0. Amodio, E. et al. SARS-CoV-2 viral load, IFNλ polymorphisms and the course of COVID-19: An observational study. J. www.nature.com/scientificreports/ COVID-19 vaccine breakthrough infections reported to CDC—United States, January 1–April 30, 2021 Morb. Mortal. Wkly Rep. 70, 792. https://doi.org/10.15585/mmwr.mm7021e3 (2021). y p p g 5. Mallapaty, S. India’s massive COVID surge puzzles scientists. Nature 592, 667–668. https://doi.org/10.1038/d41586-021-01059-y (2021). (2021). 6. Goldman, J. D. et al. Reinfection with SARS-CoV-2 and failure of humoral immunity: A case report. MedRxiv. https://doi.org/10 1101/2020 09 22 20192443 (2020) 6. Goldman, J. D. et al. Reinfection with SARS-CoV-2 and failure of humoral immunity: A case report. MedRxiv. https://doi.org/10 1101/2020.09.22.20192443 (2020). 27. Mulder, M. et al. Reinfection of severe acute respiratory syndrome coronavirus 2 in an immunocompromised patient: A case report. Clin. Infect. Dis. 73, e2841–e2842. https://doi.org/10.1093/cid/ciaa1538 (2020). 28. Abu-Raddad, L. J. et al. Assessment of the risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfectio an intense reexposure setting. Clin. Infect. Dis. 73, e1830–e1840. https://doi.org/10.1093/cid/ciaa1846 (2020). p g f p g ( ) 9. Iwasaki, A. What reinfections mean for COVID-19. Lancet. Infect. Dis 21, 3–5. https://doi.org/10.1016/S1473-3099(20)30783-0 (2021). 30. Colson, P., Finaud, M., Levy, N., Lagier, J.-C. & Raoult, D. Evidence of SARS-CoV-2 re-infection with a different genotype. J. Infect. 82, 84–123. https://doi.org/10.1016/j.jinf.2020.11.011 (2021). 31. Selhorst, P. et al. Symptomatic severe acute respiratory syndrome coronavirus 2 reinfection of a healthcare worker in a Belgian Nosocomial Outbreak Despite Primary Neutralizing Antibody Response. Clin. Infect. Dis. 73, e2985–e2991. https://doi.org/10. 1093/cid/ciaa1850 (2020). 32. Tillett, R. L. et al. Genomic evidence for reinfection with SARS-CoV-2: A case study. Lancet. Infect. Dis 21, 52–58. https://doi.org/ 10.1016/S1473-3099(20)30764-7 (2021). 3. Gupta, V. et al. Asymptomatic reinfection in 2 healthcare workers from India with genetically distinct severe acute respiratory syndrome Coronavirus 2. Clin. Infect. Dis. 73, e2823–e2825. https://doi.org/10.1093/cid/ciaa1451 (2020).ii y f p g 4. Prado-Vivar, B. et al. COVID-19 re-infection by a phylogenetically distinct SARS-CoV-2 variant, first confirmed event in South America. SSRN https://doi.org/10.2139/ssrn.3686174 (2020).i p g 35. To, K. et al. COVID-19 re-infection by a phylogenetically distinct SARS-coronavirus-2 strain confirmed by whole genome sequenc- ing. Clin. Infect. Dis. 73, e2946–e2951. https://doi.org/10.1093/cid/ciaa1275 (2020). g f p g 6. Van Elslande, J. et al. Symptomatic SARS-CoV-2 reinfection by a phylogenetically distinct strain. Clin. Infect. Diseases. https://doi org/10.1093/cid/ciaa1330 (2020). g 7. Deng, X. et al. Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant. Cell 184, 3426-3437.e3428 https://doi.org/10.1016/j.cell.2021.04.025 (2021). p g j ( ) 8. Singh, J. et al. Structure-function analyses of new SARS-CoV-2 variants B. 1.1. 7, B. 1.351 and B. 1.1. References Shirin, T. et al. Antibody responses after COVID-19 infection in patients who are mildly symptomatic or asymptomatic in Bang- ladesh. Int. J. Infect. Dis. 101, 220–225. https://doi.org/10.1016/j.ijid.2020.09.1484 (2020). f p g j j 12. Huang, A. T. et al. A systematic review of antibody mediated immunity to coronaviruses: Kinetics, correlates of protection, and association with severity. Nat. Commun. 11, 1–16. https://doi.org/10.1038/s41467-020-18450-4 (2020). association with severity. Nat. Commun. 11, 1–16. https://doi.org/ 13. BNO-News. (bnonews.com, COVID-19 reinfection tracker, 2020). 13. BNO-News. (bnonews.com, COVID-19 reinfection tracker, 2020). 14. CDC. (Centers for Disease Control and Prevention, Common Investigation Protocol for Investigating Suspected SARS-CoV-2 Reinfection, 2020). 5. Tang, C. Y. et al. Reinfection with two genetically distinct SARS-CoV-2 viruses within 19 days. J. Med. Virol. https://doi.org/10 1002/jmv.27154 (2021). j 16. Rubin, D. et al. SARS-CoV-2 infection in public school district employees following a district-wide vaccination program—Philadel- phia County, Pennsylvania, March 21–April 23, 2021. Morb. Mortal. Wkly Rep. 70, 1040. https://doi.org/10.15585/mmwr.mm703 0e1 (2021).f ( ) 17. Abu-Raddad, L. J. et al. Protection afforded by the BNT162b2 and mRNA-1273 COVID-19 vaccines in fully vaccinated cohorts with and without prior infection. medRxiv https://doi.org/10.1101/2021.07.25.21261093 (2021). Scientific Reports \| (2022) 12:1438 \| https://doi.org/10.1038/s41598-022-05325-5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 18. Bergwerk, M. et al. COVID-19 breakthrough infections in vaccinated health care workers. N. Engl. J. Med. https://doi.org/10.1056/ NEJMoa2109072 (2021).fi 18. Bergwerk, M. et al. COVID-19 breakthrough infections in vaccinated health care workers. N. Engl. J. Med. https://doi.org/10.1056/ NEJMoa2109072 (2021). 19. Voysey, M. et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: An interim analysis of f d i d t ll d t i l i B il S th Af i d th UK L t 397 99 111 htt //d i /10 1016/S0140 6736(20) J ( ) 19. Voysey, M. et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: An interim analy four randomised controlled trials in Brazil, South Africa, and the UK. Lancet 397, 99–111. https://doi.org/10.1016/S0140-6736 32661-1 (2021).f four randomised controlled trials in Brazil, South Africa, and the UK. Lancet 397, 99 111. https://doi.org/10.1016/S0140 6736(20) 32661-1 (2021). 20. Bernal, J. L. et al. Effectiveness of COVID-19 vaccines against the B.1.617.2 variant. medRxiv. https://doi.org/10.1101/2021.05.22. 21257658 (2021) 0. Bernal, J. L. et al. Effectiveness of COVID-19 vaccines against the B.1.617.2 variant. medRxiv. https://doi.org/10.1101/2021.05.22 21257658 (2021).fi ( ) 21. Emary, K. R. et al. Efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 variant of concern 202012/01 (B. 1. 1. 7): An exploratory analysis of a randomised controlled trial. Lancet 397, 1351–1362. https://doi.org/10.1016/S0140-6736(21) 00628-0 (2021).fi 22. Madhi, S. A. et al. Efficacy of the ChAdOx1 nCoV-19 COVID-19 vaccine against the B. 1.351 variant. N. Engl. J. Med. 384, 1885–1898. https://doi.org/10.1056/NEJMoa2102214 (2021). p g 3. Brown, C. M. et al. Outbreak of SARS-CoV-2 infections, including COVID-19 vaccine breakthrough infections, associated with large public gatherings—Barnstable County, Massachusetts, July 2021. Morb. Mortal. Wkly Rep. 70, 1059 (2021). 4. Team, C. C.-V. B. C. I. COVID-19 vaccine breakthrough infections reported to CDC—United States, January 1–April 30, 2021 Morb Mortal Wkly Rep 70 792 https://doi org/10 15585/mmwr mm7021e3 (2021) 23. Brown, C. M. et al. Outbreak of SARS-CoV-2 infections, including COVID-19 vaccine breakthrough infections, associated with large public gatherings—Barnstable County, Massachusetts, July 2021. Morb. Mortal. Wkly Rep. 70, 1059 (2021). 24. Team, C. C.-V. B. C. I. COVID-19 vaccine breakthrough infections reported to CDC—United States, January 1–April 30, 2021. Morb. Mortal. Wkly Rep. 70, 792. https://doi.org/10.15585/mmwr.mm7021e3 (2021). large public gatherings—Barnstable County, Massachusetts, July 2021. Morb. Mortal. Wkly Rep. 70, 1059 (2021). 4. Team, C. C.-V. B. C. I. www.nature.com/scientificreports/ New COVID-19 cases and hospitalizations among adults, by vaccination status—New York, May 3–July 25 2021. Morb. Mortal. Wkly Rep. 70, 1150–1155 (2021). y p 5. China-CDC. Laboratory testing for COVID-19. (2020). https://www.chinacdc.cn/en/COVID19/202011/P0202011195302723867 86.pdf. Accessed 16 Jan 2022. Acknowledgementsh g The study was funded by the Bill and Melinda Gates Foundation (BMGF Investment ID INV-017556) and the Global Affairs Canada (GAC, Agreement no. 7392700), and approved by the icddr,b institutional review board. icddr,b acknowledges with gratitude the commitment of the BMGF and DFATD to its research efforts. icddr,b is also grateful to the Government of Bangladesh, Canada, Sweden, and the UK for providing core/unrestricted support. Author contributions M.R. developed methodology, conceived and coordinated the study, and wrote the paper. S.R. cleaned and finalised the dataset for analysis, did the descriptive analyses, interpreted the results, and drafted the first manu- script; he also built the questionnaires for reinfected individuals, set up and ran the data collection systems, and linked participant records. M.M.R. and Mo.M. performed laboratory procedures, including rRT-PCR for SARS- CoV-2 testing, genome sequencing, and interpretation of the laboratory data. M.N.B. built the questionnaires for reinfected individuals, conducted extended interviews, and linked participant records. M.E.H. and M.Z.R. coordinated all laboratory procedures and verified the data. M.S. and Mu.M. were involved in study participant enrolment, COVID-19 patient management through icddr,b staff clinic, clinical data collection for in-patient and out-patient group, cleaned and finalised the clinical investigation dataset for analysis, did the descriptive analyses. M.J.C., T.A., S.E.A. coordinated all clinical investigation and COVID-19 patient management systems in the institute and verified clinical data. M.E.H., M.Z.R., M.J.C., T.A., and S.E.A. critically reviewed the manu- script and provided intellectual input. All authors reviewed subsequent drafts of the manuscript and approved the final version. All authors had full access to all the data in the study and accepted the responsibility to submit for publication. www.nature.com/scientificreports/ Clin. Med 9, 3315. https://doi.org/10.3390/jcm9103315 (2020). p g j 1. den Hartog, G. et al. Persistence of antibodies to SARS-CoV-2 in relation to symptoms in a nationwide prospective study. Clin Infect. Diseases. https://doi.org/10.1093/cid/ciab172 (2021).t 52. Dan, J. M. et al. Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science https://doi.org/10. 1126/science.abf4063 (2021). 53. Gaebler, C. et al. Evolution of antibody immunity to SARS-CoV-2. Nature 591, 639–644. https://doi.org/10.1038/s41586-021- 03207-w (2021).t 4. Leidi, A. et al. Risk of reinfection after seroconversion to SARS-CoV-2: A population-based propensity-score matched cohor study. Clin. Infect. Diseases. https://doi.org/10.1093/cid/ciab495 (2021). y f p g 55. Hansen, C. H., Michlmayr, D., Gubbels, S. M., Mølbak, K. & Ethelberg, S. Assessment of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in 2020: A population-level observational study. Lancet 397, 1204–1212. https://doi.org/10.1016/S0140-6736(21)00575-4 (2021). https://doi.org/10.1038/s41598-022-05325-5 Scientific Reports \| (2022) 12:1438 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ 6. Rodda, L. B. et al. Functional SARS-CoV-2-specific immune memory persists after mild COVID-19. Cell 184, 169-183.e117 https://doi.org/10.1016/j.cell.2020.11.029 (2021). p g j 7. Seow, J. et al. Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans. Nat. Microbiol. 5, 1598–1607. https://doi.org/10.1038/s41564-020-00813-8 (2020).it p g ( ) 58. Havervall, S. et al. SARS-CoV-2 induces a durable and antigen specific humoral immunity after asymptomatic to mild COVID-19 infection. medRxiv. https://doi.org/10.1101/2021.01.03.21249162 (2021). p g 8. Havervall, S. et al. SARS-CoV-2 induces a durable and antigen specific humoral immunity after asymptomatic to mild COVID-19 infection. medRxiv. https://doi.org/10.1101/2021.01.03.21249162 (2021). g infection. medRxiv. https://doi.org/10.1101/2021.01.03.21249162 p g 9. Stamatatos, L. et al. mRNA vaccination boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection. Science 372, 1413–1418. https://doi.org/10.1126/science.abg9175 (2021).ti , p // g/ / g ( ) 60. Reynolds, C. J. et al. Prior SARS-CoV-2 infection rescues B and T cell responses to variants after first vaccine dose. Science 372, 1418–1423. https://doi.org/10.1126/science.abh1282 (2021).f , p g g ( ) 60. Reynolds, C. J. et al. Prior SARS-CoV-2 infection rescues B and T cell responses to variants after first vaccine dose. Science 372, 1418–1423. https://doi.org/10.1126/science.abh1282 (2021).f p g 61. Prendecki, M. et al. Effect of previous SARS-CoV-2 infection on humoral and T-cell responses to single-dose BNT162b2 vaccine. Lancet 397, 1178–1181. https://doi.org/10.1016/S0140-6736(21)00502-X (2021). p g 61. Prendecki, M. et al. Effect of previous SARS-CoV-2 infection on humoral and T-cell responses to single-dose BNT162b2 vaccine. Lancet 397, 1178–1181. https://doi.org/10.1016/S0140-6736(21)00502-X (2021). g . Prendecki, M. et al. Effect of previous SARS-CoV-2 infection o p g 2. Kanozia, R. & Arya, R. “Fake news”, religion, and COVID-19 vaccine hesitancy in India, Pakistan, and Bangladesh. Media Asia https://doi.org/10.1080/01296612.2021.1921963 (2021). p g 3. Roberts, A. T., Piani, F., Longo, B., Andreini, R. & Meini, S. Reinfection of SARS-CoV-2–analysis of 23 cases from the literature Infect. Dis. 53, 479–485. https://doi.org/10.1080/23744235.2021.1905174 (2021). p g 63. Roberts, A. T., Piani, F., Longo, B., Andreini, R. & Meini, S. Reinfection of SARS-CoV-2–analysis of 23 cases from the literature. Infect. Dis. 53, 479–485. https://doi.org/10.1080/23744235.2021.1905174 (2021). f , p g ( ) 64. Rosenberg, E. S. et al. New COVID-19 cases and hospitalizations among adults, by vaccination status—New York, May 3–July 25, 2021. Morb. Mortal. Wkly Rep. 70, 1150–1155 (2021). f p g 4. Rosenberg, E. S. et al. © The Author(s) 2022 Additional informationh Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-022-05325-5. Correspondence and requests for materials should be addressed to M.R. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022 https://doi.org/10.1038/s41598-022-05325-5 Scientific Reports \| (2022) 12:1438 \|
https://openalex.org/W3034049350	https://bmcpublichealth.biomedcentral.com/track/pdf/10.1186/s12889-020-08943-8	English	null	Influence of quality of intensive care on quality of life/return to work in survivors of the acute respiratory distress syndrome: prospective observational patient cohort study (DACAPO)	BMC public health	2,020	cc-by	7,457	Apfelbacher et al. BMC Public Health (2020) 20:861 https://doi.org/10.1186/s12889-020-08943-8 Apfelbacher et al. BMC Public Health (2020) 20:861 https://doi.org/10.1186/s12889-020-08943-8 Open Access Influence of quality of intensive care on quality of life/return to work in survivors of the acute respiratory distress syndrome: prospective observational patient cohort study (DACAPO) Christian Apfelbacher1† , Susanne Brandstetter2, Sebastian Blecha3, Frank Dodoo-Schittko2, Magdalena Brandl2, Christian Karagiannidis4, Michael Quintel5, Stefan Kluge6, Christian Putensen7, Sven Bercker8, Björn Ellger9, Thomas Kirschning10, Christian Arndt11, Patrick Meybohm12, Steffen Weber-Carstens13, the DACAPO study group and Thomas Bein3† Abstract Background: Significant long-term reduction in health-related quality of life (HRQoL) is often observed in survivors of the acute respiratory distress syndrome (ARDS), and return to work (RtW) is limited. There is a paucity of data regarding the relationship between the quality of care (QoC) in the intensive care unit (ICU) and both HRQoL and RtW in ARDS survivors. Therefore, the aim of our study was to investigate associations between indicators of QoC and HRQoL and RtW in a cohort of survivors of ARDS. Methods: To determine the influence of QoC on HRQoL and RtW 1 year after ICU-discharge, ARDS patients were recruited into a prospective multi-centre patient cohort study and followed up regularly after discharge. Patients were asked to complete self-report questionnaires on HRQoL (Short Form 12 physical component scale (PCS) and mental component scale (MCS)) and RtW. Indicators of QoC pertaining to volume, structural and process quality, and general characteristics were recorded on ICU level. Associations between QoC indicators and HrQoL and RtW were investigated by multivariable linear and Cox regression modelling, respectively. B values and hazard ratios (HRs) are reported with corresponding 95% confidence intervals (CIs). (Continued on next page) Correspondence: christian.apfelbacher@med.ovgu.de; thomas.bein@ukr.de †Christian Apfelbacher and Thomas Bein contributed equally to this work. 1Institute of Social Medicine and Health Systems Research, Medical Faculty, Otto von Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany 3Department of Anesthesia & Operative Intensive Care, University Hospital Regensburg, 93042 Regensburg, Germany Full list of author information is available at the end of the article 3Department of Anesthesia & Operative Intensive Care, University Hospital Regensburg, 93042 Regensburg, Germany Full list of author information is available at the end of the article Study design and sample Methods of the DACAPO (Surviving ARDS: the influence of quality of care and individual patient characteristics on health-related quality of life) study have been described in detail elsewhere [14, 15]. Briefly, adult patients with diag- nosed ARDS according to the Berlin definition [16] were recruited in 61 intensive care units (ICUs) across Germany into a cohort study with three postal follow-ups (3, 6 and 12 months). Ethical approval was obtained from the ethics committee of the University of Regensburg (file number: 13–101-0262) and additionally from the ethics committees overseeing the respective study sites. With mortality estimated up to 45%, the focus of ARDS research has been on mortality for a long time. With decreasing mortality rates [5] however, the interest in long-term outcomes such as mental health, return to work (RtW) and health-related quality of life (HRQoL) of ARDS patients increased [6–10]. p A multi-centre national study in the U.S. showed that nearly half of previously employed ARDS survivors were jobless at 12 months after ARDS and that this was accom- panied by substantial lost earnings [11]. If one aims at im- proving long-term outcome for these patients, it is important to shed light on possible determinants of out- comes such as HRQoL and RtW. We performed a system- atic literature review [12] summarising the existing evidence regarding the determinants of HRQoL or RtW in ARDS patients, including 24 highly heterogeneous observa- tional studies. One of the main findings was that the core focus of published research was on clinical and care-related determinants (performance in pulmonary function testing, duration of ICU treatment etc.) which mainly showed small, non-significant effects on HRQoL and RtW. Despite the evident role of the care provided to patients with ARDS in the ICU, surprisingly, the role of quality of care (QoC) for long-term HRQoL and RtW has not been investigated thus far while ARDS mortality has been investigated in rela- tion to university level of care [13]. (Continued from previous page) Results: 877 (of initially 1225 enrolled) people with ARDS formed the DACAPO survivor cohort, 396 were finally followed up to 1 year after discharge. The twelve-month survivors were characterized by a reduced HRQoL with a greater impairment in the physical component (Md 41.2 IQR [34–52]) compared to the mental component (Md 47.3 IQR [33–57]). Overall, 50% of the patients returned to work. The proportion of ventilated ICU patients showed significant negative associations with both 12 months PCS (B = −11.22, CI −20.71; −1,74) and RtW (HR = 0,18, CI 0, 04;0,80). All other QoC indicators were not significantly related to outcome. Conclusions: Associations between ICU QoC and long-term HrQoL and RtW were weak and largely non-significant. Residual confounding by case mix, treatment variables before or during ICU stay and variables pertaining to the post intensive care period (e.g. rehabilitation) cannot be ruled out. Trial registration: Clinicaltrials.govNCT02637011. (December 22, 2015, retrospectively registered) Trial registration: Clinicaltrials.govNCT02637011. (December 22, 2015, retrospectively registered) Trial registration: Clinicaltrials.govNCT02637011. (December 22, 2015, retrospectively registered) Keywords: ARDS, Quality of care, Volume, ICU, Health-related quality of life, Return to work Keywords: ARDS, Quality of care, Volume, ICU, Health-related quality of life, Return to work Background Against this background, the hypothesis underlying the study presented in this paper was that better QoC (defined by quality indicators) received during the acute ICU stay was associated with better HRQoL and a higher rate of RtW in survivors of ARDS. Thus, our aim was to identify QoC indicators predictive of HRQoL and RtW. Acute respiratory syndrome (ARDS) is characterised by re- spiratory failure with either a direct pulmonary (e.g. pneu- monia) or extra-pulmonary cause (e.g. sepsis) that requires treatment in intensive care including mechanical ventilation [1]. Approximately 10% of all patients admitted to the ICU develop ARDS [2]. This represents a considerable amount of patients. Further, cost associated with ARDS are very high. For instance, a study from the UK estimated mean so- cietal cost over 1 year including initial ICU treatment cost at £ 44.077 [3]. A US study estimated the median cost due to hospitalization post-discharge at $ 18.756 [4]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 9 Apfelbacher et al. BMC Public Health (2020) 20:861 Measurements Outcomes The Short Form-12 self-report questionnaire (SF-12) was used to measure HRQoL [17]. Scores for the Phys- ical Component (PCS-12) and the Mental Component Scales (MCS-12) range from 0 to 100 (higher values in- dicating better HRQoL). Fifty is the mean value for the general population (German norm values [18]). RtW was captured as self-report, asking whether and when people had returned to their previous or another job (only for persons in employment before admission to ICU). We included data from all participants who returned valid questionnaires up to 13 months after discharge from ICU. Mortality status and date of death were assessed ei- ther through reports from the patients’ caregivers or through local population registries. Apfelbacher et al. BMC Public Health (2020) 20:861 Apfelbacher et al. BMC Public Health (2020) 20:861 Page 3 of 9 Page 3 of 9 Outcome: mortality We examined the influence of the exposure variables on 1- year mortality in ICU survivors. The set of covariates for the fully adjusted logistic regression models was determined as described for HRQoL and RtW. Odds ratios (ORs) and 95% CIs were computed. A p-value of < 0.05 was consid- ered statistically significant. Analyses were performed using Stata 13.1 (Stata Corporation, College Station, TX, U.S.A.). Descriptive results We preliminarily included 1900 ARDS patients from 61 ICUs across Germany between September 2014 and April 2016. One thousand two hundred twenty five pa- tients with informed consent formed the initial ICU sample (Fig. 1). Eight hundred seventy-seven patients formed the DACAPO survivor cohort. Four hundred eighty-one patients (54.8%) were lost to follow up for various reasons (e.g.inability to participate). Information on death or survival could not be obtained for 66 pa- tients. At 1 year after discharge, 19.8% had died and 396 persons were followed up. Potentially confounding variables y g Variables related to socio-demographic, clinical and care aspects were assessed as potentially confounding vari- ables. The socio-demographic variables included age, sex, living situation (living with versus without a part- ner), nationality (German, other), and health insurance (statutory, private, other). An education score was de- rived from information on the participants’ educational and professional levels (according to Lampert et al. [21]). The score ranges between 1 and 7, with higher values in- dicating higher educational and/or professional achieve- ments. General medical characteristics were captured with regard to body mass index (BMI: kg/m2), cause of ARDS (pulmonary / extra-pulmonary), Simplified Acute Physiology Score II (SAPS-II) [22], and Sequential Organ Failure Assessment (SOFA) [23] score, as well as self- reported physician-diagnosed mental disorder before treatment in the ICU (any, none). The Berlin definition of ARDS was modified in terms of the classification of the severity of hypoxemia (PaO2/FIO2 ratio [P/F ratio]) since the classification into two levels (P/F ratio ≤150 mmHg versus > 150 mmHg) may allow better selectivity between mild and severe cases [24, 25]. Outcome: return to work (RtW) We used Cox proportional hazards models to analyse RtW. The minimally adjusted models had sex, age, and ARDS-severity as covariates. The fully adjusted multivari- ate Cox models included all covariates that showed signifi- cant effects with either outcome or exposure in univariate Cox regression analyses. Observations were censored if RtW did not occur within 395 days following ICU dis- charge. Hazard ratios (HRs) with 95% CIs were computed. Outcome: health-related quality of life (HRQoL) Outcome: health-related quality of life (HRQoL) The twelve-month analysis was the primary analysis. For each follow-up (3, 6 and 12 months) and each expos- ure–outcome combination non-adjusted, minimally ad- justed, and fully adjusted linear regression models were used. The minimally adjusted model contained sex, age, and ARDS severity as covariates; all socio-demographic and medical variables which were significantly (p-value < 0.05) associated with exposures or outcome at any follow-up period were included in the fully adjusted re- gression models. Non-standardized regression coeffi- cients with 95% confidence intervals (CIs) were computed as were standardized regression coefficients. Exposures consistently close to zero on the basis of the fully uncon- ditional model and non-significant p-values for the like- lihood ratio tests indicating non-deterioration in model fit if the random intercept was restricted to zero (data not shown). For this reason, fixed-effects linear, logistic and Cox regression models were applied. p QoC was assessed at the level of the participating ICUs once during the period of the study. It was operational- ized as structural quality (head of the ICU with add- itional training in intensive care medicine), process quality (implementation of daily multi-professional ward rounds with documentation of daily therapy goals), volume (number of ventilated patients per year), and general char- acteristics (membership of the hospital in the ARDS Net- work Germany). These pre-specified indicators showed limited statistical variance and were thus complemented by additional quality indicators comprising the proportion of physicians with completed specialized ICU training, the availability of weekly microbiological ward rounds, the proportion of ventilated patients on all patients, and gen- eral level of care (university hospital versus other hospital). Both the pre-specified and the additional quality indica- tors were largely based on the published list of German quality indicators in intensive care medicine [14, 19, 20] and together with generally accepted indicators such as volume and level of care have been used in the compre- hensive QoC assessment in the DACAPO study. Statistical analysis Owing to the data structure (patients are nested within different ICUs), we used hierarchical linear modeling, testing whether there was systematic variance between the second-level units (ICUs) in the primary outcome at the three follow-ups. For all outcomes, these analyses yielded an intraclass correlation coefficient (ICC) Apfelbacher et al. BMC Public Health (2020) 20:861 Page 4 of 9 Fig. 1 Patient flow. a For all patients who were lost to follow-up survival was assessed via local municipal population registries. b Written informed consent and patient data were transferred to the study centre with a delay of more than 12 months; thus, follow-up measurement was not possible within the scheduled follow-up period Fig. 1 Patient flow. a For all patients who were lost to follow-up survival was assessed via local municipal population registries. b Written informed consent and patient data were transferred to the study centre with a delay of more than 12 months; thus, follow-up measurement was not possible within the scheduled follow-up period The general characteristics and the organizational and structural QoC indicators of the participating ICUs were as follows (Table 1): 70% of study centres were members of the German ARDS network and 28 centres were university institutions. The median number of patients ventilated per year was 493 and the median percent of patients who were ventilated was 44%. The twelve- month ARDS survivors reported a median physical SF-12 (SF-12 PCS) of 41 (IQR 35–52) and a mental SF-12 (SF-12 MCS) of 47 (IQR 33–57) Fig. 2. No The socio-demographic and general medical charac- teristics of the respondents for the twelve-month follow-up are described in an additional file (Table 4, additional file 1). In about 60% the diagnosis was made in a DACAPO ICU, the remainder was trans- ferred. The ARDS was predominantly caused by direct (pulmonary) conditions. The majority of patients (75%) suffered from ‘moderate-to-severe’ ARDS (P/F ratio < 150). A lifetime diagnosis of a mental disorder was reported in 15%. Volume Proportion of ventilated patients on all patients, Md (IQR) Statistical analysis Table 1 Characteristics of participating ICUs ICUs N Volume Number of ventilated patients per year/100, Md (IQR) 48 4.93 (3.87–8.35) Proportion of ventilated patients on all patients, Md (IQR) 47 0.44 (0.30–0.59) Process quality Daily multiprofessional ward rounds with documentation of daily therapy goals, N (%) 51 51 (100) Weekly microbiological ward rounds, N (%) 52 37 (71.2) Structural quality Proportion of physicians with completed specialised training on all physicians, Md (IQR) 52 0.25 (0.16–0.33) Direction with additional training “intensive medicine”, N (%) 52 51 (98.1) General characteristics Member of ARDS-network, N (%) 53 37 (69.8) Level of care: University hospital, N (%) 53 28 (52.8) Table 1 Characteristics of participating ICUs Table 1 Characteristics of participating ICUs Additional analytical results In the fully adjusted analysis of the three-month follow-up (Table 6, additional file 3), a higher per- centage of patients ventilated was significantly associ- ated with a decreased PCS-12. There was a trend towards a decreased PCS-12 for patients treated in university hospitals (p = 0.054). No significant General characteristics Apfelbacher et al. BMC Public Health (2020) 20:861 Page 5 of 9 Fig. 2 SF-12 values of survivors of ARDS after 12 months (a). Probability of not returning to work (b) Fig. 2 SF-12 values of survivors of ARDS after 12 months (a). Probability of not returning to work (b) ICUs that had a higher percentage of ventilated patients was significantly associated with a decreased PCS-12. The analysis of the effects of QoC on RtW after 12 months (Cox regression analyses, Table 2) showed a significantly decreased hazard of RtW for patients treated in institutions with a higher proportion of ventilated of patients but no significant association was found for all further variables. substantial increase in SF-12 values was observed be- tween 3 and 12 months. Slightly over half of the pa- tients returned to work after 1 year. Analytical results The main analysis refers to the effect of QoC on HRQoL (Table 5, additional file 2) and RtW (Table 2) after 12 months, and additional analyses were computed for three (Table 6, additional file 3) and 6 months (Table 7, additional file 4) and mortality (Table 3). Main analytical results There was no significant association between most QoC variables and HRQoL at 12 months (Table 5, additional file 2). However, in the fully adjusted analysis, treatment in Table 2 Cox regression analyses of RtW on quality of care in 12 months respondents Table 2 Cox regression analyses of RtW on quality of care in 12 months respondents 12 months follow up: RtW unadjusted minimally adjusted a fully adjusted b N Haz. Ratio (CI) p-value N Haz. Ratio (CI) p-value N Haz. Ratio (CI) p-value Volume N of ventilated patients per year/100 165 0.982 (0.941–1.024) 0.400 138 0.985 (0.940–1.032) 0.525 125 0.975 (0.924–1.029) 0.360 Proportion of ventilated patients on all patients 159 0.366 (0.115–1.163) 0.088 135 0.350 (0.097–1.266) 0.110 122 0.182 (0.041–0.803) 0.024 Process quality Weekly microbiological ward rounds 175 0.843 (0.485–1.466) 0.546 148 0.743 (0.378–1.459) 0.388 134 0.787 (0.338–1.831) 0.578 Structural quality Proportion of physicians with completed specialised training on all physicians 175 2.674 (0.459–15.571) 0.274 148 1.230 (0.141–10.726) 0.852 134 4.829 (0.436–53.482) 0.199 General characteristics Member of ARDS network 175 0.550 (0.311–0.972) 0.040 148 0.614 (0.341–1.106) 0.104 134 0.656 (0.312–1.381) 0.267 Level of Care: University hospital 175 0.829 (0.514–1.339) 0.444 148 0.845 (0.490–1.459) 0.545 134 0.724 (0.392–1.338) 0.303 a adjusted for age, sex, severity of ARDS; b adjusted for age, sex, severity of ARDS, SOFA score, SAPS-II score, diagnosis of ARDS (participating vs. other ICU), education score Apfelbacher et al. Interpretation, in relation to literature associations were observed for any QoC parameter and 6 months HRQoL (Table 7, additional file 4). Our results need careful interpretation. A study by Ray- mondos et al. [13] showed that the hospital mortality risk of ARDS patients was considerably higher in pa- tients who were treated in non-university hospitals com- pared to university institutions. However, the present study did not demonstrate a similar effect for HRQoL or RtW 1 year after discharge. A systematic review [26] demonstrated that critically ill patients generally benefit from institutions with high volume regarding mortality with more substantial effects in high risk patients, and there was evidence that this relationship is in part medi- ated by key hospital or ICU organizational factors. We could not confirm this evidence. Based on a logistic regression analysis among ICU sur- vivors twelve-month mortality risk (Table 3) was signifi- cantly elevated for patients treated in institutions with a higher number of ventilated patients per year. Addition- ally, patients treated in university hospitals had a signifi- cantly increased twelve-month mortality risk (OR 1.946, p = 0.032) compared to non-university institutions. A trend was seen for a reduced mortality risk in patients treated in ICUs with a higher percentage of ICU special- ist physicians. Main analytical results BMC Public Health (2020) 20:861 Page 6 of 9 Table 3 Logistic regression analyses of mortality on quality of care in ICU survivors 12 months follow up unadjusted minimally adjusted a fully adjusted b N Odds Ratio (CI) p-value N Odds Ratio (CI) p-value N Odds Ratio (CI) p-value Volume N of ventilated patients per year/100 767 1.044 (1.013–1.076) 0.005 660 1.053 (1.017–1.090) 0.004 544 1.048 (1.008–1.090) 0.018 Proportion of ventilated patients on all patients 749 1.476 (0.583–3.737) 0.411 646 1.228 (0.435–3.463) 0.698 533 1.856 (0.558–6.178) 0.313 Process quality Weekly microbiological ward rounds 811 1.386 (0.869–2.212) 0.171 702 1.483 (0.861–2.552) 0.155 575 1.579 (0.835–2.988) 0.160 Structural quality Proportion of physicians with completed specialised training on all physicians 811 0.058 (0.009–0.390) 0.003 702 0.072 (0.009–0.604) 0.015 575 0.098 (0.009–1.019) 0.052 General characteristics Member of ARDS network 814 1.141 (0.645–2.019) 0.651 705 1.269 (0.687–2.342) 0.447 577 1.632 (0.765–3.485) 0.205 Level of Care: University hospital 814 1.636 (1.029–2.602) 0.037 705 1.638 (0.981–2.736) 0.059 577 1.946 (1.059–3.576) 0.032 a adjusted for age, sex, severity of ARDS; b adjusted for age, sex, severity of ARDS, BMI, cause of ARDS, SAPS-II score, SOFA score, diagnosis of ARDS (participating vs. other ICU), nationality Discussion Key findings It must be noted though that in contrast to mortality, HRQoL is a complex construct containing individual as- pects (multiple dimensions, often operationalized as so- cial, somatic and psychological variables [27]. Our above mentioned systematic review [12] demonstrated signifi- cant associations with HRQoL after ARDS only for de- terminants which were closely related to the scales of the HRQoL instruments and which were measured at the same time as HRQoL. The main finding of our study was that of all the quality in- dicators investigated – taking important confounding fac- tors into account – only the proportion of ventilated patients on all patients showed a significant (negative) asso- ciation with both twelve-months physical HRQoL (PCS-12) and RtW. Secondary findings were a negative significant association of the proportion of ventilated patients on all patients and positive significant associations between the number of ventilated patients per year and university hospital level of care with post ICU mortality risk. No other quality indicator was significantly associated with the outcomes of interest. The DACAPO survivor cohort was characterized by a reduced HRQoL compared to the general population, with a greater impairment in the physical component compared to the mental component. 50% of the surviving patients who were in employment before ARDS had returned to work one year after trans- ferred from the ICU. We were unable to consider variables pertaining to the period after discharge although the post ICU period may play an important role in terms of HRQoL. For instance, a prospective one-year follow-up of 126 patients who re- ceived prolonged mechanical ventilation by Unroe et al. [28] showed that these experienced multiple ‘trajectories’ after their transfer from the ICU, resulting in frequent read- missions or transitions to various healthcare institutions. It might be argued that we did not find strong associa- tions between our exposures of interest (i.e. parameters of QoC) and the different outcomes because 1) the exposures Apfelbacher et al. BMC Public Health (2020) 20:861 Page 7 of 9 Page 7 of 9 showed little variability and 2) effects may have been seen if intermediary outcomes such as length of ventilation or pre- vention of multiorgan failure would have been considered. However, when it became clear that the quality indicators that were chosen initially did not show sufficient variability, we considered further quality indicators which were related to those that were chosen initially. These indicators did in- deed show variability. Future research F h h Further, in this paper, we only selected some QIs from the full set. One idea for future research might be to use the full set of QIs to develop a scoring system predictive of patient-level outcome. Such an attempt however is beyond the scope of this paper. Given the importance of next of kin / family during ICU stay [32], it would be interesting to additionally look at patient-family satisfaction and patient-family engage- ment in future research. Strenghts and limitations In conclusion, most indicators of acute QoC were not significantly associated with one-year HRQoL or RtW in ARDS survivors. Post-ICU exposure of ARDS survivors may have attenuated the assumed effects of high-volume care. Overall, we cannot rule out residual confounding by case mix, treatment variables before or during ICU stay and variables pertaining to the post ICU period. We were successful in the conduct of a prospective pa- tient cohort study with regular follow-ups. Unfortu- nately, the number of people lost to follow-up was considerable which may have introduced attrition bias. ICU mortality was not even used as outcome in second- ary analysis because the recruitment strategies used fo- cused on the survivor cohort as baseline. For instance, only surviving patients or patients with a legal guardian providing informed consent could be included at some sites, while consent by next-of-kin was acceptable at other sites. Any analysis of factors predictive of ICU mortality would therefore be seriously biased. Discussion Key findings Second, our research interest was precisely to study modifiable institutional-level indicators of quality of care in relation to patient-level outcomes. We are coming from a public health/health care research perspec- tive in which we can improve health or achieve better dis- ease outcomes, even if we do not know the mechanisms linking exposure to outcome. In light of this background, we did not attempt to explore pathways between QIs, HRQoL, RtW or mortality through intermediary outcomes. Treatment-related exposures not assessed on the institu- tional but on the individual patient level (parameters of the intensity of acute care management and critical events) have been investigated in a separate paper [29]. rounds and they are difficult to be monitored continu- ously in clinical practice. Usually QIs for outcome include standardized mortality (ICU, hospital, 60- or 90-days) or the incidence of decubiti [31]. No previous investigation used QIs for an assessment of the association with HRQoL. In our study we had to use established QIs only assuming that they have value to describe sufficiently the effect of care on patient-reported outcomes. Supplementary information l f pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-08943-8. Additional file 1: Excel File. Table 4. Socio-demographic and general med- ical characteristics of 12 months respondents. a derived from educational and professional level22, considered for regression analyses. SAPS-II score: Simplified Acute Physiology Score II without Glasgow Coma Scale; SOFA score: Sequential Organ Failure Assessment; BMI: Body Mass Index. bas assessed at admission at the DACAPO ICU, c without consideration of Glasgow Coma Scale. Although we adjusted for ARDS severity in our min- imally adjusted models, and further corrected for SAPS- II and SOFA scores in the fully adjusted regression models, residual confounding may still be present in re- lation to ARDS severity/case-mix which might explain our findings. Treatment received before or after ICU care was also not corrected for which might have further contributed to residual confounding. Additional file 2: Excel File. Table 5. Linear Regression Analyses of Health- related Quality of Life after 12 months on Quality of Care. a adjusted for age, sex, severity of ARDS; b adjusted for age, sex, severity of ARDS, BMI, Educa- tion score, SAPS-II score, SOFA score, diagnosis of ARDS (participating vs. other ICU), self-reported physician-diagnosed mental disorder before ARDS diagnosis; Notes: ARDS = acute respiratory distress syndrome; MCS-12 = mental component scale of short-form 12 questionnaire; PCS-12 = physical component scale of short-form 12 questionnaire. Measuring QoC in the critical care setting is challen- ging. In a review by Flaatten et al. [30] 63 quality indica- tors (QI) measuring quality of structure, process and outcome of care were identified, which are in use with a large variation between countries and no single QI was common for all. QIs for structure predominantly refer to the qualification and quantity of health care professionals, as well as the number of ventilated patients per year [31] process quality indicators are more complex, numerous and they refer to actual recommendations of guidelines (ventilation strategies, nutrition, transfusion strategy etc.). These indicators must be defined by experts or Delphi Additional file 3: Excel File. Table 6. Linear Regression Analyses of Health-related Quality of Life after 3 months on Quality of Care. a adjusted for age, sex, severity of ARDS; b adjusted for age, sex, severity of ARDS, BMI, Education score, SAPS-II score, SOFA score, diagnosis of ARDS (par- ticipating vs. Acknowledgements W d b d l We are indebted to all the intensive care specialists and study nurses throughout Germany, who, with great commitment, recruited patients for the DACAPO study: y Johannes Bickenbach, Thorben Beeker, Tobias Schürholz, Jessica Pezechk (Aachen); Jens Schloer (Amberg); Ulrich Jaschinski, Ilse Kummer (Augsburg); Oliver Kuckein (Bamberg); Steffen Weber-Carstens, Anton Goldmann, Stefan Angermair, Krista Stoycheva, Jörg Brederlau, Nadja Rieckehr, Gabriele Schrei- ber, Henriette Haennicke (Berlin); Friedhelm Bach, Immo Gummelt, Silke Haas, Catharina Middeke, Ina Vedder, Marion Klaproth (Bielefeld); Michael Adamzik, Jan Karlik, Stefan Martini, Luisa Robitzky (Bochum); Christian Putensen, Thomas Muders, Ute Lohmer (Bonn); Rolf Dembinski (Bremen); Petra Schäff- ner, Petra Wulff-Werner (Deggendorf); Elke Landsiedel-Mechenbier, Daniela Nickoleit-Bitzenberger, Ann-Kathrin Silber (Dortmund); Maximilian Ragaller, Marcello Gama de Abreu, Alin Ulbricht, Linda Reisbach (Dresden); Kai Zachar- owski, Patrick Meybohm, Simone Lindau, Haitham Mutlak (Frankfurt am Main); Alexander Hötzel, Johannes Kalbhenn (Freiburg); Christoph Metz, Ste- fan Haschka (Freising); Stefan Rauch (Göppingen); Michael Quintel, Lars-Olav Harnisch, Sophie Baumann, Andrea Kernchen (Göttingen); Sigrun Friesecke, Sebastian Maletzki (Greifswald); Stefan Kluge, Olaf Boenisch, Daniel Frings, Bir- git Füllekrug, Nils Jahn, Knut Kampe, Grit Ringeis, Brigitte Singer, Robin Wüs- tenberg (Hamburg); Jörg Ahrens, Heiner Ruschulte, Andre Gerdes, Matthias Groß, Olaf Wiesner, Aleksandra Bayat-Graw (Hannover); Thorsten Brenner, Felix Schmitt, Anna Lipinski (Heidelberg); Dietrich Henzler, Klaas Eickmeyer, Juliane Krebs, Iris Rodenberg (Herford); Heinrich Groesdonk, Kathrin Meiers, Karen Salm, Thomas Volk (Homburg); Stefan Fischer, Basam Redwan (Ibben- büren); Martin Schmölz, Kathrin Schumann-Stoiber, Simone Eberl (Immen- stadt); Gunther Lenz, Thomas von Wernitz-Keibel, Monika Zackel (Ingolstadt); Frank Bloos, Petra Bloos, Anke Braune, Anja Haucke, Almut Noack, Steffi Kola- nos, Heike Kuhnsch, Karina Knuhr-Kohlberg (Jena); Markus Gehling (Kassel); Mathias Haller, Anne Sturm, Jannik Rossenbach (Kempten); Dirk Schädler, Ste- fanie D’Aria (Kiel); Christian Karagiannidis, Stephan Straßmann, Wolfram Wind- isch, Thorsten Annecke, Holger Herff (Köln); Michael Schütz (Langen); Sven Bercker, Hannah Reising, Mandy Dathe, Christian Schlegel (Leipzig); Katrin Lichy (Ludwigsburg); Wolfgang Zink, Jana Kötteritzsch (Ludwigshafen); Marc Bodenstein, Susanne Mauff, Peter Straub (Mainz); Christof Strang, Florian Prätsch, Thomas Hachenberg (Magdeburg); Thomas Kirschning, Thomas Frie- drich, Dennis Mangold (Mannheim); Christian Arndt, Tilo Koch (Marburg); Hendrik Haake, Katrin Offermanns (Mönchengladbach); Patrick Friederich, Florian Bingold, Michael Irlbeck, Bernhard Zwissler, Ines Kaufmann, Ralph Bogdanski, Barbara Kapfer, Markus Heim, Günther Edenharter (München); Björn Ellger, Daniela Bause (Münster); Götz Gerresheim (Neumarkt i.d.OPf); Dorothea Muschner, Michael Christ, Arnim Geise (Nürnberg); Martin Beiderlin- den, Thorsten Heuter (Osnabrück); Alexander Wipfel (Passau); Werner Kargl, Marion Harth, Christian Englmeier, Thomas Bein, Sebastian Blecha, Dr. Abbreviations ARDS: Acute respiratory distress syndrome; CRF: Case report form; ICU: Intensive care Unit; IQR: Interquartile range; HRQoL: Health-related quality of life; MCS-12: Mental component summary; Md: Median; PCS- 12: Physical component summary; PTSD: Post-traumatic stress disorder; QoC: Quality of care; QoL: Quality of life; RtW: Return to work; SAPS- II: Simplified acute physiology score II; SF-12: Short form 12 survey; SOFA: Sequential organ failure assessment Supplementary information l f other ICU), self-reported physician-diagnosed mental dis- order before ARDS diagnosis Notes: ARDS = acute respiratory distress syndrome; MCS-12 = mental component scale of short-form 12 question- naire; PCS-12 = physical component scale of short-form 12 questionnaire. Additional file 3: Excel File. Table 6. Linear Regression Analyses of Health-related Quality of Life after 3 months on Quality of Care. a adjusted for age, sex, severity of ARDS; b adjusted for age, sex, severity of ARDS, BMI, Education score, SAPS-II score, SOFA score, diagnosis of ARDS (par- ticipating vs. other ICU), self-reported physician-diagnosed mental dis- order before ARDS diagnosis Notes: ARDS = acute respiratory distress syndrome; MCS-12 = mental component scale of short-form 12 question- naire; PCS-12 = physical component scale of short-form 12 questionnaire. Additional file 4: Excel File. Table 7. Linear Regression Analyses of Health-related Quality of Life after 6 months on Quality of Care. a adjusted for age, sex, severity of ARDS; b adjusted for age, sex, severity of ARDS, BMI, Education score, SAPS-II score, SOFA score, diagnosis of ARDS Page 8 of 9 Page 8 of 9 Page 8 of 9 Apfelbacher et al. BMC Public Health (2020) 20:861 for critically reviewing the intellectual content of an earlier version of this manuscript. Medical writing support was sought from topcorrect, Germany. (participating vs. other ICU), self-reported physician-diagnosed mental dis- order before ARDS diagnosis. Notes: ARDS = acute respiratory distress syn- drome; MCS-12 = mental component scale of short-form 12 (participating vs. other ICU), self-reported physician-diagnosed mental d order before ARDS diagnosis. Notes: ARDS = acute respiratory distress sy drome; MCS-12 = mental component scale of short-form 12 questionnaire; PCS-12 = physical component scale of short-form 12 questionnaire. (participating vs. other ICU), self-reported physician-diagnosed mental dis- order before ARDS diagnosis. Notes: ARDS = acute respiratory distress syn- drome; MCS-12 = mental component scale of short-form 12 questionnaire; PCS-12 = physical component scale of short-form 12 questionnaire. Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available due to confidentiality of patient data but are available from the corresponding author on reasonable request. Acknowledgements W d b d l Kathrin Thomann-Hackner, Marius Zeder (Regensburg); Markus Stephan (Stuttgart); Martin Glaser (Traunstein); Helene Häberle (Tübingen); Hendrik Bracht, Chris- tian Heer, Theresa Mast (Ulm); Markus Kredel, Ralf Müllenbach (Würzburg). Further, we are grateful to previous members of the Regensburg DACAPO study team (medical documentation: Phillip Sebök, study physician: Kathrin Thomann-Hackner), to the members of the Advisory Board of the DACAPO- Study (Julika Loss, Bernhard Graf, Michael Leitzmann, Michael Pfeifer all Re- gensburg) and to our student assistants (Simon Bein, Vreni Brunnthaler, Car- ina Forster, Stefanie Hertling, Sophie Höhne, Carolin Schimmele, Elisa Valletta, Philipp Drewitz and Chiara Eberle). We are grateful to Arthur Slutsky, Toronto, Funding Th DACA The DACAPO study was funded by a research grant from the German Federal Ministry of Education and Research (01GY1340). Grant holders were TB (University Hospital Regensburg, principal investigator) and CA1 (University of Regensburg, co-principal investigator). SB1, FDS, MB and SB2 were funded by this grant for parts of or the entire study period. All other authors received payments from the grant to support patient recruitment. The funding body had no role in the design of the study, nor in the collec- tion, analysis, and interpretation of the data, nor in writing the manuscript. Authors’ contributions CA1 and TB conceived the study with the help of SB1, SB2, FDS, SWC and MQ. CA1, SB1, FDS and MB were responsible for data management, quality assurance, conduct of the follow-up and statistical analyses. CA1 and TB wrote the manuscript with the help of SB1, FDS, CK and SWC. SK, CP, SB3, BE, TK, CA2, PM were involved in recruitment and practical implementation of the study. All authors critically read and approved the article. Competing interests B C Q S CP SB TB, CK, MQ, SK, CP, SB3, BE, TK, CA2, PM, and SWC are members of the German ARDS-Network. TB: received honoraria for lectures from Xenios Com- pany, Germany. MQ: received honoraria for lectures from Maquet, Company, and Xenios Company, Germany. All other authors declare: no relationships/ conditions/circumstances that present a potential conflict of interest. Ethics approval and consent to participate The study was approved by the Ethics Committee of the University of Regensburg (file number: 13–101-0262) and by the ethics committees responsible for the participating hospitals. All participants provided a written informed consent form. Author details 1 f 1Institute of Social Medicine and Health Systems Research, Medical Faculty, Otto von Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany. 2Medical Sociology, Institute of Epidemiology and Preventive Medicine, University of Regensburg, 93051 Regensburg, Germany. 3Department of Anesthesia & Operative Intensive Care, University Hospital Regensburg, 93042 Regensburg, Germany. 4Department of Pneumology and Critical Care Medicine, Cologne-Merheim Hospital, ARDS and ECMO Centre, Kliniken der Stadt Köln, Witten/Herdecke University Hospital, 51109 Cologne, Germany. 5Department of Anaesthesiology, Emergency and Intensive Care Medicine, University Medicine, 37075 Göttingen, Germany. 6Department of Intensive Care Medicine, University Medical Centre, Hamburg-Eppendorf, 20246 Hamburg, Germany. 7Department of Anesthesiology and Operative Intensive Care, University Hospital Bonn, 53127 Bonn, Germany. 8Department of Anaesthesiology and Intensive Care Medicine, University Hospital Leipzig, 04103 Leipzig, Germany. 9Department of Anesthesiology and Intensive Care, Klinikum Dortmund, 44137 Dortmund, Germany. 10Department of Anesthesiology and Intensive Care, University Hospital Mannheim, 68167 Mannheim, Germany. 11Department of Anesthesiology and Operative Intensive Care, University Hospital Marburg, 35042 Marburg, Germany. 12Department of Anesthesiology, Intensive Care Medicine, and Pain Therapy, University Hospital Würzburg, 97080 Würzburg, Germany. 13Department of Anaesthesiology and Intensive Care Medicine, Charité –University Medicine Berlin, 10117 Berlin, Germany. Page 9 of 9 Page 9 of 9 Page 9 of 9 Apfelbacher et al. BMC Public Health (2020) 20:861 Apfelbacher et al. BMC Public Health (2020) 20:861 Apfelbacher et al. BMC Public Health (2020) 20:861 Received: 11 April 2019 Accepted: 18 May 2020 References Respir Care. 2017;62(1):113–22. 6. Myhren H, Ekeberg O, Stokland O. Health-related quality of life and return to work after critical illness in general intensive care unit patients: a 1-year follow-up study. Crit Care Med. 2010;38(7):1554–61. y 5. Maca J, Jor O, Holub M, Sklienka P, Bursa F, Burda M, et al. Past and Present ARDS Mortality Rates: A Systematic Review. Respir Care. 2017;62(1):113–22. 27. Gaudry S, Messika J, Ricard JD, Guillo S, Pasquet B, Dubief E, et al. Patient- important outcomes in randomized controlled trials in critically ill patients: a systematic review. Ann Intensive Care. 2017;7(1):28. 6. Myhren H, Ekeberg O, Stokland O. Health-related quality of life and return to work after critical illness in general intensive care unit patients: a 1-year follow-up study. Crit Care Med. 2010;38(7):1554–61. 28. Unroe M, Kahn JM, Carson SS, Govert JA, Martinu T, Sathy SJ, et al. One-year trajectories of care and resource utilization for recipients of prolonged mechanical ventilation: a cohort study. Ann Intern Med. 2010;153(3):167–75. 28. Unroe M, Kahn JM, Carson SS, Govert JA, Martinu T, Sathy SJ, et al. One-year trajectories of care and resource utilization for recipients of prolonged mechanical ventilation: a cohort study. Ann Intern Med. 2010;153(3):167–75. y 7. Herridge MS, Tansey CM, Matte A, Tomlinson G, Diaz-Granados N, Cooper A, et al. Functional disability 5 years after acute respiratory distress syndrome. N Engl J Med. 2011;364(14):1293–304. 7. Herridge MS, Tansey CM, Matte A, Tomlinson G, Diaz-Granados N, Cooper A, et al. Functional disability 5 years after acute respiratory distress syndrome. N Engl J Med. 2011;364(14):1293–304. 29. Bein T, Weber-Carstens S, Apfelbacher C, Brandstetter S, Blecha S, Dodoo- Schittko F, et al. The quality of acute intensive care and the incidence of critical events have an impact on health-related quality of life in survivors of the acute respiratory distress syndrome - a nationwide prospective multicenter observational study. German Med Sci. 2020;18:Doc01. 29. Bein T, Weber-Carstens S, Apfelbacher C, Brandstetter S, Blecha S, Dodoo- Schittko F, et al. The quality of acute intensive care and the incidence of critical events have an impact on health-related quality of life in survivors of the acute respiratory distress syndrome - a nationwide prospective multicenter observational study. German Med Sci. 2020;18:Doc01. 8. Hashem MD, Nallagangula A, Nalamalapu S, Nunna K, Nausran U, Robinson KA, et al. Patient outcomes after critical illness: a systematic review of qualitative studies following hospital discharge. Crit Care. 2016;20:345. 9. References Jutte JE, Needham DM, Pfoh ER, Bienvenu OJ. Psychometric evaluation of the hospital anxiety and depression scale 3 months after acute lung injury. J Crit Care. 2015;30(4):793–8. 30. Flaatten H. The present use of quality indicators in the intensive care unit. Acta Anaesthesiol Scand. 2012;56(9):1078–83. 30. Flaatten H. The present use of quality indicators in the intensive care unit. Acta Anaesthesiol Scand. 2012;56(9):1078–83. 10. Biehl M, Kashyap R, Ahmed AH, Reriani MK, Ofoma UR, Wilson GA, et al. Six- month quality-of-life and functional status of acute respiratory distress syndrome survivors compared to patients at risk: a population-based study. Crit Care. 2015;19:356. 31. de Vos M, Graafmans W, Keesman E, Westert G, van der Voort PH. Quality measurement at intensive care units: which indicators should we use? J Crit Care. 2007;22(4):267–74. 31. de Vos M, Graafmans W, Keesman E, Westert G, van der Voort PH. Quality measurement at intensive care units: which indicators should we use? J Crit Care. 2007;22(4):267–74. 32. Schneeberger A, Brandstetter S, Bein T, Blecha S, Apfelbacher C. Stressors and strains of next of kin of patients with ARDS in intensive care: a qualitative interview study using a stress-strain approach. Intensive Crit Care Nurs. 2019;24:102783. 11. Kamdar BB, Huang M, Dinglas VD, Colantuoni E, von Wachter TM, Hopkins RO, et al. Joblessness and lost earnings after acute respiratory distress syndrome in a 1-year National Multicenter Study. Am J Respir Crit Care Med. 2017;196(8):1012–20. 12. Dodoo-Schittko F, Brandstetter S, Blecha S, Thomann-Hackner K, Brandl M, Knuttel H, et al. Determinants of quality of life and return to work following acute respiratory distress syndrome. Deutsches Arzteblatt Int. 2017;114(7):103–9. Received: 11 April 2019 Accepted: 18 May 2020 21. Lampert T, Kroll L, Muters S, Stolzenberg H. Measurement of socioeconomic status in the German health interview and examination survey for adults (DEGS1). Bundesgesundheitsblatt, Gesundheitsforschung, Gesundheitsschutz. 2013;56(5–6):631–6. Gesundheitsschutz. 2013;56(5–6):631–6. References 22. Le Gall JR, Lemeshow S, Saulnier F. A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study. JAMA. 1993;270(24):2957–63. 1. Ferguson ND, Fan E, Camporota L, Antonelli M, Anzueto A, Beale R, et al. The Berlin definition of ARDS: an expanded rationale, justification, and supplementary material. Intensive Care Med. 2012;38(10):1573–82. 23. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonca A, Bruining H, et al. The SOFA (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. On behalf of the working group on sepsis-related problems of the European Society of Intensive Care Medicine. Intensive Care Med. 1996;22(7):707–10. 2. Bellani G, Laffey JG, Pham T, Fan E, Brochard L, Esteban A, et al. Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries. JAMA. 2016;315(8):788–800. 2. Bellani G, Laffey JG, Pham T, Fan E, Brochard L, Esteban A, et al. Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries. JAMA. 2016;315(8):788–800. 3. Marti J, Hall P, Hamilton P, Lamb S, McCabe C, Lall R, et al. One-year resource utilisation, costs and quality of life in patients with acute respiratory distress syndrome (ARDS): secondary analysis of a randomised controlled trial. J Intensive Care. 2016;4:56. 3. Marti J, Hall P, Hamilton P, Lamb S, McCabe C, Lall R, et al. One-year resource utilisation, costs and quality of life in patients with acute respiratory distress syndrome (ARDS): secondary analysis of a randomised controlled trial. J Intensive Care. 2016;4:56. 24. Fan E, Brodie D, Slutsky AS. Acute respiratory distress syndrome: advances in diagnosis and treatment. JAMA. 2018;319(7):698–710. 25. Maiolo G, Collino F, Vasques F, Rapetti F, Tonetti T, Romitti F, et al. Reclassifying acute respiratory distress syndrome. Am J Respir Crit Care Med. 2018;197(12):1586–95. 4. Ruhl AP, Huang M, Colantuoni E, Karmarkar T, Dinglas VD, Hopkins RO, et al. Healthcare utilization and costs in ARDS survivors: a 1-year longitudinal national US multicenter study. Intensive Care Med. 2017;43(7):980–91. 26. Nguyen YL, Wallace DJ, Yordanov Y, Trinquart L, Blomkvist J, Angus DC, et al. The volume-outcome relationship in critical care: a systematic review and meta-analysis. Chest. 2015;148(1):79–92. national US multicenter study. Intensive Care Med. 2017;43(7):980–91. 5. Maca J, Jor O, Holub M, Sklienka P, Bursa F, Burda M, et al. Past and Present ARDS Mortality Rates: A Systematic Review. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 13. Raymondos K, Dirks T, Quintel M, Molitoris U, Ahrens J, Dieck T, et al. Outcome of acute respiratory distress syndrome in university and non- university hospitals in Germany. Crit Care. 2017;21:122. 14. Brandstetter S, Dodoo-Schittko F, Blecha S, Sebok P, Thomann-Hackner K, Quintel M, et al. Influence of quality of care and individual patient characteristics on quality of life and return to work in survivors of the acute respiratory distress syndrome: protocol for a prospective, observational, multi- Centre patient cohort study (DACAPO). BMC Health Serv Res. 2015;15:563. 15. Dodoo-Schittko F, Brandstetter S, Brandl M, Blecha S, Quintel M, Weber- Carstens S, et al. German-wide prospective DACAPO cohort of survivors of the acute respiratory distress syndrome (ARDS): a cohort profile. BMJ Open. 2018;8(4):e019342. 16. Force ADT, Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, et al. Acute respiratory distress syndrome: the Berlin definition. JAMA. 2012;307(23):2526–33. 17. Ware JE Jr, Kosinski M, Keller SD. A 12-item short-form health survey: construction of scales and preliminary tests of reliability and validity. Med Care. 1996;34(3):220–33. 18. Bullinger M, Kirchberger I. Der SF-36 Fragebogen zum Gesundheitszustand: Handbuch für die deutschsprachige Fragebogenversion. Boston: Medical Outcomes Trust; 1995. 19. Braun JP, Kumpf O, Deja M, Brinkmann A, Marx G, Bloos F, et al. The German quality indicators in intensive care medicine 2013--second edition. German Med Sci. 2013;11:Doc09. 20. Kumpf O, Braun JP, Brinkmann A, Bause H, Bellgardt M, Bloos F, et al. Quality indicators in intensive care medicine for Germany - third edition 2017. German Med Sci. 2017;15:Doc10.
https://openalex.org/W2043106704	https://europepmc.org/articles/pmc3318856?pdf=render	English	null	Dental-derived Stem Cells and whole Tooth Regeneration: an Overview	Journal of clinical medicine research	2,009	cc-by	7,027	J Clin Med Res • 2009;1(2):63-71 J Clin Med Res • 2009;1(2):63-71 Review Dental-derived Stem Cells and whole Tooth Regeneration: an Overview Aous Dannan being composed of enamel, dentin, pulp and periodontium. Tooth development is characterized by a series of reciprocal epithelial mesenchymal interactions that result in differentia- tion and the spatial organization of cells to form organs [1, 2]. Since gene expression comparisons during teeth develop- ment have shown only slight differences between human and mouse teeth, mice have been used as the major animal model for studying tooth development. Human genetic diseases that encompass loss of teeth also contribute to the understanding of tooth formation [3, 4]. Abstract The need for new dental tissue-replacement therapies is evi- dent in recent reports which reveal startling statistics regarding the high incidence of tooth decay and tooth loss. Recent advances in the identification and characterization of dental stem cells, and in dental tissue-engineering strategies, suggest that bioengineering approaches may successfully be used to regenerate dental tissues and whole teeth. Interest in dental tissue-regeneration applications continues to increase as clinically relevant methods for the genera- tion of bioengineered dental tissues, and whole teeth, continue to improve. This paper is concerned about dental-derived stem cells and their characterization. Additionally, since conventional dental treatments partially serve the purpose for replacing missing teeth and always include possible failure rates, the potential of dental- derived stem cells in promoting whole tooth regeneration is also discussed. Modern dentistry for replacing missing teeth utilizes metal implants capped with a ceramic crown [5]. Although these prostheses serve the purpose, factors that interfere with osseointegration may cause surgery failure [6]. With ad- vances in stem cell biology and emerging concepts of tissue engineering [7], biological teeth [8] may become an alterna- tive for replacing missing teeth. The idea is to cultivate stem cells with odontogenic induction signals through epithelial- mesenchymal interactions, thereby programming the stem cells to adopt dental lineages and, with the help of scaffold matrix, to become part of a tooth. Keywords: Dental stem cells; Tissue engineering; Tooth regenera- tion Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org Introduction In order to understand how tooth regeneration should be induced, a deep knowledge of the normal tooth develop- ment is always a prerequisite. Teeth are structures that develop on the maxilla and mandible of mammals and serve eating, defense and pho- netic purposes. Although the morphology of teeth varies de- pending on species and location, they are similar in structure, Generally, tooth development is initiated by the dental epithelium and proceeds through five distinct morphological stages: bud, cap, bell, crown, and root [2, 9]. The coordinated development of tooth supporting structures, including peri- odontal ligament (PDL) and alveolar bone, begins around the bell stage. The tooth germ is first identifiable as a local- ized thickening and proliferation of the oral epithelium. The dental epithelium forms a bud that extends into the under- lying dental mesenchyme, marking the first stage of tooth development. The dental epithelium subsequently undergoes significant proliferative activity, extending around the pe- riphery to form a cap-like structure. Manuscript accepted for publication March 26, 2009. doi:10.4021/jocmr2009.03.1230 Manuscript accepted for publication March 26, 2009. Department of Periodontology, Faculty of Dental Medicine, Witten/Herd- ecke University, Witten, Germany. Email: aousdannan@yahoo.com Manuscript accepted for publication March 26, 2009. Department of Periodontology, Faculty of Dental Medicine, Witten/Herd- ecke University, Witten, Germany. Email: aousdannan@yahoo.com doi:10.4021/jocmr2009.03.1230 During this process, the non-proliferating enamel knot 63 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2009;1(2):63-71 Aous Dannan signaling center [10] becomes identifiable, as epithelial cells organize themselves into three distinct regions, namely the outer epithelium, the inner epithelium, and central cell lay- ers called the stratum intermedium and stellate reticulum. The ectomesenchymal cells of the dental papilla condense beneath the invaginating dental epithelium, eventually giv- ing rise to dentin and pulp tissues. The dental follicle forms around the enamel organ and dental papilla, eventually form- ing the periodontal tissues. Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divi- sions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extra embryonic cell types. signaling center [10] becomes identifiable, as epithelial cells organize themselves into three distinct regions, namely the outer epithelium, the inner epithelium, and central cell lay- ers called the stratum intermedium and stellate reticulum. The ectomesenchymal cells of the dental papilla condense beneath the invaginating dental epithelium, eventually giv- ing rise to dentin and pulp tissues. The dental follicle forms around the enamel organ and dental papilla, eventually form- ing the periodontal tissues. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a close- ly related family of cells (e.g. hematopoietic stem cells dif- ferentiate into red blood cells, white blood cells, platelets, etc.). The bell stage is characterized by continued prolifera- tion and histo-differentiation of the dental epithelium. Inner dental epithelial cells assume a cuboidal shape and produce high levels of glycogen, adjacent stratum intermedium pro- duces high levels of alkaline phosphatase, and the stellate reticulum assumes a distinctive star shape, surrounded by the outer epithelial cell layer. Unipotent cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells). Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, where single cells are characterized by their ability to differentiate and self-renew [12, 13]. Manuscript accepted for publication March 26, 2009. As well, stem cells can be isolated based on a dis- tinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells will behave in a similar manner in vivo. Considerable debate exists whether some proposed adult cell populations are truly stem cells. As tooth development proceeds through differentiation stages, dental mesenchyme-derived odontoblasts differenti- ate and elaborate the dentin matrix, and epithelial cell-de- rived ameloblasts cells secrete the enamel matrix for enamel production. After the tooth crown has formed, tooth root structures develop from the rudimentary Hertwig’s epithelial root sheath, forming dentin, cementum, periodontal ligament and alveolar bone. In dental research, there are two major categories of stem cells which are discussed for clinical application: em- bryonic stem cells and somatic (or adult) stem cells. Iso- lation and use of human embryonic stem cells is ethically controversial, but first evaluations are on the way for actual treatments [14]. In naturally formed teeth, enamel - the only mineral- ized tooth tissue derived from the dental epithelium - exhib- its no regenerative properties, while the remaining mineral- ized periodontal and dental tissues, including dentin, pulp, cementum, periodontal ligament and alveolar bone, all of which are formed from neural crest-derived dental ecto- mesenchyme, each exhibit a certain degree of regenerative capability which is supposed to be, partially, related to the existence of stem cells. Somatic stem cells have a limitation in their potentials of differentiation. However, it is thought that somatic or adult stem cells are a better option for dentistry, as these cells are easily accessible, and their use does not bring up ethical concerns [15]. Location of stem cells within dental tissues Stem cells are one of the most fascinating areas of bi- ology today. But like many expanding fields of scientific inquiry, research on stem cells raises scientific questions as rapidly as it generates new discoveries. Recently, it has been found that specific populations of stem cells and/or progenitor cells could be isolated from three main dental resources, namely the dental follicle, the dental pulp and the developed periodontal ligament. Stem cells differ from other kinds of cells in the body. All stem cells, regardless of their source, have three general properties: they are capable of dividing and renewing them- selves for long periods; they are unspecialized; and they can give rise to specialized cell types [11]. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org Stem cells in the dental follicle One important biological function of the dental fol- licle is the coordination of tooth eruption [16]. Moreover this tissue harbors progenitor cells for the periodontal ligament [17]. The differentiation and function of dental follicle cells are controlled by a network of regulatory molecules includ- ing growth factors and cytokines. It is thought that the dental follicle cells near the forming root (innermost) differentiate into cementum forming cementoblasts and that cells towards the alveolar bone (outermost) differentiate to osteoblasts Unlike muscle cells, blood cells, or nerve cells -which do not normally replicate themselves- stem cells may rep- licate many times. When cells replicate themselves many times over it is called proliferation. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal. 64 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2009;1(2):63-71 Dental Stem Cells secreting bone matrix. Dental follicle cells found centrally between the cementoblast and osteoblast precursor cells de- velop into fibroblasts producing the extracellular matrix of the periodontal ligament. secreting bone matrix. Dental follicle cells found centrally between the cementoblast and osteoblast precursor cells de- velop into fibroblasts producing the extracellular matrix of the periodontal ligament. and dental papilla cells promote tooth morphogenesis by stimulating a subpopulation of mesenchymal cells to differ- entiate into odontoblasts, which in turn form primary dentin. These odontoblasts are thought to arise from the prolifera- tion and differentiation of a precursor population, residing somewhere within the pulp tissue [26]. Mechanisms, which regulate follicle cell differentia- tion, are, in the beginning, to be understood. Cementogene- sis during tooth development is dependent on root formation. Root formation begins by proliferation of the outer and inner enamel epithelium forming the bilayered Hertwig’s epithe- lial sheath. Epithelial stimuli derived from this root sheath seem to be responsible for the differentiation of follicle pre- cursor cells into cementoblasts [18, 19]. It is known that in certain conditions, cultures of pulp cells derived from early developing dental root tissue and pulp tissue can develop an odontoblast-like appearance with the capacity to form mineralized nodules in vitro [27], a trait normally attributed to cultures of bone or bone marrow cells [28, 29]. Stem cells in the dental follicle It was demonstrated that Bone Morphogenetic Pro- tein-2 (BMP-2) promotes differentiation of immortalized dental follicle cells towards an osteoblast and/or cemento- blast phenotype [20]. Moreover, dental follicle progenitor cells were isolated from bovine tooth germs by digestion with bacterial collagenase [21, 22]. The differentiation capacity of these cells was proved by in vivo tests with mice. Here, cells formed a cementum-like matrix in contrast to bovine PDL fibroblasts or bovine alveolar osteoblasts. In contrast to in vivo tests, no differentiation potential was observed in long term cultures in the presence of dexamethasone. It has been speculated that adult dental pulp tissue might also contain a population of multipotential stem cells [30], and that postnatal dental pulp contains cells that are clonogenic, highly proliferative, and capable of regenerating a tissue, properties that effectively define them as stem cells. It has also been shown that Dental Pulp Stem Cells (DPSCs) were capable of forming ectopic dentin and associated pulp tissue in vivo [31]. Stromal-like cells were re-established in culture from primary DPSCs transplants and retransplanted into immunocompromised mice to generate a dentin-pulp- like tissue, demonstrating their self renewal capability [31]. Later after, Morsczeck and co-workers [23] reported the isolation of precursor cells derived from dental follicle of human third molar teeth. These fibroblast-like, colony- forming and plastic adherent cells expressed putative stem cell markers; Notch-1 and Nestin. It has also been shown that long-term cultures with dexamethasone produced compact calcified nodules or appeared as plain membrane structures of different dimensions consisting of a connective tissue like matrix encapsulated by a mesothelium-like cellular struc- ture. Therefore, these results demonstrated that cultured pre- cursor cells are unique undifferentiated lineage committed cells residing in the periodontium prior or during tooth erup- tion. In a recent study, it was hypothesized that stem cells may be present in the dental follicle and be capable of dif- ferentiating into cells of the periodontium [24]. At the same field of another study, it was demonstrated that human third molar pad possesses neural crest-derived cells that represent multipotent stem/progenitor cells [25]. For future studies, however, the dental follicle’s stem cells will need to be fur- ther characterized. Stem cell cultures have been successfully established from both dental pulp tissue and periodontal ligament by Varga and co-workers [32] who demonstrated that both cell cultures showed typical fibroblast-like morphology, showing clonogenic activity. STRO-1 immunoreactivity in both cell cultures was also detected. Stem cells in the dental follicle Taken together, these results demonstrated that DPSCs possess stem-cell-like qualities, including self-renewal capa- bility and multi-lineage differentiation. Another study has provided evidence that DPSCs and their osteoblast differentiated cells can be safely recovered after long-term cryopreservation [33]. All these features and abilities make these cells attractive for therapeutic 3-dimen- sional tissue reconstruction, with the potential of tailoring storage and recovery to the needs of the patient.i In the same field, it has been found that exfoliated hu- man deciduous tooth contains multipotent stem cells [Stem cells from Human Exfoliated Deciduous teeth (SHED)] [34]. SHED were identified to be a population of highly prolifera- tive, clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, and odonto- blasts. After in vivo transplantation, SHED were found to be able to induce bone formation, generate dentin, and sur- vive in mouse brain along with expression of neural mark- ers. SHED are not only derived from a very accessible tissue resource but are also capable of providing enough cells for potential clinical application. Thus, it has been shown that exfoliated teeth may be an unexpected unique resource for stem-cell therapies, including autologous stem-cell trans- plantation and tissue engineering. Stem cells in the periodontium The periodontal ligament is a group of specialized connective tissue fibers that essentially attach a tooth to the alveolar bone within which it sits. These fibers help the tooth withstand the naturally substantial compressive forces which occur during chewing and remain embedded in the bone. Another function of the PDL is to serve as a source of proprioception, or sensory innervations, so that the brain can detect the forces being placed on the teeth and react ac- cordingly. In addition to the PDL fibers, there is another set of fibers, known as the gingival fibers, which attach the teeth to their adjacent gingival tissue. Both the gingival fibers, as well as the PDL fibers, are composed primarily of type I col- lagen. Immunohistochemical staining and Western blot analysis showed that cultured PDLSCs express an array of cementoblastic/osteoblastic markers, including alkaline phosphatase, bone sialoprotein, osteocalcin and transform- ing growth factor-β receptor type I. These PDLSCs were transplanted into artificially created periodontal defects in the mandibular molars in rats. Histological evaluations 6–8 weeks after implantation showed that these cells had the ca- pacity to generate a thin layer of cementum-like tissue on the surface of the hydroxyapatite/tricalcium phosphate ceramic particles carrier, along with condensed collagen fibers that resembled Sharpey’s fibers [61]. The periodontal ligament is derived embryologically from the ectomesenchymal tissue of the dental follicle that surrounds the developing tooth in its bony crypt. At the time of tooth eruption the cells and collagen fibers in the dental follicle, i.e. the future periodontal ligament, are orientated primarily with their long axis parallel to the root surface. Re- modeling of the follicle into a periodontal ligament begins at the cemento-enamel junction and proceeds in an apical direction. The presence of MSCs in the periodontal ligament is also supported by other findings where a population of MSCs from the periodontal ligament has been isolated and charac- terized showing the ability to express a variety of stromal cells markers (CD90, CD29, CD44, CD166, CD105, CD13) [60, 64]. The clinical potential for the use of PDLSCs has been further enhanced by the demonstration that these cells can be isolated from cryopreserved periodontal ligaments, thus providing a ready source of MSCs [62]. The periodontal ligament contains a unique assortment of cells that are capable of generating and maintaining three distinct tissues, namely the ligament itself as well as the mineralized tissues on either side of it, i.e. Stem cells in the dental pulp The dental pulp is the part in the center of a tooth made up of living soft tissue and cells called odontoblasts. The cen- tral region of the coronal and radicular pulp contains large nerve trunks and blood vessels. This area is lined peripher- ally by a specialized odontogenic area which has three layers which are (from innermost to outermost): cell rich zone, cell free zone and odontoblastic layer. During tooth formation, interactions between epithelial In a trial to model the epithelial-mesenchymal interac- 65 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2009;1(2):63-71 Aous Dannan PDL contains heterogeneous cell populations [38, 39] that can differentiate into either cementum-forming cells (cementoblasts) or bone-forming cells (osteoblasts) [40-43]. Recent findings suggest that PDL cells have many osteo- blast-like properties, including the capacity to form miner- alized nodules in vitro, expression of the bone-associated markers alkaline phosphatase and bone sialoprotein, and response to bone-inductive factors such as parathyroid hor- mone, insulin-like growth factor 1, bone morphogenetic pro- tein 2 and transforming growth factor β1 [44-49]. The pres- ence of multiple cell types within PDL has led to speculation that this tissue might contain progenitor cells that maintain tissue homoeostasis and regeneration of periodontal tissue [40, 50-52]. tions which take place during tooth germ differentiation and to focus on the importance of DPSCs at this level, Kadar and co-workers [35] used cultures of mesenchymal cells iso- lated from adult human dental pulp and undifferentiated epi- thelial cells from salivary origin, and it has been shown that coculturing of DPSCs and epithelial cells resulted in dramat- ic changes of cell shape and migrating activity of DPSCs, while no obvious morphological change can be observed in the epithelial cells. Szlavik and co-workers tried to characterize the neu- ronal differentiation in a culture of DPSCs and SHED, and it was shown that induction of neuronal differentiation can be achieved by retinoic acid in the pulp originated cultures. When cultured together with undifferentiated epithelial cells, DPSC and SHED cells showed similar signs of neu- ronal differentiation suggesting the high plasticity and trans- differentiation potency of these cell populations [36]. Stem cells in the dental pulp Using a methodology similar to that utilized to iso- late Mesenchymal Stem Cells (MSCs) from deciduous and adult dental pulp, multipotent postnatal stem cells from hu- man periodontal ligament or Periodontal Ligament Stem Cells (PDLSCs) have also been isolated and described [32, 53-63]. Cultured cells were expanded from single cell sus- pensions derived from periodontal ligament tissue and the presence of stem cells was determined using antibodies such as STRO-1 and CD146. Under defined cultured conditions, PDLSCs were able to differentiate into cementoblast-like cells, adipocytes and collagen-forming cells. Moreover, it has been suggested that functional evi- dence for neuronal differentiation of human DPSCs in vitro may exist [37]. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org Stem cells in the periodontium the cementum and the alveolar bone. The major cell types of the periodon- tal ligament include: Fibroblasts, macrophages and undiffer- entiated ectomesenchymal cells, cementoblasts and cemen- toclasts, osteoblasts and osteoclasts, cell rests of Malassez, vascular and neural elements. Moreover, putative stem cells in both healthy and dis- eased periodontal ligament could be identified [53]. They were mainly located in the paravascular region and small clusters of cells were also found in the extra-vascular region. Wider distributions of these cells were detected in sections of diseased ligament. In a recent study, our research group described a meth- od for serum-free culture, rapid expansion and subsequent 66 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2009;1(2):63-71 Dental Stem Cells efficient induction of neuronal phenotype in the so-called periodontium-derived neural stem cells (PdNSCs) [65]. The isolated, cultured, highly proliferative cells were positive for the neural stemness markers Nestin and Sox2. In addition, the PdNSCs migrated when exposed to chemokines that are reported to be migration-inducing in neural stem cells iso- lated from subventicular zone. Retinoic acid treatment ef- ficiently induced neuronal fate in PdNSCs as shown by the high levels (> 90%) of neuron-specific markers. However, the capacity of these periodontium-derived stem cells to function in vivo remains unresolved. generate composite scaffolds that degrade within a period of a few weeks up to 1 year [72]. Poly-Lactic acid sponges can support the growth of chondrocytes in a uniform cellular distribution, their utility has been demonstrated in cartilage tissue regeneration [72], and polyglycolic acid and polylac- tic acid have been shown to support the growth of biopsied neonatal intestine cells into functional, small intestinal tissue [73]. Optimized polymer fabrication techniques have been used to generate three-dimensional structures composed of an intercommunicating network of pores, where the result- ing morphology and mechanical properties of the scaffold walls were found to influence tissue engineering applica- tions. However, each type of scaffold has unique features that provide flexibility for a variety of tissue-engineering applications. More recently, many studies have investigated the be- havior of stem cells in the periodontium during wound heal- ing of bone defects [66], and their involvement in periodontal regeneration [67]. These studies showed that cells with char- acteristics of putative mesenchymal stem cells were found in regenerating periodontal tissues, implying their involvement in periodontal regeneration. Stem cells in the periodontium Moreover, mesenchymal stem cells and hematopoietic stem cells in the bone marrow were not involved in the regeneration of the periodontium. Cells that migrated from the residual periodontal ligament regen- erated new alveolar bone at early stages. From early studies, we know it is possible to regener- ate tooth crowns if suitable environments are provided, such as in vitro organ culture, grafts on chick chorio-allantoic membrane, ocular grafts, subcutaneous transplants or kidney capsules [74-78]. These culture sites provide nutrients and oxygen to nurture tooth germs. Thus, several choices exist for cultivating small-sized primordia, such as those of teeth, before they can be implanted into their anatomical sites. It could be stated that within the periodontal ligament of both healthy and diseased teeth, cells have been identi- fied consistent with their identification as putative stem cells [68-70]. The presence of an inflammatory reaction associ- ated with periodontitis may enhance the number of these cells, and their role in periodontal tissue regeneration might be of great importance as a step on the road of dental tissue regeneration. Optimally, the setting should reproduce cells in a three dimensional organization, support the differentiating func- tion, and avoid xenograft rejection. According to Yen and Sharpe [79], two current ways of tooth tissue engineering are now thinkable. The first method includes growing of dissociated tooth germs on a tooth- shaped scaffold and produce small complex tooth-like struc- tures. The second method includes growing of epithelial and mesenchymal (stem) cells, either from tooth germs or other sources, in organ culture and, through epithelial-mesenchy- mal interactions, forming organized teeth. In our opinion, however, the first method seems to be more plausible since its elements are more controllable. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org References undifferentiated SCAP, which is downregulated following odontogenic differentiation. These data support the notion that SCAP are a unique population of postnatal stem cells. 1. Fleischmajer R. Epithelial-mesenchymal interactions. Science 1967;157:1472-1482. Although dental pulp contains DPSCs with dentin/ pulp regenerative capacity, developing tissue-derived SCAP showed a superior tissue regeneration potential than that of DPSCs. SCAP collected from just one tooth are capable of providing a large number of stem cells probably sufficient for human transplantation because they have high prolifera- tive potential, reflected in high telomerase activity [81]. 2. Thesleff I. Epithelial-mesenchymal signalling regulating tooth morphogenesis. J Cell Sci 2003;116:1647-1648. 3. Lin D, Huang Y, He F, Gu S, Zhang G, Chen Y, Zhang Y. Expression survey of genes critical for tooth devel- opment in the human embryonic tooth germ. Dev Dyn 2007;236:1307-1312. Although newly formed bio-roots show a lower com- pressive strength than that of natural swine root dentin, they seemed capable of supporting porcelain crown and resulted in normal functions. It may be possible to improve the com- pressive strength and hardness of the bio-roots by selecting optimal bioengineered materials and by optimizing the im- planted stem cell numbers and quality. 4. Tucker A, Sharpe P. The cutting-edge of mammalian de- velopment; how the embryo makes teeth. Nat Rev Genet 2004;5:499-508. 5. Crubezy E, Murail P, Girard L, Bernadou JP. False teeth of the Roman world. Nature 1998;391:29. 6. Esposito M, Hirsch JM, Lekholm U, Thomsen P. Bio- logical factors contributing to failures of osseointegrat- ed oral implants. (II). Etiopathogenesis. Eur J Oral Sci 1998;106:721-764. This hybrid strategy of autologous dental stem cell en- gineering may be applicable to human tooth regeneration. Furthermore, functional tooth restoration in swine may shed light on human tooth regeneration in the future because of the close similarities between swine and human dental tis- sues. 7. Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920-926. 8. Sharpe PT, Young CS. Test-tube teeth. Sci Am 2005;293:34-41. We believe that the model of Sonoyama and co-work- ers provided a unique imagination of how to use dental stem cells to create a functional tooth, which is, practically, the most important unit in replacing missed teeth. However, in order to optimize such models, further investigations should be carried in order to test the stem cells-scaffold interrela- tionships. 9. Thesleff I, Partanen AM, Vainio S. References Epithelial-mesen- chymal interactions in tooth morphogenesis: the roles of extracellular matrix, growth factors, and cell surface receptors. J Craniofac Genet Dev Biol 1991;11:229-237. 10. Thesleff I, Keranen S, Jernvall J. Enamel knots as sig- naling centers linking tooth morphogenesis and odonto- blast differentiation. Adv Dent Res 2001;15:14-18. 11. Blau HM, Brazelton TR, Weimann JM. The evolv- ing concept of a stem cell: entity or function? Cell 2001;105:829-841. Conclusions 12. Friedenstein AJ, Deriglasova UF, Kulagina NN, Panasuk AF, Rudakowa SF, Luria EA, Ruadkow IA. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 1974;2:83-92. Complex human tissues harbor stem cells and/or pre- cursor cells, which are responsible for tissue development and repair. Recently, dental tissues such as periodontal liga- ment, dental papilla or dental follicle have been identified as easily accessible sources of undifferentiated cells. 13. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoi- etic organs. Exp Hematol 1976;4:267-274. Dental stem cells have been isolated according to their anatomical locations, colony-forming ability, expression of stem cell markers, and regeneration of pulp/dentin and/or periodontium/cement structures in vivo. These dental-de- rived stem cells are currently under increasing investigation as sources for tooth regeneration and repair. 14. Ohazama A, Modino SA, Miletich I, Sharpe PT. Stem- cell-based tissue engineering of murine teeth. J Dent Res 2004;83:518-522. 15. Morsczeck C, Reichert TE, Vollner F, Gerlach T, Driemel O. The state of the art in human dental stem cell research. Mund Kiefer Gesichtschir 2007. Although many challenges remain, stem-cell-based tis- sue engineering of teeth could be a choice for the replace- ment of missing teeth in the future. 16. Wise GE, Frazier-Bowers S, D’Souza RN. Cellular, mo- lecular, and genetic determinants of tooth eruption. Crit Rev Oral Biol Med 2002;13:323-334. Whole tooth regeneration As bioengineered tooth crown formation requires the interactions of both dental epithelial cell progenitors and mesenchymal cell progenitors (as in natural tooth forma- tion), the ability to bioengineer a tooth of specified size and shape will depend on the ability to first identify, and then guide, the interactions of both types of cells. Methods to guide the interactions of epithelial and mesenchymal postna- tal dental stem cells to form dentin and enamel layers char- acteristic of natural teeth, using modified scaffold materials and designs, for example, must be developed. The impor- tance of scaffold materials and design for tissue engineering has long been recognized. Scaffold porosity, biocompatibil- ity and biodegradability, the ability to support cell growth, and use as a controlled gene- and protein-delivery vehicle [71] are all highly significant properties. A variety of hydro- philic polymers have been synthesized that provide cell sup- port and guidance. Importantly, scaffold materials provide a three-dimensional macromolecular structure to guide the final shape of bioengineered tissues. Poly-L-lactic acid and poly lactic co-glycolic acid co-polymers have been used to Considering the regeneration of a functional and living tooth, Sonoyama and co-workers conducted an interesting study to explore the potential for reconstructing a functional tooth in miniature pigs (mini-pigs) [80], in which a bio-root periodontal complex is built up by postnatal stem cells in- cluding stem cells from root apical papilla (SCAP) and PDLSCs, to which an artificial porcelain crown is affixed. It has been shown that the root apical papilla contained mesenchymal stem cells that appear to have a greater capacity for dentin regeneration than DPSCs. These findings suggest that developing tissue may contain a good stem cell resource for tissue regeneration. SCAP represent a novel population of multipotent stem cells as demonstrated by their capacity to develop into odontoblast-like cells and adipocytes in vi- tro. This cell population was found to express high levels of survivin and telomerase, which are both important molecules in mediating cell proliferation. In addition, CD24, marker for 67 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2009;1(2):63-71 Aous Dannan Acknowledgements Cell Biol Int 2005;29:567-575. 38. Lekic P, Rojas J, Birek C, Tenenbaum H, McCulloch CA. Phenotypic comparison of periodontal ligament cells in vivo and in vitro. J Periodontal Res 2001;36:71- 79. 24. Yao S, Pan F, Prpic V, Wise GE. Differentiation of stem cells in the dental follicle. J Dent Res 2008;87:767-771. 25. Degistirici O, Jaquiery C, Schonebeck B, Siemonsmeier J, Gotz W, Martin I, Thie M. Defining properties of neu- ral crest-derived progenitor cells from the apex of hu- man developing tooth. Tissue Eng Part A 2008;14:317- 330. 39. Murakami Y, Kojima T, Nagasawa T, Kobayashi H, Ishi- kawa I. Novel isolation of alkaline phosphatase-positive subpopulation from periodontal ligament fibroblasts. J Periodontol 2003;74:780-786. 26. Ruch JV. Odontoblast commitment and differentiation. Biochem Cell Biol 1998;76:923-938. 40. Gould TR, Melcher AH, Brunette DM. Migration and division of progenitor cell populations in periodontal ligament after wounding. J Periodontal Res 1980;15:20- 42. 27. Couble ML, Farges JC, Bleicher F, Perrat-Mabillon B, Boudeulle M, Magloire H. Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif Tissue Int 2000;66:129-138. 41. Isaka J, Ohazama A, Kobayashi M, Nagashima C, Taki- guchi T, Kawasaki H, Tachikawa T, et al. Participation of periodontal ligament cells with regeneration of alveo- lar bone. J Periodontol 2001;72:314-323.i 28. Cheng SL, Yang JW, Rifas L, Zhang SF, Avioli LV. Dif- ferentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 1994;134:277-286. 42. McCulloch CA, Bordin S. Role of fibroblast subpopula- tions in periodontal physiology and pathology. J Peri- odontal Res 1991;26:144-154. 29. Gronthos S, Graves SE, Ohta S, Simmons PJ. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood 1994;84:4164-4173. 43. McCulloch CA, Melcher AH. Cell density and cell gen- eration in the periodontal ligament of mice. Am J Anat 1983;167:43-58. 30. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625- 13630. 44. Lekic PC, Rajshankar D, Chen H, Tenenbaum H, Mc- Culloch CA. Transplantation of labeled periodontal liga- ment cells promotes regeneration of alveolar bone. Anat Rec 2001;262:193-202. 31. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, DenBesten P, et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002;81:531- 535. 45. Acknowledgements 17. Luan X, Ito Y, Dangaria S, Diekwisch TG. Dental fol- licle progenitor cell heterogeneity in the developing mouse periodontium. Stem Cells Dev 2006;15:595-608. The authors declare no conflicts of interest related to this article. 18. Diekwisch TG. The developmental biology of cemen- 18. Diekwisch TG. The developmental biology of cemen- Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org 68 Dental Stem Cells J Clin Med Res • 2009;1(2):63-71 tum. Int J Dev Biol 2001;45:695-706. tum. Int J Dev Biol 2001;45:695-706. cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tis- sue repair. J Cell Physiol 2006;208:319-325. 19. Saygin NE, Giannobile WV, Somerman MJ. Molecu- lar and cell biology of cementum. Periodontol 2000 2000;24:73-98. 34. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S. SHED: stem cells from human exfoliated de- ciduous teeth. Proc Natl Acad Sci U S A 2003;100:5807- 5812. 20. Zhao M, Xiao G, Berry JE, Franceschi RT, Reddi A, So- merman MJ. Bone morphogenetic protein 2 induces den- tal follicle cells to differentiate toward a cementoblast/ osteoblast phenotype. J Bone Miner Res 2002;17:1441- 1451. 35. Kadar K, Vag, J., Szlavik, V., Varga, G. Cocultured DPSC and HSG cells: a Model for Epithelial-mesenchy- mal Communication. J Dent Res 2006;85, Spec Iss C. 21. Handa K, Saito M, Tsunoda A, Yamauchi M, Hattori S, Sato S, Toyoda M, et al. Progenitor cells from dental fol- licle are able to form cementum matrix in vivo. Connect Tissue Res 2002;43:406-408. 36. Szlavik V, Kadar, K., Natz, E., Kiraly, M., Grimm, W.- D., Widera, D., Kaltschmidt, B., Kaltschmidt, C., Vag, J., Gera, I., Varga, G. Neuronal Differentiation of Cul- tured Human Dental Pulp Cells. J Dent Res 2007;86, Spec Iss A. 22. Handa K, Saito M, Yamauchi M, Kiyono T, Sato S, Teranaka T, Sampath Narayanan A. Cementum matrix formation in vivo by cultured dental follicle cells. Bone 2002;31:606-611. 37. Kiraly M, , Kadar, K., Porcsalmy, B., Molnar, B., Pa- taki, A., Dank, T., Jelitai, M., Grimm, W.-D., Zsembery, A., Gera, I., Varga, G. Functional Evidence for Neuronal Differentiation of Human DPSCs In Vitro. J Dent Res 2008;87, Spec Iss B. 23. Morsczeck C, Moehl C, Gotz W, Heredia A, Schaffer TE, Eckstein N, Sippel C, et al. In vitro differentiation of human dental follicle cells with dexamethasone and insulin. Acknowledgements Marcopoulou CE, Vavouraki HN, Dereka XE, Vrotsos IA. Proliferative effect of growth factors TGF-beta1, PDGF-BB and rhBMP-2 on human gingival fibroblasts and periodontal ligament cells. J Int Acad Periodontol 2003;5:63-70. 32. Varga G, Molnar B, Kadar K, Ovari G, Windisch P, Gera I. Adult Stem Cells in Primary Culture From Human Dental Tissues. J Dent Res 2005;84, Spec Iss B. 46. Ohno S, Doi T, Fujimoto K, Ijuin C, Tanaka N, Tanimoto K, Honda K, et al. RGD-CAP (betaig-h3) exerts a nega- tive regulatory function on mineralization in the human 33. Papaccio G, Graziano A, d’Aquino R, Graziano MF, Pirozzi G, Menditti D, De Rosa A, et al. Long-term Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org 69 J Clin Med Res • 2009;1(2):63-71 Aous Dannan periodontal ligament. J Dent Res 2002;81:822-825. 61. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364:149-155. 47. Ouyang H, McCauley LK, Berry JE, D’Errico JA, Strayhorn CL, Somerman MJ. Response of immortal- ized murine cementoblasts/periodontal ligament cells to parathyroid hormone and parathyroid hormone-related protein in vitro. Arch Oral Biol 2000;45:293-303. 62. Seo BM, Miura M, Sonoyama W, Coppe C, Stanyon R, Shi S. Recovery of stem cells from cryopreserved peri- odontal ligament. J Dent Res 2005;84:907-912. 48. Pitaru S, Pritzki A, Bar-Kana I, Grosskopf A, Savion N, Narayanan AS. Bone morphogenetic protein 2 induces the expression of cementum attachment protein in hu- man periodontal ligament clones. Connect Tissue Res 2002;43:257-264. 63. Tomokiyo A, Maeda H, Fujii S, Wada N, Shima K, Aka- mine A. Development of a multipotent clonal human periodontal ligament cell line. Differentiation 2007. 64. Trubiani O, Di Primio R, Traini T, Pizzicannella J, Scar- ano A, Piattelli A, Caputi S. Morphological and cyto- fluorimetric analysis of adult mesenchymal stem cells expanded ex vivo from periodontal ligament. Int J Im- munopathol Pharmacol 2005;18:213-221. 49. Zhao M, Berry JE, Somerman MJ. Bone morphogenetic protein-2 inhibits differentiation and mineralization of cementoblasts in vitro. J Dent Res 2003;82:23-27. 50. Beertsen W, McCulloch CA, Sodek J. The periodontal ligament: a unique, multifunctional connective tissue. Periodontol 2000 1997;13:20-40. 65. Widera D, Grimm WD, Moebius JM, Mikenberg I, Piechaczek C, Gassmann G, Wolff NA, et al. Acknowledgements Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal sur- gery. Stem Cells Dev 2007;16:447-460. 51. Boyko GA, Melcher AH, Brunette DM. Formation of new periodontal ligament by periodontal ligament cells implanted in vivo after culture in vitro. A preliminary study of transplanted roots in the dog. J Periodontal Res 1981;16:73-88. 66. Ohta S, Yamada S, Matuzaka K, Inoue T. The behavior of stem cells and progenitor cells in the periodontal liga- ment during wound healing as observed using immuno- histochemical methods. J Periodontal Res 2008. 52. Liu HW, Yacobi R, Savion N, Narayanan AS, Pitaru S. A collagenous cementum-derived attachment protein is a marker for progenitors of the mineralized tissue-forming cell lineage of the periodontal ligament. J Bone Miner Res 1997;12:1691-1699. 67. Lin NH, Gronthos S, Bartold PM. Stem cells and peri- odontal regeneration. Aust Dent J 2008;53:108-121. 68. Grimm W-D, Arnold WH, Becher S, Dannan A, Gassmann G, Kaltschmidt B, Levermann S, et al. Stam- mzellbasierte Therapieformen in der parodontalen Re- generation Zahnärztliche Mitteilungen 2008;98:40-48. 53. Chen SC, Marino V, Gronthos S, Bartold PM. Location of putative stem cells in human periodontal ligament. J Periodontal Res 2006;41:547-553. 54. Dannan A, Grimm W-D. [Periodontal Ligament Stem Cells (PLSCs); A New Horizon in the Periodontal Tis- sue Regeneration]. Dental Medium 2008;16:27-29. 69. Grimm W-D, Arnold WH, Becher S, Dannan A, Gassmann G, Levermann S, Widera D, et al. Ex vivo- Expansion von adulten parodontalen Stammzellen- ein neuer Weg der regenerativen Parodontalchirurgie. Par- odontologie Nachrichten 2007;4:6-8. 55. Fujii S, Maeda H, Wada N, Tomokiyo A, Saito M, Aka- mine A. Investigating a clonal human periodontal liga- ment progenitor/stem cell line in vitro and in vivo. J Cell Physiol 2008. 70. Grimm W-D, Dannan A, Gassmann G, Kaltschmidt B, Varga G, Widera D, Kaltschmidt C. Periodontium de- rived stem cells as multipotential adult progenitor cell. J Dent Res 2007;86, Spec Iss A. 56. Gay IC, Chen S, MacDougall M. Isolation and charac- terization of multipotent human periodontal ligament stem cells. Orthod Craniofac Res 2007;10:149-160. 71. Murphy WL, Mooney DJ. Controlled delivery of induc- tive proteins, plasmid DNA and cells from tissue engi- neering matrices. J Periodontal Res 1999;34:413-419. 57. Gronthos S, Mrozik K, Shi S, Bartold PM. Ovine peri- odontal ligament stem cells: isolation, characteriza- tion, and differentiation potential. Calcif Tissue Int 2006;79:310-317. 72. Mikos AG, Lyman MD, Freed LE, Langer R. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org Acknowledgements Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture. Biomaterials 1994;15:55-58. 58. Ivanovski S, Gronthos S, Shi S, Bartold PM. Stem cells in the periodontal ligament. Oral Dis 2006;12:358-363. 73. Choi RS, Riegler M, Pothoulakis C, Kim BS, Mooney D, Vacanti M, Vacanti JP. Studies of brush border en- zymes, basement membrane components, and electro- physiology of tissue-engineered neointestine. J Pediatr Surg 1998;33:991-996; discussion 996-997. 59. Molnár B, Kadar K, Kiraly M, Porcsalmy B, Somogyi E, Hermann P, Wolf-Dieter G, et al. [Isolation, cultivation and characterisation of stem cells in human periodontal ligament]. Fogorv Sz 2008;101:155-161. 74. Hay MF. The development in vivo and in vitro of the lower incisor and molars of the mouse. Arch Oral Biol 1961;3:86-109. 60. Nagatomo K, Komaki M, Sekiya I, Sakaguchi Y, No- guchi K, Oda S, Muneta T, et al. Stem cell properties of human periodontal ligament cells. J Periodontal Res 2006;41:303-310. 75. Morio I. Recombinant study of the mouse molar cervi- 75. Morio I. Recombinant study of the mouse molar cervi- 70 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2009;1(2):63-71 Dental Stem Cells cal loop and dental papilla by renal transplantation. Arch Oral Biol 1985;30:557-561. and dental sac tissues in ocular grafts. Arch Oral Biol 1981;26:303-307. 79. Yen AH, Sharpe PT. Stem cells and tooth tissue engi- neering. Cell Tissue Res 2008;331:359-372. 76. Slavkin HC, Bavetta LA. Odontogenesis in vivo and in xenografts on chick chorio-allantois. I. Collagen and hexosamine biosynthesis. Arch Oral Biol 1968;13:145- 154. 80. Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, Liu H, et al. Mesenchymal stem cell-mediat- ed functional tooth regeneration in swine. PLoS ONE 2006;1:e79. 77. Yamada M, Bringas P, Jr., Grodin M, MacDougall M, Cummings E, Grimmett J, Weliky B, et al. Chemically- defined organ culture of embryonic mouse tooth organs: morphogenesis, dentinogenesis and amelogenesis. J Biol Buccale 1980;8:127-139. 81. Shi S, Gronthos S, Chen S, Reddi A, Counter CM, Robey PG, Wang CY. Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression. Nat Biotechnol 2002;20:587-591. 78. Yoshikawa DK, Kollar EJ. Recombination experi- ments on the odontogenic roles of mouse dental papilla 71 71 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org
https://openalex.org/W2737695031	https://digital.csic.es/bitstream/10261/289776/1/crebneuron.pdf	English	null	CREB Regulates Distinct Adaptive Transcriptional Programs in Astrocytes and Neurons	Scientific reports	2,017	cc-by	11,555	CREB Regulates Distinct Adaptive Transcriptional Programs in Astrocytes and Neurons PEN Received: 21 July 2016 Accepted: 12 June 2017 Published online: 25 July 2017 Luis Pardo1, Luis Miguel Valor3, Abel Eraso-Pichot1, Angel Barco2, Arantxa Golbano1, Giles E. Hardingham4,5, Roser Masgrau1 & Elena Galea1,6 The cyclic AMP response element binding protein (CREB) is a primary hub of activity-driven genetic programs in neurons controlling plasticity, neurogenesis and survival. By contrast, the gene networks coordinated by CREB in astrocytes are unknown despite the fact that the astrocytic CREB is also activity-driven and neuroprotective. Herein we identified the transcriptional programs regulated by CREB in astrocytes as compared to neurons using, as study materials, transcriptome databases of astrocyte exposed to well-known activators of CREB-dependent transcription as well as publicly available transcriptomes of neuronal cultures. Functional CREB signatures were extracted from the transcriptomes using Gene Ontology, adult-brain gene lists generated by Translating Ribosome Affinity Purification (TRAP) and CREB-target gene repositories. We found minimal overlap between CREB signatures in astrocytes and neurons. In astrocytes, the top triad of functions regulated by CREB consists of ‘Gene expression’, ‘Mitochondria’, and ‘Signalling’, while in neurons it is ‘Neurotransmission’, ‘Signalling’ and ‘Gene expression’, the latter two being represented by different genes from those in astrocytes. The newly generated databases will provide a tool to explore novel means whereby CREB impinges on brain functions requiring adaptive, long-lasting changes by coordinating transcriptional cascades in astrocytes. Since its discovery in the late 1980s, the cyclic AMP-response element-binding protein (CREB) is arguably the most widely studied transcription factor, its two best documented functions being the coordination of liver metabolism during cycles of feeding and fasting1, and the regulation of neuronal plasticity—defined as long-term adaptive changes—in several scenarios: development, memory acquisition and consolidation, addiction, circa- dian rhythms and natural regeneration after injury2–5. Two lines of evidence support the notion that CREB regulates astrocytic functions as well. First, CREB-dependent transcription is activated in astrocytes by noradrenaline (NE), ATP, forskolin (FSK), tamoxifen and cinnamon6–8. NE and ATP act in a protein kinase C-dependent but calcium- and cyclic AMP-independent fashion, FSK activates adenylate cyclase and hence increases cyclic AMP content, whereas tamoxifen and cin- namon act via protein kinase A. Second, the targeted expression of a constitutively active CREB construct (VP16-CREB) in astrocytes is neuroprotective in focal acute brain injury9. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. Core signatures of ast-CREB-dependent gene candidates, defined as the set of genes common to all stimuli, were generated from the overlapping sections among DEG-Q1-TRAP NE/FSK/VP16-CREB, bioinformatic l i f 18 d d b f i i CRE i 19 Lik i h f CREB g y y g gy ( )t 5) Identification of direct ast-CREB gene target candidates and comparison with neu-CREB targets. Core signatures of ast-CREB-dependent gene candidates, defined as the set of genes common to all stimuli, were generated from the overlapping sections among DEG-Q1-TRAP NE/FSK/VP16-CREB, bioinformatic analysis of promoters18, and a database of genes containing CRE sites19. Likewise, the core of neu-CREB signature was obtained from common DEGs from FSK and VP16-CREB lists in neurons10 (ArrayExpress database; accession number E-MEXP-3167). t 5) Identification of direct ast-CREB gene target candidates and comparison with neu-CREB targets. Core signatures of ast-CREB-dependent gene candidates, defined as the set of genes common to all stimuli, were generated from the overlapping sections among DEG-Q1-TRAP NE/FSK/VP16-CREB, bioinformatic analysis of promoters18, and a database of genes containing CRE sites19. Likewise, the core of neu-CREB signature was obtained from common DEGs from FSK and VP16-CREB lists in neurons10 (ArrayExpress database; accession number E-MEXP-3167). VP16-CREB promotes CREB-dependent transcription in astrocytes. We have previously shown that NE and FSK induce CREB-dependent transcription in astrocytes6; here we confirmed that VP16-CREB does as well. Astrocytes infected with Ad2/5-CMV-VP16-CREB at MOIs of 5–30 efficiently transduced VP16-CREB, as deduced by the robust expression of VP16 mRNA and protein detected with Western blotting, immunocyto- chemistry and qPCR (Fig. 1c–e). That VP16-CREB activates CREB-dependent transcription was confirmed with luciferase assays, which showed a CRE-based activation comparable to that induced by FSK and NE (Fig. 1f), and by a ∼12-fold up-regulation of the Fos gene (Fig. 1e, right). Altogether, these analyses support the use of virally transduced VP16-CREB as a tool to induce CREB-dependent transcription in astrocytes. The viruses were used henceforth at a MOI of 5. Global transcriptome changes upon activation of ast-CREB-dependent transcription. The number of DEGs (p < 0.05) was 3279 genes in FSK-stimulated astrocytes and 2667 in the NE-treated group— relative to untreated controls—and 9884 in VP16-CREB over-expressing astrocytes with respect to Null con- ditions. We selected the genes present in the first quartile of the TRAP list of cortical astrocytes, ordered by expression level. CREB Regulates Distinct Adaptive Transcriptional Programs in Astrocytes and Neurons PEN y p j y That is, both neuronal CREB and astrocytic CREB (hereafter neu-CREB and ast-CREB) are (i) activity-dependent (i.e., regulated by neurotransmitters), (ii) neuroprotective and (iii) activated by different sig- nalling pathways. However, while the transcriptional programs regulated by neu-CREB have been well character- ized in vitro and in vivo10–12, there is little information about the gene targets of ast-CREB and the cellular origin of CREB-dependent transcriptional programs in vivo. Tools do exist to specifically activate CREB in neurons or astrocytes, but the related gene profiles have been characterized in whole brains without separating individual cells11, 13. 1Institut de Neurociències and Unitat de Bioquímica, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain. 2Instituto de Neurociencias, Universidad Miguel Hernández/Consejo Superior de Investigaciones Científicas, Sant Joan d’Alacant, 03550, Alicante, Spain. 3Unidad de Investigación, Hospital Universitario Puerta del Mar, Av. Ana de Viya 21, 11009, Cádiz, Spain. 4UK Dementia Research Institute at The University of Edinburgh, Edinburgh Medical School, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK. 5Deanery of Biomedical Sciences, Edinburgh Medical School, University of Edinburgh, Edinburgh, EH8 9XD, UK. 6ICREA, Pg. Lluís Companys 23, 08010, Barcelona, Spain. Correspondence and requests for materials should be addressed to L.P. (email: luis.pardo82@gmail.com) or E.G. (email: Elena.Galea@uab.es) ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 1 www.nature.com/scientificreports/ Herein we set out to identify the functional programs coordinated by CREB in astrocytes as compared to neurons. We generated transcriptome databases of rat cortex primary astrocyte cultures treated with NE or FSK in order to, respectively, activate cyclic AMP-independent and dependent pathways, or used over-expression of VP16-CREB, which promotes global expression of CREB-dependent genes regardless of upstream kinases14, and should thus faithfully unravel the complete population of ast-CREB-target genes. Since CREB-dependent transcription appears to be highly dependent on the stimulation context15, 16, we reasoned that the comparison of different transcriptomes, together with a stringent data mining using publicly available databases of adult genes, would lead us to the core functional signatures of ast-CREB as compared to neu-CREB. We found that ast-CREB and neu-CREB core signatures are utterly different, supporting a functional division of CREB in the brain between astrocytes and neurons. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. 1) CREB-dependent transcription was activated in primary cortical rat astrocyte cultures with 10 μM NE, 1 μM FSK or VP16-CREB (multiplicity of infection or MOI of 5). Culture composition was 95% astrocytes (GFAP-positive cells) and 5% microglia (Iba1-positive cells) (Fig. 1b). Neurons (NeuN-positive cells) and fibroblasts (Vimentin positive/GFAP negative cells) were not detected. i g 2) Transcriptomic changes were characterized with Agilent DNA microarrays. 3) To minimize the impact of genetic signatures of neonatal stages and culture conditions on the resulting differentially expressed genes (DEG) (p < 0.05) we took two actions. First, we selected for the ensuing anal- yses DEGs more likely to be relevant in vivo. The lists were generated by filtration of DEG lists through the first quartile (Q1) of the translational ribosome affinity purification (TRAP), a repository of adult astrocyte genes that only contains genes undergoing effective translation17. Second, we examined whether DEG-Q1- TRAP lists were enriched in transcriptomes from adult brains with targeted expression of VP16-CREB in astrocytes9. 3) To minimize the impact of genetic signatures of neonatal stages and culture conditions on the resulting differentially expressed genes (DEG) (p < 0.05) we took two actions. First, we selected for the ensuing anal- yses DEGs more likely to be relevant in vivo. The lists were generated by filtration of DEG lists through the first quartile (Q1) of the translational ribosome affinity purification (TRAP), a repository of adult astrocyte genes that only contains genes undergoing effective translation17. Second, we examined whether DEG-Q1- TRAP lists were enriched in transcriptomes from adult brains with targeted expression of VP16-CREB in astrocytes9.h y 4) The DEG-Q1-TRAP lists for NE, FSK and VP16-CREB were processed independently to identify function- al signatures by enrichment analysis using Gene Ontology (GO) with ClueGO v1.4 and ReviGO softwares.i y 4) The DEG-Q1-TRAP lists for NE, FSK and VP16-CREB were processed independently to identify function- al signatures by enrichment analysis using Gene Ontology (GO) with ClueGO v1.4 and ReviGO softwares. 5) Identification of direct ast-CREB gene target candidates and comparison with neu-CREB targets. Core i y 4) The DEG-Q1-TRAP lists for NE, FSK and VP16-CREB were processed independently to identify function- al signatures by enrichment analysis using Gene Ontology (GO) with ClueGO v1.4 and ReviGO softwares. 5) Identification of direct ast-CREB gene target candidates and comparison with neu-CREB targets. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. The VP16-CREB is localized in the nucleus as expected for a CREB-like factor. Astrocytes and VP16-CREB were respectively identified by GFAP and VP16 immunostaining (viral infection at a MOI of 5). Infection with the Null virus showed no VP16-CREB expression. Images are representative of 3 independent experiments. (d) Western blots using anti-VP16 or anti-actin antibodies—the latter to control for equal protein load. VP16-CREB is 65 kd. Expression of VP16-CREB is MOI-dependent. The blot is representative of at least 3 independent experiments and is a cropped image. The full-length blot is Figure 1. Outline and characterization of VP16-CREB over-expression in astrocytes. (a) Procedure outline. (b) Characterization of cellular composition. Cellular markers were GFAP (astrocytes), Iba1 (microglia), NeuN (neurons) and Vimentin, which labels both astrocytes (GFAP positive cells) and fibroblasts (GFAP negative cells). With respect to total DAPI counts, 95% of the cells were astrocytes, 5% microglia and no neurons or fibroblasts were detected−all Vimentin positive cells were GFAP positive as well. (c–f) Confirmation of VP16-CREB transduction. Astrocytes were incubated with Ad2/5-CMV-VP16-CREB or Ad2/5-CMV (Null) at MOIs within 1–30. (c) Immunodetection. The VP16-CREB is localized in the nucleus as expected for a CREB-like factor. Astrocytes and VP16-CREB were respectively identified by GFAP and VP16 immunostaining (viral infection at a MOI of 5). Infection with the Null virus showed no VP16-CREB expression. Images are representative of 3 independent experiments. (d) Western blots using anti-VP16 or anti-actin antibodies—the latter to control for equal protein load. VP16-CREB is 65 kd. Expression of VP16-CREB is MOI-dependent. The blot is representative of at least 3 independent experiments and is a cropped image. The full-length blot is presented in Supplementary Figure 1. (e) Left, robust up-regulation of VP16-CREB mRNA assessed by PCR in Ad2/5-CMV-VP16-CREB infected astrocytes but not in Null-infected ones. Data are means ± SEM of n = 3 independent determinations. (*) p < 0.001, Student’s T-test. Right, VP16-CREB induced Fos upregulation in Ad2/5-CMV-VP16-CREB infected astrocytes at a MOI of 5. Fos was not detected upon stimulation with NE and FSK because these stimuli are transient and Fos is a short-lived gene. Data are means ± SEM of 3 independent experiments; () p < 0.001, one-way ANOVA followed by Bonferroni. (f) Functional confirmation of VP16-CREB expression. VP16-CREB stimulates CRE-dependent transcription in luciferase-reporter assays (MOI = 5). FSK (1 μM) and NE (10 μM) were assayed in parallel as positive controls using 6-hour incubations. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. The resulting genes will be referred to as Q1-TRAP genes and the discarded as REST (final lists in Supplementary data S2). The process reduced by approximately 3-times the number of genes per group, with the final numbers being: 3355 VP16-CREB, 1049 FSK, and 822 NE. Finally, we analyzed with PSCAN software the localization of direct CREB-targets throughout the transcriptomes. PSCAN scans gene promoters looking for motifs of transcription-factor binding sites—herein CRE sites—and statistically assesses which motifs are over-represented18. p Figure 2 shows the transcriptome profiles in the three groups before and after the Q1-TRAP filtration. VP16-CREB caused a dramatic change in the transcriptome as compared to NE and FSK, as shown by histo- grams of gene distribution, heatmaps and Venn diagrams (Fig. 2a,b). The proportion of up-regulated (UP) vs down-regulated (DOWN) genes was similar in the FSK and NE groups (47% vs 53%) and slightly deviated towards the UP section in the VP16-CREB group (64% vs 46%). That is, there was a slight predominance of UP genes after sustained activation, and the UP section reached larger fold-changes and had more significant p-values (Fig. 2a). Overall, the FSK and NE groups were very similar (Pearson’s correlation = 0.72), while the VP16-CREB group was markedly different from the other two (Pearson’s correlation = 0.18 vs FSK; 0.21 vs NE) (Fig. 2b, bottom right). These correlations were confirmed by the overlap of differentially expressed genes: 78% of the genes in the NE group were in the FSK group, and, conversely, 63% of the FSK genes were also in the NE group. The VP16-CREB group in turn included 79% of the genes present in the NE and FSK groups combined, whereas 61% of the genes were unique to VP16-CREB over-expression (Fig. 2b, Venn diagrams). ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 2 www.nature.com/scientificreports/ Figure 1. Outline and characterization of VP16-CREB over-expression in astrocytes. (a) Procedure outline. (b) Characterization of cellular composition. Cellular markers were GFAP (astrocytes), Iba1 (microglia), NeuN (neurons) and Vimentin, which labels both astrocytes (GFAP positive cells) and fibroblasts (GFAP negative cells). With respect to total DAPI counts, 95% of the cells were astrocytes, 5% microglia and no neurons or fibroblasts were detected−all Vimentin positive cells were GFAP positive as well. (c–f) Confirmation of VP16-CREB transduction. Astrocytes were incubated with Ad2/5-CMV-VP16-CREB or Ad2/5-CMV (Null) at MOIs within 1–30. (c) Immunodetection. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. Top, gene clustering according to log2FC by Euclidean distance in the initial transcriptome (ALL genes) and in the list of genes filtered by Q1-TRAP list of adult astrocyte genes. Bottom, Venn diagrams of differentially expressed genes and Pearson correlations. There are major transcriptome changes upon VP16-CREB over-expression as compared to stimulation with FSK or NE, and the latter groups are highly similar. The same patterns are observed in ALL and TRAP genes. (c) PSCAN analysis of CRE-enrichment probability in ‘all’ and Q1-TRAP-filtered genes using promoters of mouse and human orthologs. The probability of CRE occurrence is highlighted in a spectrum of reds. Gray shades are transcriptome regions not analyzed. Rows are different matrices containing variations of CRE motifs. CRE- containing genes are enriched in the UP portion in all lists. (d) PSCAN analysis comparing ‘all’, ‘Q1-TRAP’ and ‘rest’, or remaining genes. CRE-containing genes are enriched in Q1-TRAP genes as compared to ‘rest’ genes. Figure 2. Transcriptome profiles. (a) Gene distribution according to fold change, adjusted p-values, and direction of change (UP or DOWN) Orange shades are regions of significance (adjusted p-value<0 05) The PSCAN analysis revealed increased probability of CRE-site occurrence in the UP sections of all transcrip- tomes (Fig. 2c,d). The fact that highly similar results were obtained using mouse and human sequences supports the high conservation of CRE sites across species and demonstrates that no bias was introduced in using mouse genes. Of the three stimuli, VP16-CREB was the most efficient means to trigger the CREB-dependent transcrip- tional program, confirming that, in astrocytes as elsewhere, CREB is mostly a transcription activator.hi p gi g y y p The refinement of the global (ALL) list using the Q1-TRAP list did not introduce a bias in the transcriptome profiles since the distribution patterns of ALL genes were conserved in the Q1-TRAP gene group (Fig. 2a,b). FSK and NE groups were still highly similar (62% of the FSK genes were in the NE group and 77% of the genes in the NE group were in the FSK group); the VP16-CREB group included 95% of the genes in the NE and FSK groups; the distribution between UP and DOWN genes was approximately 50%, and CRE-sites were also enriched in the UP section of the Q1-TRAP list (Fig. 2c,d). Interestingly, CRE-sites were more enriched in the UP section of the Q1-TRAP list than in the UP section of the ALL list (Fig. 2c,d). Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. Data are means ± SD of an assay with four replicates representative of at least 5 independent experiments, (**/&&&) p < 0.001, one-way ANOVA followed by Bonferroni. Figure 1. Outline and characterization of VP16-CREB over-expression in astrocytes. (a) Procedure outline. ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 3 www.nature.com/scientificreports/ Figure 2. Transcriptome profiles. (a) Gene distribution according to fold change, adjusted p-values, and direction of change (UP or DOWN). Orange shades are regions of significance (adjusted p-value < 0.05). TRAP genes are genes in the first quartile (Q1) of the TRAP list. Bins of 250 genes were used to calculate the median of fold change and p-value, and to count the number of TRAP genes in each bin. (b) Heatmaps, Venn diagrams and Pearson correlations of differentially expressed genes. Top, gene clustering according to log2FC by Euclidean distance in the initial transcriptome (ALL genes) and in the list of genes filtered by Q1-TRAP list of adult astrocyte genes. Bottom, Venn diagrams of differentially expressed genes and Pearson correlations. There are major transcriptome changes upon VP16-CREB over-expression as compared to stimulation with FSK or NE, and the latter groups are highly similar. The same patterns are observed in ALL and TRAP genes. (c) PSCAN analysis of CRE-enrichment probability in ‘all’ and Q1-TRAP-filtered genes using promoters of mouse and human orthologs. The probability of CRE occurrence is highlighted in a spectrum of reds. Gray shades are transcriptome regions not analyzed. Rows are different matrices containing variations of CRE motifs. CRE- containing genes are enriched in the UP portion in all lists. (d) PSCAN analysis comparing ‘all’, ‘Q1-TRAP’ and ‘rest’, or remaining genes. CRE-containing genes are enriched in Q1-TRAP genes as compared to ‘rest’ genes. re 2. Transcriptome profiles. (a) Gene distribution according to fold change, adjusted p-values, and i Figure 2. Transcriptome profiles. (a) Gene distribution according to fold change, adjusted p-values, and direction of change (UP or DOWN). Orange shades are regions of significance (adjusted p-value < 0.05). TRAP genes are genes in the first quartile (Q1) of the TRAP list. Bins of 250 genes were used to calculate the median of fold change and p-value, and to count the number of TRAP genes in each bin. (b) Heatmaps, Venn diagrams and Pearson correlations of differentially expressed genes. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. This suggests that CREB-dependent genes are among the genes of highest expression in adult astrocytes. In vivo validation. In order to validate our strategy for extraction of adult genetic signatures by using Q1-TRAP, we compared DEG-Q1-TRAP lists with the transcriptomes of Gfa2-TtA/TetO-VP16-CREB trans- genic mice, which express VP16-CREB in astrocytes linked to the activity of a gfap-based promoter, such that VP16-CREB expression is conditional to astrogliosis9. In the existing databases, VP16-CREB expression was achieved by acute injury caused by cryolesion (GSE68187). Using Gene Set Enrichment Analysis (GSEA)20, we compared the transcriptomes of transgenic versus wild-type mice subjected to cryolesion (TC-WTC group) with the in vitro gene lists (NE-CT, FSK-CT, VP16-Null) filtered by Q1-TRAP. GSEA showed a positive enrichment of in vitro gene lists in the portion of up-regulated genes in the group TC/WTC (‘positively correlated’ segment in Fig. 3a, statistics in Table 1), meaning that genes up-regulated upon NE/FSK treatment and VP16-CREB expres- sion are also up-regulated in the cortex of transgenic mice upon targeted activation of CREB dependent tran- scription in astrocytes. The highest enrichment score (NES) was found—as expected—in VP16-CREB-Q1-TRAP (Table 1), while NE and FSK showed similar NES—although FSK enrichment was not statistically significant (Table 1). Conversely, the NES of in vitro genes in the down-regulated portion of TC-WTC was negative ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 4 www.nature.com/scientificreports/ p / (‘negatively correlated’, Fig. 3b, Table 1), meaning that down-regulated genes, which plausibly represen secondary to the primary wave of CREB dependent transcription are comparable in vitro and in vivo T Figure 3. GSEA-based comparison of ast-CREB-dependent transcription in vitro and in vivo. Enrichm DEG-Q1-TRAP databases in transcriptomes from mouse cortices with targeted expression of VP16-C in astrocytes (Gfa2-TtA/TetO-VP16-CREB mice). Transgen expression in mice was triggered by brain GSEA comparisons were carried out between in vitro databases and the up-regulated (a) and down-re (b) fraction of transgenic vs wild-type mice (TC-WTC). The upper parts of the plots show enrichment black lines represent the genes present in both lists (hits) and the bottom parts show the data sets rank gene expression (logFC). Figure 3. GSEA-based comparison of ast-CREB-dependent transcription in vitro and in vivo. Enrichment of DEG-Q1-TRAP databases in transcriptomes from mouse cortices with targeted expression of VP16-CREB in astrocytes (Gfa2-TtA/TetO-VP16-CREB mice). Transgen expression in mice was triggered by brain injury. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. support the use of in vitro tools to gain insight into direct and indirect transcriptional programs of CREB in astro- cytes. For the sake of clarity, the group TC-WTC DOWN is equivalent to WTC-WT UP (Table 1) because the transgene reverts genotypical and phenotypical changes in lesioned wild-type mice back to normal9. support the use of in vitro tools to gain insight into direct and indirect transcriptional programs of CREB in astro- cytes. For the sake of clarity, the group TC-WTC DOWN is equivalent to WTC-WT UP (Table 1) because the transgene reverts genotypical and phenotypical changes in lesioned wild-type mice back to normal9. Functional signature of ast-CREB. The transcriptional programs coordinated by ast-CREB were charac- terized with a GO-based functional enrichment analysis considering categories in Cellular Compartment (CC) and Biological Process (BP) to obtain complementary views. GO was preferred over pathway analysis as with KEGG because we estimate that GO included 85% of our DGEs while KEGG only 40%, leaving a great deal of DEGs uncharacterized. Categories were ranked by significance (−log10 P-value) after redundancy was elimi- nated with ReviGO, and the final categories were manually collapsed in larger themes (Figs 4 and 5). The higher the value, the better represented the category. The complete list of GOs and associated p-values, as well as pathway analysis with KEGG—which largely mirrors the results of GO-BP for V16-CREB, are shown in Supplementary data S3. data S3. Ast-CREB has a profound impact on several functions in astrocytes. Leaving ‘Cytosol’ out because it is too generic, the functional signature comprises seven categories: 1) ‘Gene expression’: This includes ‘Gene expression’ in BP and ‘Nucleus’ in CC, and represents transcription, mRNA processing and translation. Of note, transcription factors are also annotated in categories like ‘RNA processing’. According to significance values, ‘Gene expression’/’Nucleus’ categories are more predominant in the UP section (‘Gene expression’ indeed does not appear in BP-DOWN). The presence of GO catego- ries in ‘Nucleus’ in CC-DOWN in FSK and NE suggests that the transient nature of the latter stimuli may launch a negative feedback of gene expression, absent in the sustained stimulation by VP16-CREB.h 2) ‘Mitochondrial functions’: ‘Mitochondria’ is a clear hit in GO-CC. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. GSEA comparisons were carried out between in vitro databases and the up-regulated (a) and down-regulated (b) fraction of transgenic vs wild-type mice (TC-WTC). The upper parts of the plots show enrichment scores, black lines represent the genes present in both lists (hits) and the bottom parts show the data sets ranked by gene expression (logFC). (‘negatively correlated’, Fig. 3b, Table 1), meaning that down-regulated genes, which plausibly represent cascades secondary to the primary wave of CREB-dependent transcription, are comparable in vitro and in vivo. The results ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 5 www.nature.com/scientificreports/ GENE SET DATA SET NES NOM p-val TC-WTC UP FSK-CT 1.54 0.062 TC-WTC UP NE-CT 2.10 0.004 TC-WTC UP VP16-CREB 4.14 0.000 TC-WTC DOWN FSK-CT −3.12 0.000 TC-WTC DOWN NE-CT −3.15 0.000 TC-WTC DOWN VP16-CREB −7.07 0.000 WTC-WT UP FSK-CT −2.16 0.004 WTC-WT UP NE-CT −2.49 0.000 WTC-WT UP VP16-CREB −4.50 0.000 Table 1. GSEA of in vitro DEG-Q1-TRAP lists in Gfa2-TtA/TetO-VP16-CREB mice. In vitro databases were compared with UP and DOWN genes in injured transgenic (TC) vs injured WT mice (WTC), and with UP genes in WTC vs WT mice. NES: normalized enrichment score. NOM p-val: normalized p-value. GENE SET DATA SET NES NOM p-val TC-WTC UP FSK-CT 1.54 0.062 TC-WTC UP NE-CT 2.10 0.004 TC-WTC UP VP16-CREB 4.14 0.000 TC-WTC DOWN FSK-CT −3.12 0.000 TC-WTC DOWN NE-CT −3.15 0.000 TC-WTC DOWN VP16-CREB −7.07 0.000 WTC-WT UP FSK-CT −2.16 0.004 WTC-WT UP NE-CT −2.49 0.000 WTC-WT UP VP16-CREB −4.50 0.000 Table 1. GSEA of in vitro DEG-Q1-TRAP lists in Gfa2-TtA/TetO-VP16-CREB mice. In vitro databases were compared with UP and DOWN genes in injured transgenic (TC) vs injured WT mice (WTC), and with UP genes in WTC vs WT mice. NES: normalized enrichment score. NOM p-val: normalized p-value. Table 1. GSEA of in vitro DEG-Q1-TRAP lists in Gfa2-TtA/TetO-VP16-CREB mice. In vitro databases were compared with UP and DOWN genes in injured transgenic (TC) vs injured WT mice (WTC), and with UP genes in WTC vs WT mice. NES: normalized enrichment score. NOM p-val: normalized p-value. Table 1. GSEA of in vitro DEG-Q1-TRAP lists in Gfa2-TtA/TetO-VP16-CREB mice. In vitro databases were compared with UP and DOWN genes in injured transgenic (TC) vs injured WT mice (WTC), and with UP genes in WTC vs WT mice. NES: normalized enrichment score. NOM p-val: normalized p-value. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. The categories are more represented in UP compared to DOWN and include nuclear-encoded genes related to mitochondrial functions such as oxidative phosphorylation, tricarboxylic acid cycle (TCA), fatty-acid oxidation, redox homeostasis, repli- cation, transcription, translation, apoptosis regulation, fusion/fission, chaperones, nucleotide homeostasis, and amino acid metabolism, the latter being related to energy metabolism since amino acid catabolism provides intermediates for TCA. 3) ‘Vesicle dynamics’: A highly enriched category is ‘intracellular bounded organelle’, which includes other highly enriched categories like ‘Nucleus’, ‘Mitochondria’, ‘Ribosome’, as well as the endosome system and genes related to SNARE complexes. 4) ‘Stress response’: Pathways include responses against oxidative stress, apoptosis and unfolded protein response, the latter perhaps related to ‘Proteasome’, also present in GO-CC. The significances are generally higher in UP than in DOWN, suggesting that the net tendency is up-regulation of these pathways.h 5) ‘Metabolism: This is highly heterogeneous and includes functions like lipid metabolism and nucleic acid metabolic processes (also represented in ‘Gene expression’) and respiration (also represented in ‘Mitochondria’).hf 6) ‘Signaling’: The most heterogeneous one, featuring different subcategories per group.f ) g gh g , gf g p g 7) ‘Proliferation/differentiation/cytoskeleton’: Categories are clearly DOWN. In summary, ast-CREB triggers intense activity of the transcriptional and translational machinery, associated with general activation of mitochondria functions, vesicle trafficking, modulation of signaling pathways, protec- tion against stress and arrested proliferation. Among the most prominent functions in FSK/NE are amino acid metabolism (‘oxoacid metabolic process’, ‘carboxylic acid metabolic process’) and cytoskeleton dynamics (‘actin cytoskeleton’), while the most relevant functions in VP16-CREB are related to mRNA processing (‘RNA process- ing’) and proteins (‘translation’, ‘protein transport’, ‘proteosome’). Ast-CREB and neu-CREB regulate distinct programs. In parallel we set out to identify direct ast-CREB targets upstream from global functional changes. The premises were: (i) gene targets should be concentrated in the UP sections of the post-Q1-TRAP transcriptomes as predicted by the PSCAN analysis (Fig. 2), (ii) harbor ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 6 www.nature.com/scientificreports/ Figure 4. Functional enrichment analysis with GO Biological Pathway. Heatmaps show -log10 p-values for each enriched GO Biological Pathway (BP) term (described in the right) in the pair-wise comparisons NE- CT; FSK-CT; VP16-Null. GOs were manually grouped into larger categories (left) for better understanding of functional changes among conditions. p-values correspond to an enrichment/depletion two-sided hypergeometric statistical test, corrected for multiple comparisons by Bonferroni post-hoc. Figure 4. Functional enrichment analysis with GO Biological Pathway. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. p-values correspond to an enrichment/depletion two-sided hypergeometric statistical test, corrected for multiple comparisons by Bonferroni post-hoc. Figure 5. Functional enrichment analysis with GO Cellular Compartment. Heatmaps show -log10 p-values for each enriched GO Cellular Compartment (CC) term (described in the right) in the pair-wise comparisons NE-CT; FSK-CT; VP16-Null. GOs were manually grouped into larger categories (left) for better understanding of functional changes among conditions. p-values correspond to an enrichment/depletion two-sided hypergeometric statistical test, corrected for multiple comparisons by Bonferroni post-hoc. Figure 5. Functional enrichment analysis with GO Cellular Compartment. Heatmaps show -log10 p-values for each enriched GO Cellular Compartment (CC) term (described in the right) in the pair-wise comparisons NE-CT; FSK-CT; VP16-Null. GOs were manually grouped into larger categories (left) for better understanding of functional changes among conditions. p-values correspond to an enrichment/depletion two-sided hypergeometric statistical test, corrected for multiple comparisons by Bonferroni post-hoc. folding, modification and trafficking’. New categories with more than 4 genes that were not detected or not at the top in the previous screen are ‘Plasticity’ and ‘Hormones’. folding, modification and trafficking’. New categories with more than 4 genes that were not detected or not at the top in the previous screen are ‘Plasticity’ and ‘Hormones’. At least half of the genes in ‘Gene expression’ are transcription factors. Other nuclear genes are related to ‘RNA export’, ‘DNA replication’ and ‘Translation’. ‘Mitochondria’ comprises genes regulating trycarboxylic acid chain, electron chain, fatty-acid oxidation or mitochondrial translation. Ndufa5, a highly conserved NADH: ubiquinone of complex I in the electron chain is the top hit. ‘Signalling’ includes the GTPase Gem (top hit). In ‘Metabolism’, the most predominant function is lipid metabolism, as represented by Dgat, a key protein in the synthesis of triglycerides. ‘Vesicles’ include genes related to SNARE-dependent exocytosis (Vamp2, Gabarapl2) or to general endosome trafficking (Golph3, Rhobtb3, Reps1). Finally, ‘Redox homeostasis/Anti-oxidant response’ includes Hagh, a thiolesterase responsible for the hydrolysis of S-lactoyl-glutathione to reduced glutathione and D-lactate, protective oxido-reductases like Cb1, Cb3 and Grhp, which detoxifies advanced glycation end prod- ucts and, finally, Gpx4, a glutathione peroxidase with a preference for reducing lipid hydroperoxides. ‘Plasticity’ encompasses genes that have been shown to control cell-fate specification and adaptive changes in neurons, and/ or to be up-regulated in injury. Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. Heatmap each enriched GO Biological Pathway (BP) term (described in the right) in the p CT; FSK-CT; VP16-Null. GOs were manually grouped into larger categories (lef of functional changes among conditions. p-values correspond to an enrichment/ hypergeometric statistical test, corrected for multiple comparisons by Bonferron Figure 4. Functional enrichment analysis with GO Biological Pathway. Heatmaps show -log10 p-values for each enriched GO Biological Pathway (BP) term (described in the right) in the pair-wise comparisons NE- CT; FSK-CT; VP16-Null. GOs were manually grouped into larger categories (left) for better understanding of functional changes among conditions. p-values correspond to an enrichment/depletion two-sided hypergeometric statistical test, corrected for multiple comparisons by Bonferroni post-hoc. CRE-sites in their promoters and (iii) be up-regulated by FSK, NE and VP16-CREB thus representing the core targets of ast-CREB independently of specific regulatory contexts. Th f l l d ll h d f h h The transcriptomes of FSK vs control, NE vs control and VP16-CREB vs Null shared 413 genes of which 125 were up-regulated in the three conditions; 71 are listed in the Salk Institute database as CREB-dependent genes19. We ranked the genes according to their average fold-change in the FSK, NE and VP16-CREB transcriptomes and listed 13 categories (Supplementary data S4). Most of the categories largely agree with the ones unbias- edly identified in the functional enrichment analyses, namely ‘Gene expression’, ‘Mitochondria’ and ‘Signalling’, ‘Metabolism’, ‘Vesicles’, ‘Redox homeostasis/Antioxidant response’, which is allied to ‘Stress response’, and ‘Protein The transcriptomes of FSK vs control, NE vs control and VP16-CREB vs Null shared 413 genes of which 125 were up-regulated in the three conditions; 71 are listed in the Salk Institute database as CREB-dependent genes19. We ranked the genes according to their average fold-change in the FSK, NE and VP16-CREB transcriptomes and listed 13 categories (Supplementary data S4). Most of the categories largely agree with the ones unbias- edly identified in the functional enrichment analyses, namely ‘Gene expression’, ‘Mitochondria’ and ‘Signalling’, ‘Metabolism’, ‘Vesicles’, ‘Redox homeostasis/Antioxidant response’, which is allied to ‘Stress response’, and ‘Protein ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 7 www.nature.com/scientificreports/ entificreports/ Figure 5. Functional enrichment analysis with GO Cellular Compartment. Heatmaps show -log10 p-values for each enriched GO Cellular Compartment (CC) term (described in the right) in the pair-wise comparisons NE-CT; FSK-CT; VP16-Null. GOs were manually grouped into larger categories (left) for better understanding of functional changes among conditions. Discussionh The purpose of this study was to identify molecular and functional targets of ast-CREB in order to facilitate the study of astrocyte-based adaptive functions of CREB in the brain. A most important finding is that the core signature of CREB differs in astrocytes and neurons. While 75% of the gene signature of neu-CREB encodes for genes related to neurotransmission, signalling and transcription, 75% of the gene signature of ast-CREB controls transcription, mitochondrial functions, lipid metabolism, signalling through MAPK, SNARE function and pro- tective responses encompassing redox protection and repair ‘plasticity’ genes. This difference notwithstanding, the enrichment of CRE-sites in the genes most highly expressed in mature cells—both in neurons and astro- cytes—supports that CREB plays as important a function in the adult brain.f y pp p y p It is worth noting that the stringent criteria used to generate signatures (i.e., that genes be differentially expressed by all stimuli and present in the Q1-TRAP list) may leave bona fide CREB target genes out. In other words, the existence of core genes does not preclude the context-specific activation of CREB-target genes out- side the core. These would include genes activated by cyclic AMP-dependent and independent pathways, here represented by the FSK and NE transcriptomes, which, unexpectedly, were highly similar, suggesting a common downstream step in the signalling pathways, or genes activated by yet unknown physiological pathways, as rep- resented in the VP16-CREB transcriptome, which reveals the ast-CREB ‘regulon’. That said, what the signatures represent are the genes with the greatest probability of being regulated by CREB in astrocytes or neurons, this being a necessary distinction when exploring the role of CREB in whole brains. g y g An important implication of the results is that they reveal novel ways by which CREB may modulate adap- tive processes in the brain such as memory via astrocytes. A cornerstone of neuroscience is that learning and memory are the result of the induction of specific genes in neurons in response to experience. CREB is the pro- totypical memory-related gene, and fulfils the task in large part because it activates other transcription factors such as Fos, Nr4a1, Nr4a2 and Jun, thereby triggering a broad downstream program which, through structural and functional changes in neurons, ultimately modifies the activity of neuronal circuits10, 12, 27. www.nature.com/scientificreports/ www.nature.com/scientificreports/ astrocyte de-differentiation, neuroprotection and circuit remodeling. The category includes genes that protect the blood-brain barrier (Rdh10) or promote axon growth (Ninj1)21, 22. It also includes genes like Nkd1 and Galnt119, respectively involved in Wtn and Notch signalling, pathways also involved in brain injury23, 24, and Rgs2, recently shown to be involved in signaling and synaptic plasticity in neurons25. ‘Hormones’ is represented by Dio2, which encodes for a protein highly abundant in adult astrocytes that activates the thyroid hormone and hence may exert a wide set of actions in the brain26; other genes are related to steroid metabolism (Dexi, Hsd17B3). The top gene in ‘Protein trafficking/chaperones’ is the heat shock protein70, Hspa2. fi g p p p Finally, we manually curated the candidate lists in the FSK, NE and VP16-CREB groups in the search for differentially expressed genes associated with well-recognized astrocytic functions such as glutamate/glutamine cycle, neurotransmitter recycling, glutathione metabolism, potassium clearance, maintenance of electrochemical gradients by Na+/K+ ATPases, ion transport, ApoE metabolism, exocytosis via SNAREs, production of growth factors and production of synapse-inducing proteins (Supplementary data S5). The only genes differentially expressed in the three groups, that is, core genes, were genes encoding for SNARE proteins (Vamp2, Vamp5, Snap- 25) and Gpx4. In summary, of all the canonical astrocyte functions the ones that may be regulated by ast-CREB functions are ‘Vesicle dynamics’ and ‘Redox homeostasis’. y We next compared the transcriptional programs of ast-CREB and neu-CREB. To produce comparable mate- rials we processed neuronal lists in the same manner as we did with astrocytes. Thus, the differentially expressed genes of neuronal cultures infected with lentiviruses that transduce VP16-CREB or exposed to FSK10 were filtered using the first quartile of the TRAP list to select genes with the highest probability of being relevant to adult func- tion. Figure 6a–c shows the gene distribution in neurons over-expressing VP16-CREB according to expression and statistical significance thereof. Unlike astrocytes, there is some predominance of down-regulated genes that is maintained after the Q1-TRAP filtration (Fig. 6a). However, as with astrocytes, PSCAN revealed enrichment of CRE-sites in the genes derived from the Q1-TRAP list, and more so in the UP group of the differentially expressed. The lower the p-value the greater the density of CRE-sites (Fig. www.nature.com/scientificreports/ 6c,d).i ph p g y ( g ) To generate the core molecular signature of neu-CREB, we identified CREB-target genes in the UP genes com- mon to FSK and VP16-CREB, using the repository of the Salk Institute. There were 93 genes common to FSK and VP16-CREB. Of the 93 genes, 60 were up-regulated in both VP16-CREB and FSK lists, of which 39 appeared in the Salk Institute databases. As expected, core genes regulated by neu-CREB were the ones traditionally associated with CREB in the brain: Fos, nuclear receptors (Nr4a1, Nr4a2), neuropeptides (Tac1, Npy, Sst) and dual specificity phosphatase (Dusp1) (Supplementary data S4). The functional categories over-represented in the molecular core of neu-CREB were ‘Neurotransmission’ (12 genes related to well-known neurotransmitters), ‘Signalling’ (11 genes related to kinases/phosphatases) and ‘Gene expression’ (6 out of 7 genes encoding for transcription factors), the top ranking genes being Tac1, Dusp1 and Nr4a1. These categories account for 75% of the molecular signature of neu-CREB. Less represented categories are ‘Cytoskeleton’ (Clstn3), and ‘Carriers’, with the glucose carrier Scl2a3. A t CREB d CREB i t th b littl bl Th l I t p g y ( ) g Ast-CREB and neu-CREB core signatures thus bear little resemblance. The only common genes are Impact, which controls translation, and Emerin, a nuclear gene related to actin. Shared categories like ‘Gene expression’ and ‘Signalling’ are represented by different genes. ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x Results Analysis outline. The study was carried out in the five stages depicted in Fig. 1a. Since the genes are in the top quartile of the TRAP astrocyte list, and hence highly expressed in adult brain, they may play a role in restorative/repair phenomena including astrocyte proliferation, ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 8 Discussionh The high rep- resentation of transcription factors in the ast-CREB-dependent transcriptomes suggests that ast-CREB is also a hub of activity-driven genetic programs, although through different transcription factors. However, although a wealth of data demonstrates that astrocytes regulate neurotransmission28, it is barely recognized that adaptive changes driven by gene expression may happen in astrocytes themselves. Astrocytes have been studied solely in the context of the short-term regulation of synaptic events29. Indeed, the available GO classification and path- way enrichment repositories like KEGG mirror the current neuron-centric conceptualization of the brain, and hence lack categories related to astrocyte-specific functions and excitability as shown by calcium responses30. This ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 9 www.nature.com/scientificreports/ Figure 6. Molecular signatures of CREB in neurons and astrocytes. (a) Histograms showing the distribution of genes according to fold change, adjusted p-values, and direction of change (UP or DOWN) in neurons over- expressing VP16-CREB. The whole transcriptome was ranked by the t statistics from more to less significance of change in UP and DOWN sections. Q1-TRAP genes were also mapped below all the transcriptomes, as in Fig. 2. Similar results were observed in the FSK group (not shown). (b) PSCAN analysis of CRE-enrichment probability in neurons over-expressing VP16-CREB, using the same procedure as in Fig. 2. CRE-containing genes are enriched in the UP portion, indicating that CREB is mostly a transcriptional activator in neurons. Only the top50 in either UP or DOWN portions were scanned for CRE sites, producing the same bias towards up-regulation. Similar results were observed in the FSK group. (c) The same PSCAN analysis without considering bins but entire sets of genes: ‘all’, all the genes; ‘up’ and ‘down’, all the up-regulated and down- regulated genes independently of the significance of change; ‘TRAP’, only the Q1-TRAP list; ‘rest’, the remaining genes. The up-regulated Q1-TRAP genes were enriched in CRE sites. (d) Core up-regulated astrocyte genes filtered through the Q1-TRAP list and the CREB-target-gene database (Salk institute) were compared with neuronal databases obtained from neurons treated with FSK or VP16-CREB10 processed in like manner. Venn diagrams showed almost no overlapping between both cell types. Categories were manually inferred from gene function according to the literature. The full lists are in Supplementary data S4. Chart slices correspond to the percentage of genes in each category. Figure 6. Molecular signatures of CREB in neurons and astrocytes. Discussionh (a) Histograms showing the distribution of genes according to fold change, adjusted p-values, and direction of change (UP or DOWN) in neurons over- expressing VP16-CREB. The whole transcriptome was ranked by the t statistics from more to less significance of change in UP and DOWN sections. Q1-TRAP genes were also mapped below all the transcriptomes, as in Fig. 2. Similar results were observed in the FSK group (not shown). (b) PSCAN analysis of CRE-enrichment probability in neurons over-expressing VP16-CREB, using the same procedure as in Fig. 2. CRE-containing genes are enriched in the UP portion, indicating that CREB is mostly a transcriptional activator in neurons. Only the top50 in either UP or DOWN portions were scanned for CRE sites, producing the same bias towards up-regulation. Similar results were observed in the FSK group. (c) The same PSCAN analysis without considering bins but entire sets of genes: ‘all’, all the genes; ‘up’ and ‘down’, all the up-regulated and down- regulated genes independently of the significance of change; ‘TRAP’, only the Q1-TRAP list; ‘rest’, the remaining genes. The up-regulated Q1-TRAP genes were enriched in CRE sites. (d) Core up-regulated astrocyte genes filtered through the Q1-TRAP list and the CREB-target-gene database (Salk institute) were compared with neuronal databases obtained from neurons treated with FSK or VP16-CREB10 processed in like manner. Venn diagrams showed almost no overlapping between both cell types. Categories were manually inferred from gene function according to the literature. The full lists are in Supplementary data S4. Chart slices correspond to the percentage of genes in each category. means that the existing databases are obsolete. Their update will require that astrocytic molecules and pathways controlling circuit long-term plasticity be sufficiently conceptualized and characterized to give rise to novel cate- gories. For now, we posit three mechanisms whereby ast-CREB may control adaptive, long-term plasticity in the brain. means that the existing databases are obsolete. Their update will require that astrocytic molecules and pathways controlling circuit long-term plasticity be sufficiently conceptualized and characterized to give rise to novel cate- gories. For now, we posit three mechanisms whereby ast-CREB may control adaptive, long-term plasticity in the brain. One is modulation of gliotransmission. Astrocytes are secretory cells: they respond to neurotransmitters by releasing so-called gliotransmitters (e.g., glutamate, ATP, D-serine) via vesicular transporters and Vamp pro- teins31. ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x Methods ll l Cell culture and treatments. Experiments have been carried out in accordance to the guidelines of the European Union Laws for the protection of experimental animals. Experimental protocols have been approved by the Animal Welfare Committee of the Autonomous University of Barcelona and the Generalitat de Catalunya. Cortical astrocyte cultures were prepared from 1-day old Sprague-Dawley rats. Briefly, rats were decapitated and cortices were rapidly removed and separated from brain meninges. The tissue was minced and incubated for 10 min at 37 °C in Ca2+-free Krebs-Ringer buffer containing 0.025% trypsin. The cells were then mechanically triturated through a glass pipette and filtered through a 40-µm nylon mesh in the presence of 0.52 mg/ml soybean trypsin inhibitor and 170 IU/ml DNAse. After centrifugation (500 g), the cells were stained with Trypan Blue exclusion, counted in a Neubauer chamber, and then resuspended (3 × 105 cells/ml) in 90% DMEM, 10% FBS, 20 U/ml penicillin, and 20 µg/ml streptomycin. The cells were seeded in appropriate plates (35 mm dishes for RNA and protein extraction, or in 24-well plates for luciferase assays and immunocytochemistry), and maintained in a humidified atmosphere of 95% air-5% CO2. The culture medium was changed two hours after seeding and at the 7th day in vitro. Cells were used when confluent, around day 10. Characterization of cellular contents was carried out by immunocytochemistry for astrocyte, microglia, neuron and fibroblast markers (see below). h d f d f h fl l b d l dd y y y y gi Treatments with 1 µM FSK and 10 µM NE were performed for 6 hours in confluent cultures by directly add- ing the agents to the culture medium. The over-expression of VP16-CREB was carried out by infection with the adenoviruses Ad2/5-CMV-VP16-CREB or Ad2/5-CMW (Null) in pre-confluent cultures using DMEM with 1% FBS without antibiotics. The SK-based plasmids were obtained from A. Barco’s laboratory, and the viruses were generated in the Core Service of Virus Production at the Universitat Autònoma de Barcelona. The viruses were tested at a range of multiplicity of infections (MOI) within 5–30. The medium was changed after 3 hours and the cells were used 18 hours post-infection. Immunocytochemistry. www.nature.com/scientificreports/ metabolism via a catecholamine signal originating in neurons34. The unifying theme of these studies is that CREB mediates adaptive metabolic changes. Here we posit that, in the brain, adaptive metabolic changes mediated by an ast-CREB/mitochondria axis influence memory formation. This is in line with the emerging notion that mito- chondria regulate synaptic plasticity by locally controlling energy status and calcium content35, 36. Of note, hits of the array are Dgat, related to triglyceride synthesis, which has been recently described as an essential requirement for fatty-acid oxidation in the mitochondria37, and Gpx4, specialized in reducing lipid-derived reactive oxygen species. All in all, the data suggest that ast-CREB has an impact on astrocyte aerobic metabolism by enhancing mitochondrial fatty-acid oxidation while protecting cells from lipid-derived oxidative stress. Enhanced mito- chondrial activation may, in turn, modify astrocyte excitability and hence circuit activity. y y y y y A third mechanism is circuit remodeling through genes involved in Wtn and Notch signalling (Nkd1, Galnt119)23, 24, or shown to regulate synaptic plasticity in neurons (Rgs2)25, raising the possibility that they con- trol astrocyte plasticity as well. y p y Still, a limitation of the study is that the analyses were performed with materials from cultured neonatal astro- cyte and neurons. Although we did find that DEGs from NE/FSK/VP16-CREB astrocytes were enriched in mice with targeted activation of CREB dependent transcription, supporting that the in vitro signature is relevant in vivo, the possibility remains that neonatal programs, or programs related to maturation, may be present in our DEGs. Along these lines, sustained activation of cyclic AMP-dependent pathways, which may be comparable to our FSK group, has been recently reported to promote maturation of astrocytes38, as evidenced by the enrich- ment of DEGs of astrocytes treated with 8Br-cAMP, an analog of cyclic AMP, in the transcriptome of mature astrocytes38, 39. However, over 70% of DEGs in ref. 38 do not overlap with the transcriptome of mature cells39, sug- gesting development-independent actions of cyclic AMP-dependent signaling in astrocytes. Moreover, another recent study reports cyclic AMP/CREB-dependent glutamatergic regulation of mature astrocytes40. www.nature.com/scientificreports/ All in all, the emerging evidence emphasizes the importance of model, and hence of context, in the CREB-dependent regula- tion of adaptive changes in astrocytes.if p g y In conclusion, we identified here a subset of genes highly expressed in adult astrocytes, differentially regu- lated upon CREB activation, and harboring CRE-sites at their promoters, indicating that they are direct targets of this transcription factor in astrocytes. Furthermore, we demonstrate that the core signatures of ast-CREB and neu-CREB target genes are different, suggesting novel ways by which CREB regulates brain adaptive func- tions. Finally, the enrichment of CRE-sites in genes highly expressed in adult astrocytes and neurons supports the relevance of CREB in the regulation of astrocyte-neuron circuits. Future research may identify specific CREB-dependent programs for different physiological contexts. Discussionh Vesicle-mediated dynamics and Vamp2 are hits in functional and molecular signatures of ast-CREB, sug- gesting that the transcription factor may tune the communication between neurons and astrocytes.h g g p y y A second mechanism is the modulation of aerobic metabolism. The idea that CREB controls astrocyte energy metabolism stems from the discovery that many ast-CREB hits are genes encoding for mitochondrial proteins. The relationship between CREB and mitochondria is not new. CREB regulates the expression of genes encod- ing for mitochondrial calcium channels32. Also, CREB-regulated transcriptional co-activator (CRTC1) promotes mitochondrial biogenesis via cofactor PGC-1alpha in muscle cells33, and down-regulates systemic mitochondrial ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 10 www.nature.com/scientificreports/ ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x Methods ll l The mixture was incubated for 15 minutes at room temperature and added to the cells. The medium was changed after 2 hours and the cells were subjected to appropriate treatment and monitored for luciferase expression 48 hours after transfection. qPCR. The RNA from treated cultures was isolated using the RNAeasy spin kit (Capsumlab) according to the manufacturer’s instructions. Genomic DNA contamination was prevented by treating samples with DNAse1 (Invitrogen). The RNA concentration was determined with the NanoDrop 1000 spectrophotometer (Thermo Scientific) and quality was tested using the Agilent 2100 Bioanalyzer (Agilent Technologies). Only samples with RIN >8 were used. One µg of RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) in a reaction of 25 µL. Samples at a 1:100 dilution were amplified in an Applied Biosystems 7500 Fast system using the Power Sybr Green PCR master mix (Life technologies). Data analysis was performed with the comparative Cq method (Pfaffl 2001) using the average value of PCR efficiencies obtained with LinRegPCR software and normalized to Gapdh. DNA microarray. DNA arrays were carried out by Bioarray® (Alicante, Spain) using SurePrint G3 Rat Gene Expression 8 × 60 K (Agilent, Santa Clara, California, USA). These microarrays include 39,430 Entrez Gene RNAs and 16,251 lincRNAs. The RNA was extracted from cell cultures as described previously. The concen- tration was determined with the NanoDrop 1000 spectrophotometer (Thermo Scientific), and the RNA quality with Tape Station, using the R6K ScreenTape kit (Agilent). Three samples of each condition (CT, FSK, NE, Null, VP16-CREB) were amplified, labeled for Cy5 or Cy3 using the Low Input Quick Amp Labeling kit (Agilent), and hybridized in the SurePrint G3 Microarray slides according to the manufacturer’s protocol. We performed two independent experiments for the treated and the infected samples, and each replicate was hybridized three times and balanced with respect to the use of Cy3 and Cy5. The microarray slides were scanned using the Agilent microarray scanner (G2565CA), and the resulting images were extracted with Agilent Feature Extraction Software (version 10.7). Bioinformatics. Raw data extraction and statistical analyses were performed using Bioconductor pack- ages in the R programming environment. Background correction was performed using the ‘normexp’ method (offset = 10) implemented in the LIMMA package to adjust local median (M) background estimates. Methods ll l For immunocytochemistry the cells were fixed in 4% paraformaldehyde for 15 minutes and, after several washes with cold PBS, blocked with 5% NGS for 30 minutes and incubated over- night in a humidified chamber with the primary antibodies against VP16 (1:150, Santa Cruz), GFAP (Dako, 1:2000), NeuN (1:1000, Merck Millipore), Iba1 (1:1000, wAKO) and Vimentin (1:500, Abcam). The next day the cells were incubated with fluorescence-labeled secondary antibodies (Life technologies, 1:1000), counterstained with the nuclear marker DAPI (1:20000, Invitrogen) and mounted with fluoromount-G (Southern biotech). The labeling was visualized with a epifluorescence microscope (Nikon Eclipse 90i) and pictures taken with a NIKON DXM1200F (Sofware ACT-1) at a resolution of 3840 × 3072 pixels and saved in TIF. Pictures were combined using Adobe Photoshop version 8.0. Western blot. The cells were lysed in cold RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 1% NP-40, 0.1% SDS, 1 mM Na3VO4, 50 mM NaF, and 1 mM PMSF) supplemented with protease and phosphatase inhibitors (Roche). Twenty-five µg of protein was run in 10% SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinyl difluoride membranes, blocked with 5% milk in ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 11 www.nature.com/scientificreports/ Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 h, and incubated overnight at 4 °C with VP16 (Santa Cruz Biotech, 1:150) or actin-β antibodies (Sigma, 1:30000). The next day the membranes were incubated with anti-IgG-horseradish peroxidase-labeled secondary antibodies (Pierce, 1/10,000) for 1 h at room temperature and detected with an enhanced chemiluminescence detection kit (ECL, Amersham Life Science). Films were revealed with Fujifoto FPM-100A, and scanned in HPScanjet G3010. Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 h, and incubated overnight at 4 °C with VP16 (Santa Cruz Biotech, 1:150) or actin-β antibodies (Sigma, 1:30000). The next day the membranes were incubated with anti-IgG-horseradish peroxidase-labeled secondary antibodies (Pierce, 1/10,000) for 1 h at room temperature and detected with an enhanced chemiluminescence detection kit (ECL, Amersham Life Science). Films were revealed with Fujifoto FPM-100A, and scanned in HPScanjet G3010. Luciferase assays. Pre-confluent astrocyte cultures were transfected with luciferase reporters using FuGene6 (Roche) following manufacturer’s instructions. Briefly, for each well in a 24-well plate, 1 µg of pCRE-luciferase and 0.5 µg of pTK-renilla were mixed with 3 µL of FuGene6 in 250 µL of DMEM 10% FBS without antibiotics. Methods ll l Background-corrected intensity data were normalized using the Loess method to remove the bias within each array, and A-value quantile normalization (Aquantile) was used to remove the bias between arrays. The princi- pal component analysis was carried out to ensure the correct separation of the samples. Normalized data were adjusted to a linear model in LIMMA to determine the target genes differentially expressed between groups. P-values were computed with empirical Bayes moderated t-statistics at the level of 0.05 to calculate differen- tial gene expression in the paired comparisons FSK-CT, NE-CT, and VP16-Null. The lists can be found in the Gene Expression Omnibus database (GSE80967). Differentially expressed genes were ranked according to Gene Symbol and t-statistic in absolute value. Mouse orthologous were extracted using the Gene Analyzer Tool from the RGD (Rat Genome Database) website (http://rgd.mcw.edu) to allow for comparisons with the Q1-TRAP and neu-CREB lists, which are from mice.i TRAP lists were retrieved from GSE13379 files. Gene expression was normalized using LIMMA. In the case of the neuronal TRAP lists (below), we averaged the profiles of the several cortical subtypes (Cck+, Pnoc+, cholin- ergic and neurons from layers 5a, 5b and 6) to minimize their functional and molecular diversity.fii Differentially expressed gene lists filtered by the first quartile of the TRAP list (5220 genes) were divided into UP and DOWN depending on the logFC and associated to GO terms (Biological Process and Cellular Compartment) with the software ClueGO v1.441. The analysis was independently conducted in the UP and DOWN lists to gain insight into the predominant direction of change, although it is worth stressing that func- tional changes normally encompass both up-regulation and down-regulation of genes due to complex home- ostatic adjustment of positive and negative feedback loops. The parameters used were enrichment/depletion: two-sided hypergeometric statistical test; correction method: Bonferroni; GO term range levels: 5–10; minimal number of genes for term selection: 3; minimal percentage of genes for term selection: 2%; κ score threshold: 0.5; general term selection method: smallest p-value; group method: κ; minimal number of subgroups included in a group: 2; minimal percentage of shared genes between subgroups: 50%. The resulting GO terms were analysed for semantic similarity (cut-off value of 0.4) with ReviGO software42 to reduce redundancy, and then manually grouped into categories based on GO ancestors and gene overlapping between terms. ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x References Roles for Regulator of G Protein Signaling Proteins in Synaptic Signaling and Plasticity Mol. Pharmacol. 89, 273–286 (2016).i 6. Chatonnet, F., Guyot, R., Benoit, G. & Flamant, F. Genome-wide analysis of thyroid hormone receptors shared and specific function in neural cells. Proc. Natl. Acad. Sci. USA 110, E766–E775 (2013). 27. Viosca, J. L., de, A. M., Jancic, D. & Barco, A. Enhanced CREB-dependent gene expression increases the excitability of neurons in the basal amygdala and primes the consolidation of contextual and cued fear memory. Learn. Mem. 16, 193–197 (2009).i 8. Martin, R., Bajo-Graneras, R., Moratalla, R., Perea, G. & Araque, A. Circuit-specific signaling in astrocyte-neuron networks in basa ganglia pathways. Science. 349, 730–734 (2015). 29. Perez-Alvarez, A., Navarrete, M., Covelo, A., Martin, E. D. & Araque, A. Structural and functional plasticity of astrocyte processes and dendritic spine interactions. J. Neurosci. 34, 12738–12744 (2014). p 30. Shigetomi, E., Patel, S. & Khakh, B. S. Probing the complexities of astrocyte calcium signaling. Trends Cell Biol. 26, 300–312 (2 30. Shigetomi, E., Patel, S. & Khakh, B. S. Probing the complexities of astrocyte calcium signaling. Trends Cell Biol. 26, 300–312 (2016). 31. Verkhratsky, A., Matteoli, M., Parpura, V., Mothet, J. P. & Zorec, R. Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion. EMBO J. 35, 239–257 (2016). g g p y g g 31. Verkhratsky, A., Matteoli, M., Parpura, V., Mothet, J. P. & Zorec, R. Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion. EMBO J. 35, 239–257 (2016). 32. Shanmughapriya, S. et al. Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU. Sci. Signal. 8, ra23 (2015). mitochondrial Ca2+ uniporter gene MCU. Sci. Signal. 8, ra23 (2015 3. Wu, Z. et al. Transducer of regulated CREB-binding proteins (TORCs) induce PGC-1alpha transcription and mitochondria biogenesis in muscle cells. Proc. Natl. Acad. Sci. USA 103, 14379–14384 (2006). 33. Wu, Z. et al. Transducer of regulated CREB-binding proteins (TORCs) induce PGC-1alph biogenesis in muscle cells. Proc. Natl. Acad. Sci. USA 103, 14379–14384 (2006). g 34. Burkewitz, K. et al. Neuronal CRTC-1 governs systemic mitochondrial metabolism and lifespan via a catecholamine signal. Cell. 160, 842–855 (2015).fi 5. Sheng, Z. H. Mitochondrial trafficking and anchoring in neurons: New insight and implications. J. Cell Biol. 204, 1087–1098 (2014) 35. Sheng, Z. H. Data availabilty. Accession number microarray data. Gene Expression Omnibus GSE80967. Data availabilty. Accession number microarray data. Gene Expression Omnibus GSE80967. References & Barco, A. Ultrastructural and transcriptional profiling of neuropathological misregulation o CREB function. Cell Death. Differ. 17, 1636–1644 (2010). ff 12. Barco, A. & Marie, H. Genetic approaches to investigate the role of CREB in neuronal plasticity and memory. Mol. Neurobiol. 44, 330–349 (2011). 13. Parra-Damas, A. et al. Crtc1 activates a transcriptional program deregulated at early Alzheimer’s disease-related stages. J. Neurosci. 34, 5776–5787 (2014). 4. Riccio, A., Ahn, S., Davenport, C. M., Blendy, J. A. & Ginty, D. D. Mediation by a CREB family transcription factor of NGF dependent survival of sympathetic neurons. Science. 286, 2358–2361 (1999). 15. Lemberger, T., Parkitna, J. R., Chai, M., Schutz, G. & Engblom, D. CREB has a context-dependent role in activity-regulated transcription and maintains neuronal cholesterol homeostasis. FASEB J. 22, 2872–2879 (2008).i 16. Cha-Molstad, H., Keller, D. M., Yochum, G. S., Impey, S. & Goodman, R. H. Cell-type-specific binding of the transcription factor CREB to the cAMP-response element. Proc. Natl. Acad. Sci. USA 101, 13572–13577 (2004).i P-response element. Proc. Natl. Acad. Sci. USA 101, 13572–13577 ( 7. Doyle, J. P. et al. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell. 135, 749–762 (2008).i 8. Zambelli, F., Pesole, G. & Pavesi, G. Pscan: finding over-represented transcription factor binding site motifs in sequences from co regulated or co-expressed genes. Nucleic Acids Res. 37, W247–W252 (2009). 19. Zhang, X. et al. Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues. Proc. Natl. Acad. Sci. USA 102, 4459–4464 (2005). 0. Subramanian et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression studies Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005). 1. Mizee, M. R. et al. Astrocyte-derived retinoic acid: a novel regulator of blood-brain barrier function in multiple sclerosis. Acta Neuropathol. 128, 691–703 (2014). p 22. Araki, T. & Milbrandt, J. Ninjurin, a novel adhesion molecule, is induced by nerve injury and promotes axonal growth. Neuron 353–361 (1996). 23. Lambert, C., Cisternas, P. & Inestrosa, N. C. Role of Wnt Signaling in Central Nervous System Injury. Mol. Neurobiol. 53, 2297– (2016). 4. Alberi, L., Hoey, S. E., Brai, E., Scotti, A. L. & Marathe, S. Notch signaling in the brain: in good and bad times. Ageing Res. Rev. 12 801–814 (2013). 5. Gerber, K. J., Squires, K. E. & Hepler, J. R. References e e e ces 1. Altarejos, J. Y. & Montminy, M. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nat. Rev. Mol. Cel Biol. 12, 141–151 (2011). ( ) 2. Ehrlich, D. E. & Josselyn, S. A. Plasticity-related genes in brain development and amygdala-dependent learning. Genes Brain Behav 15, 125–143 (2016). et al. Manipulating a “cocaine engram” in mice. J. Neurosci. 34, 141 . Hsiang, H. L. et al. Manipulating a “cocaine engram” in mice. J. N 4. Kim, J., Kwon, J. T., Kim, H. S., Josselyn, S. A. & Han, J. H. Memory recall and modifications by activating neurons with elevated CREB. Nat. Neurosci. 17, 65–72 (2014). 5. Ma, T. C., Barco, A., Ratan, R. R. & Willis, D. E. cAMP-responsive element-binding protein (CREB) and cAMP co-regulate activato protein 1 (AP1)-dependent regeneration-associated gene expression and neurite growth. J. Biol. Chem. 289, 32914–32925 (2014). 5. Ma, T. C., Barco, A., Ratan, R. R. & Willis, D. E. cAMP-responsive element-binding protein (CREB) and cAMP co-regulate activator protein 1 (AP1)-dependent regeneration-associated gene expression and neurite growth. J. Biol. Chem. 289, 32914–32925 (2014). 6. Carriba, P. et al. ATP and noradrenaline activate CREB in astrocytes via noncanonical Ca(2+) and cyclic AMP independent pathways. Glia. 60, 1330–1344 (2012). 6. Carriba, P. et al. ATP and noradrenaline activate CREB in astrocytes via noncanonical Ca(2+) and cyclic AMP independen pathways. Glia. 60, 1330–1344 (2012). p y 7. Karki, P. et al. cAMP response element-binding protein (CREB) and nuclear factor kappaB mediate the tamoxifen-induced up regulation of glutamate transporter 1 (GLT-1) in rat astrocytes. J. Biol. Chem. 288, 28975–28986 (2013). g g p y 8. Modi, K. K., Jana, M., Mondal, S. & Pahan, K. Sodium benzoate, a metabolite of cinnamon and a food additive, upregulates ciliary neurotrophic factor in astrocytes and oligodendrocytes. Neurochem. Res. 40, 2333–2347 (2015). 8. Modi, K. K., Jana, M., Mondal, S. & Pahan, K. Sodium benzoate, a metabolite of cinnamon and neurotrophic factor in astrocytes and oligodendrocytes. Neurochem. Res. 40, 2333–2347 (2015) 9. Pardo, L. et al. Targeted activation of CREB in reactive astrocytes is neuroprotective in focal acute cortical injury. Glia. 64, 853–874 (2016). 10. Benito, E., Valor, L. M., Jimenez-Minchan, M., Huber, W. & Barco, A. cAMP response element-binding protein is a primary hub of activity-driven neuronal gene expression. J. Neurosci. 31, 18237–18250 (2011).i y g p 1. Valor, L. M., Jancic, D., Lujan, R. Methods ll l g g g g To identify CREB target genes we used PSCAN18 to predict CRE-binding sites of the TRANSFAC database in the promoter region (−450/+50) of selected genes, and the database of Salk Institute19. Additional soft- ware included ‘gplots’ (http://CRAN.R-project.org/package=gplots) and ‘RColorBrewer’ packages (http:// CRAN.R-project.org/package=RColorBrewer) to compute heat maps and hierarchical clustering.i p j g p g p p g GSEA (www.broadinstitute.org/gsea/index.jsp)20. Lists of DEG filtered by Q1-TRAP were pre-ranked by old-change and analyzed for enrichment in gene sets from up-regulated or down-regulated genes in the pairwise ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 12 www.nature.com/scientificreports/ comparisons TC-WTC (VP16-CREB lesion vs WT lesion) and WTC-WT (lesion vs control) (GSE68187)9. Only enriched sets with a normalized p-value (NOM p-val) < 0.05 were considered significant. Of note, the same plat- form (Agilent) and procedure was used to identify DEG in the in vitro and in vivo microarrays. comparisons TC-WTC (VP16-CREB lesion vs WT lesion) and WTC-WT (lesion vs control) (GSE68187)9. Only enriched sets with a normalized p-value (NOM p-val) < 0.05 were considered significant. Of note, the same plat- form (Agilent) and procedure was used to identify DEG in the in vitro and in vivo microarrays. Data availabilty. Accession number microarray data. Gene Expression Omnibus GSE80967. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 8. Paco, S., Hummel, M., Pla, V., Sumoy, L. & Aguado, F. Cyclic AMP signaling restricts activation and promotes maturation and antioxidant defenses in astrocytes. BMC. Genomics. 17, 304–2623 (2016). y 39. Cahoy et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding b development and function. J Neurosci 28, 264–278 (2008). p J , ( ) 40. Hasel et al. Neurons and neuronal activity control gene expression in astrocytes to regulate their development and metabolism. Nature Commun 8, 15132 (2017). p J ( ) 40. Hasel et al. Neurons and neuronal activity control gene expression in astrocytes to regulate their development and metabolism. Nature Commun 8, 15132 (2017). 41. Bindea, G. et al. ClueGO: A Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation netw Bioinformatics 25, 1091–1093 (2009). f 42. Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. Revigo summarizes and visualizes long lists of gene ontology terms. PLoS O (2011). f 42. Supek, F., Bošnjak, M., Škunca, N. & Šmuc, T. Revigo summarizes and visualizes long lists of gene ontology terms. PLoS One 6 (2011). Acknowledgementsh g This work was supported by Ministerio de Economía y Competitividad, Spain (grant numbers BFU2012-38844 and BFU2016-79735-P to E.G.; SAF2011-22506 grant to L.M.V.; SAF2014-56197-R to A.B. an L.M.V.; PCIN- 2015-192-C02-01 and SEV-2013-0317 to A.B.); Autonomous Government of Catalonia AGAUR (grant number 2014SGR984 to E.G. and R.M.); Generalitat Valenciana (grant number Prometeo/2012/005 to A.B.); and the Brain & Behavior Research Foundation (NARSAD Independent Investigator Grant) and the Alicia Koplovitz Foundation to A.B.). L.M.V. was additionally supported by a Ramón y Cajal contract (Ministerio de Economía y Competitividad), followed by a Miguel Servet contract (Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, and Fondo Social Europeo 2014–2020). The Instituto de Neurociencias de Alicante is a “Centre of Excellence Severo Ochoa”. Author Contributions E.G. and L.P. designed the study, performed mining and bioinformatics and wrote up the manuscript. L.M.V. performed mining and bioinformatics. L.P. and A.E.P. characterized VP16-CREB expression. A.G. characterized the cellular composition of the cultures. G.E.H., A.B. and R.M. interpreted data in the context of neuronal and astrocyte biology. References Mitochondrial trafficking and anchoring in neurons: New insight and implications. J. Cell Biol. 204, 1087–1098 (2014). 36. Todorova, V. & Blokland, A. Mitochondria and synaptic plasticity in the mature and aging nervous system. Curr. Neuropharmacol. (2016). fi 36. Todorova, V. & Blokland, A. Mitochondria and synaptic plasticity in the mature and aging nervous system. Curr. Neuropharmacol. (2016). 37. Cabodevilla, A. G. et al. Cell survival during complete nutrient deprivation depends on lipid droplet-fueled beta-oxidation of fatty acids. J. Biol. Chem. 288, 27777–27788 (2013). ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 13 ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-06231-xh Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 ScIeNTIFIc REPOrTS \| 7: 6390 \| DOI:10.1038/s41598-017-06231-x 14
https://openalex.org/W4236643437	https://www.geosci-instrum-method-data-syst.net/5/437/2016/gi-5-437-2016.pdf	English	null	A coincidence detection system based on real-time software	null	2,016	cc-by	11,361	Correspondence to: Sindulfo Ayuso (sindulfo.ayuso@edu.uah.es) Correspondence to: Sindulfo Ayuso (sindulfo.ayuso@edu.uah.es) Received: 1 May 2016 – Published in Geosci. Instrum. Method. Data Syst. Discuss.: 6 June 2016 Revised: 27 August 2016 – Accepted: 7 September 2016 – Published: 26 September 2016 Received: 1 May 2016 – Published in Geosci. Instrum. Method. Data Syst. Discuss.: 6 June 2016 Revised: 27 August 2016 – Accepted: 7 September 2016 – Published: 26 September 2016 Abstract. Conventional real-time coincidence systems use electronic circuitry to detect coincident pulses (hardware co- incidence). In this work, a new concept of coincidence sys- tem based on real-time software (software coincidence) is presented. This system is based on the recurrent supervi- sion of the analogue-to-digital converters status, which is de- scribed in detail. A prototype has been designed and built using a low-cost development platform. It has been applied to two different experimental sets for cosmic ray muon de- tection. Experimental muon measurements recorded simul- taneously using conventional hardware coincidence and our software coincidence system have been compared, yielding identical results. These measurements have also been vali- dated using simultaneous neutron monitor observations. This new software coincidence system provides remarkable ad- vantages such as higher simplicity of interconnection and adjusting. Thus, our system replaces, at least, three Nuclear Instrument Modules (NIMs) required by conventional coin- cidence systems, reducing its cost by a factor of 40 and elim- inating pulse delay adjustments. tions between highly energetic CRs and the nuclei of atmo- spheric particles (Cecchini and Spurio, 2012). Muons (µ− and µ+) are particles belonging to the lepton family, and they have the same charge (negative and positive, respec- tively) as that of an electron and 207 times its mass. Coincidence counting is widely used in experimental par- ticle physics with different purposes such as reducing noise; getting directional information (Karapetyan et al., 2013); re- ducing the probability of a measurement being triggered by independent, unrelated particles; lessening the probability of independent random background events (Remmen and Mc- Creary, 2012); or identifying energetic particles in multi- element particle telescopes (see e.g. Müller-Mellin et al., 1995). Traditional particle detection systems using coincidence rely on dedicated electronic modules. When real-time opera- tion is not required, alternative approaches based on the anal- ysis of recorded pulse information can be used (e.g. Havelka et al., 2002; Brancaccio et al., 2009). These systems are based on the registration of pulse properties (e.g. amplitude voltages) and their corresponding accurate timestamps. Correspondence to: Sindulfo Ayuso (sindulfo.ayuso@edu.uah.es) The recorded data are then processed by software in order to ob- tain the coincidence counting rates. In this work we present a new software-based coincidence system capable of real-time operation in ground-based cosmic ray detection systems. Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 www.geosci-instrum-method-data-syst.net/5/437/2016/ doi:10.5194/gi-5-437-2016 © Author(s) 2016. CC Attribution 3.0 License. 1 Introduction Cosmic rays (CRs) are energetic particles that constantly rain through the Earth’s atmosphere. They are the source of a uni- form background ionizing radiation. Most of the CR energy reaches the Earth’s surface in the form of kinetic energy of relativistic muons, which are secondary products of interac- Electronic chains based on the Nuclear Instrumentation Module (NIM) standard (US NIM Committee, 1978) are widely used by many experimental particle physics laborato- ries around the world. Two of the most important advantages 1Castilla-La Mancha Neutron Monitor, Space Research Group, Parque Cientíﬁco y Tecnológico de Castilla-La Mancha, Avda. Buendía 11, 19005 Guadalajara, Spain 1Castilla-La Mancha Neutron Monitor, Space Research Group, Parque Cientíﬁco y Tecnológico de Castilla-La Mancha, Avda. Buendía 11, 19005 Guadalajara, Spain 2Physics Department, Space Research Group, Universidad de Alcalá, Ctra. Madrid-Barcelona km 33.6, 28871 Alcalá de Henares, Spain 3Computing Engineering Department, Space Research Group, Universidad de Alcalá, Ctra. Madrid-Barcelona km 33.6, 28871 Alcalá de Henares, Spain Correspondence to: Sindulfo Ayuso (sindulfo.ayuso@edu.uah.es) Sindulfo Ayuso1, Juan José Blanco1,2, José Medina1, Raúl Gómez-Herrero1,2, Oscar García-Población1,3, and Ignacio García Tejedor1,3 Sindulfo Ayuso1, Juan José Blanco1,2, José Medina1, Raúl Gómez-Herrero1,2, Oscar García-Población1,3, and Ignacio García Tejedor1,3 2.1 Muon detectors We have used two different muon detection systems based on plastic scintillators. The ﬁrst device (henceforth MD1, Fig. 1) consists of a pile of two identical detectors separated by 8.5 cm. The gap between both detection layers is partially occupied by a 30 cm × 30 cm × 5 cm lead block, which re- jects low-energy particles (Chilingarian et al., 2009b), ensur- ing that only cosmic muons are detected when coincidence between both detectors is applied. Each detector is an opaque sealed box, with a 30 cm × 30 cm × 3 cm plastic scintillator on the bottom and a photomultiplier tube (PMT) type 53 AVP (Philips, 1959) on the top, which are 18 cm apart. This dis- tance is required to be sure that the PMT observes the whole scintillator surface. The upper detector and the lead block can be moved horizontally, allowing different ﬁelds of view for testing the directionality of muon ﬂux. In contrast, recent advances in microelectronics have put on the market low-cost and small-size devices with high per- formance (Arduino, Raspberry Pi, Beaglebone Black, etc.). Some of them are open-source hardware and run open-source operating systems like Linux, which confer them with a great versatility to satisfy different user requirements. More- over, they usually include many general purpose inputs out- puts (GPIOs), which are very useful for implementing com- munication protocols with other electronic devices like one or more ADCs. The goals of this work are, ﬁrstly, the establishment of the theoretical background and conditions allowing software- based real-time coincidence detection (Sect. 3); secondly, the prototype implementation with a low-cost development plat- form and minimal and simple hardware and software designs (Sect. 4); thirdly, the validation of an operation extracting data from a muon telescope (Sect. 5); and, ﬁnally, the pro- totype testing in two practical applications (Sect. 6). In ad- dition, we will see how our prototype is able to replace at least three NIM modules used in the conventional set-up for coincidence detection. We can see in Fig. 2 the variation of the counter response with the bias voltage for both PMTs used in the experiment. Bearing in mind they work in coincidence coupled to identi- cal scintillators, we have chosen 1270 V as the optimal value because it is the point where both PMTs have the same re- sponse within the plateau. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. A coincidence detection system based on real-time software Figure 2. Counting rate vs. voltage for PMT type 53AVP (Philips). Note the plateau below 1500 V and the crossing point of both curves in 1270 V (bias voltage chosen). S. Ayuso et al.: A coincidence detection system based on real-time software 438 Figure 1. Muon telescope. Main parts. Figure 2. Counting rate vs. voltage for PMT type 53AVP (Philips). Note the plateau below 1500 V and the crossing point of both curves in 1270 V (bias voltage chosen). 2 Experimental set-up Figure 1. Muon telescope. Main parts. In this section we describe the different elements that have been used in our experiment, mainly two muon detectors and some NIM modules, and how they have been set up to achieve the results presented in this paper. of the NIM concepts are ﬂexibility and interchangeability. Although NIM modules cannot communicate with each other through the crate backplane, some modules, like analogue- to-digital converters (ADCs), provide their own interface to communicate with external devices. Nowadays, suppliers of- fer updated replacements that can read data from ADCs and transfer them to a personal computer (PC), including analy- sis and data mining software. Their main disadvantages are high cost and the fact that they are not open-source systems. 2.1 Muon detectors The second device (henceforth MD2) is made up of a large-area plastic scintillator (100 cm × 100 cm × 5 cm, polyvinyletoluene with 65 % anthracene), three PMTs gath- ering the light emerging through three of four lateral sides and a fourth small bismuth germanate (BGO) scintillator (hexagonal prism of 3 cm side and a height of 2 cm) working in coincidence with the other three PMTs. This experimental Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 2.4 ADCs The ADC communication protocol is not described in the NIM standard; however, several manufacturers (CAN- BERRA, FAST, etc.) follow the same basic protocol in their communication lines. In order to understand our software- coincidence basis, the ADC data acquisition process is brieﬂy described below. set-up operating in quadruple coincidence selects muon tra- jectories crossing the BGO, which can be moved over the sur- face in order to calibrate a position-sensitive detection sys- tem currently under development (Fig. 3). The ADC can work in two modes: coincidence mode (CO- INC) and anticoincidence mode (ANTI). When it works in COINC mode (Fig. 4), input pulse conversions must be en- abled or disabled by using the GATE input signal. If the GATE input is low, conversion will not take place. www.geosci-instrum-method-data-syst.net/5/437/2016/ Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 www.geosci-instrum-method-data-syst.net/5/437/2016/ S. Ayuso et al.: A coincidence detection system based on real-time software S. Ayuso et al.: A coincidence detection system based on real-time software 439 Figure 3. Main scintillator (100 cm × 100 cm × 5 cm). Four PMTs inside its pyramidal guides gather the light from lateral sides of the scintillator. The BGO inside the little black box can be moved over the scintillator surface. The BGO and three PMTs work in coin- cidence; thus, only the muon trajectories crossing the BGO will be detected and the amplitude of PMTs pulses will carry position infor- mation. The BGO is used to calibrate the system. The whole system is located inside a closed dark chamber. Preampl if ier Amp lif ier Detector SCA ADC GATE DAS 11001101 PC SCA: Single Channel Analyzer. DAS: Data Acquisition System. Figure 4. Data acquisition block diagram with NIM modules. ADC working in coincidence mode. SCA: Single Channel Analyzer. Figure 4. Data acquisition block diagram with NIM modules. ADC working in coincidence mode. Figure 3. Main scintillator (100 cm × 100 cm × 5 cm). Four PMTs inside its pyramidal guides gather the light from lateral sides of the scintillator. The BGO inside the little black box can be moved over the scintillator surface. The BGO and three PMTs work in coin- cidence; thus, only the muon trajectories crossing the BGO will be detected and the amplitude of PMTs pulses will carry position infor- mation. The BGO is used to calibrate the system. The whole system is located inside a closed dark chamber. 2.2 NIM ampliﬁcation chains When it works in ANTI mode, the SCA is not required; the ADC performs peak detection on the signal and provides in its output this maximum as a digital value. The Lower Level Discriminator (LLD) and Upper Level Discriminator (ULD) potentiometers set the limits for the input signal amplitude to be accepted by the ADC for conversion. If an input pulse falls within these limits, the ADC starts the conversion pro- cess. When the conversion process has ﬁnished, the Data Ready (DR) signal is activated. When an error occurs in the conversion process, the Invalid (INV) line is activated, DR stays inactive and the process is aborted (this is impor- tant for the Sect. 5 discussion). After reading data, the exter- nal system (the SAS in our scenario) activates the Data Ac- cepted (DA) line, which resets the ADC, leaving it ready for a new conversion. Once the ADC has started the conversion and up to the DA signal activation, the signal input remains disabled and therefore ignored (see Fig. 5). NIM standardization provides users with the ability to in- terchange modules and the ﬂexibility to reconﬁgure or ex- pand nuclear counting systems as their counting applications change or grow. A typical conﬁguration of a single NIM am- pliﬁcation chain with the main modules used in this work can be seen in Fig. 4. The detector signal is ampliﬁed by the preampliﬁer and ampliﬁer, which also stretches the pulses, making the ADC conversion easier. When the signal ampli- tude level is between two preconﬁgured values, the Single Channel Analyzer (SCA) generates a pulse that triggers the ADC conversion when the ADC is working in “Coincidence mode”. The Data Acquisition System (DAS) reads and pro- cesses this value and transfers the data to the PC. 2.3 Software-coincidence Acquisition System (SAS) A new device has been designed and built in order to ac- quire data from several NIM chains and perform a coinci- dence policy by means of real-time software, taking advan- tage of the characteristics of low-cost card-sized embedded- processor platforms. It is also able to store the pulse heights for each separate chain. This device has been validated for our muon detectors based on scintillators with different ar- eas (up to 1 m2). We name this acquisition system SAS (Software-coincidence Acquisition System). Its design and implementation are described in Sect. 4. As we will see in Sect. 3.2, the ADC conversion time is needed to perform the software-based coincidence detection code. In this work, the CANBERRA ADC model 8075 was used and, according to the ADC operator’s manual (CAN- BERRA Industries, 1983), the conversion time is given by (1) t[µs] = 1.5 + 0.01(N + X), (1) where N is the channel number (quantization) and X is a selected number for “digital offset” control. In this work the “digital offset” control has not been used (X = 0) and the channel number has been ﬁxed to N = 1024 Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 www.geosci-instrum-method-data-syst.net/5/437/2016/ www.geosci-instrum-method-data-syst.net/5/437/2016/ S. Ayuso et al.: A coincidence detection system based on real-time software 440 440 S. Ayuso et al.: A coincidence detection system based on real-time software t LLD Input signal ULD Pulse detection Conversion process Data ready (active low) Conversion error Conversion OK DR inactive DR active Read data Data accepted (active low) reset DA active Input enable Enabled Disabled Enabled Disabled Enabled Read Figure 5. ADC conversion process. The ADC detects a peak when the input signal rises above the LLD threshold and below the ULD threshold. The detection process ends when the input pulse falls below 90 % of its peak amplitude. In that moment, the signal input is disabled and the conversion process starts. If an error occurs in the conversion process, the DR signal remains inactive and the input signal enabled again. If the conversion process is OK, the DR is activated, and the Data Acquisition System reads the data and activates the DA signal, which causes the DA to go to inactive and the signal input to be enabled again. t LLD Input signal ULD Pulse detection Conversion process Data ready (active low) Conversion error Conversion OK DR inactive DR active Read data Data accepted (active low) reset DA active Input enable Enabled Disabled Enabled Disabled Enabled Read Figure 5. ADC conversion process. The ADC detects a peak when the input signal rises above the LLD threshold and below the ULD threshold. The detection process ends when the input pulse falls below 90 % of its peak amplitude. In that moment, the signal input is disabled and the conversion process starts. If an error occurs in the conversion process, the DR signal remains inactive and the input signal enabled again. If the conversion process is OK, the DR is activated, and the Data Acquisition System reads the data and activates the DA signal, which causes the DA to go to inactive and the signal input to be enabled again. USB Osciloscope ADC SAS Data ready signal PC Pulser Figure 6. Block diagram used to verify the ADC conversion time. PC is only used to launch and stop SAS software. USB Osciloscope ADC SAS Data ready signal PC Pulser Figure 6. Block diagram used to verify the ADC conversion time. PC is only used to launch and stop SAS software. Figure 7. CANBERRA 8075 ADC conversion time. www.geosci-instrum-method-data-syst.net/5/437/2016/ Values be- tween 7.2 and 16 µs with an error rate of ±0.2 µs. Figure 6. Block diagram used to verify the ADC conversion time. PC is only used to launch and stop SAS software. (10 bits) because higher precision is not needed, so the con- version should always take 11.74 µs. In order to verify the time before writing the ﬁrst version of the software, the conversion time with 10 bits of resolu- tion (1024 channels) was experimentally measured with the set-up shown in Fig. 6. As can be seen, the pulse generator output is connected to both the oscilloscope and the ADC, with its output in turn connected to the SAS. When the SAS completes a single reading, it asserts the Data Ready signal. The total conversion time was determined by measuring the time difference between the pulse from the pulse generator and the pulse from the Data Ready signal. Figure 7. CANBERRA 8075 ADC conversion time. Values be- tween 7.2 and 16 µs with an error rate of ±0.2 µs. 3 Coincidence Particle detection systems are often based on multiple de- tection layers operating in coincidence. These coincidence- based systems may provide relevant physical information such as particle identiﬁcation by the use of dE vs. dE or dE vs. E techniques (Del Peral et al., 1995) or by means of some shielding block between pilled detectors (Chilingar- ian et al., 2009a), the particle impact point on the detector surface (Hasebe et al., 1988) and particle energy deposition in detectors or incident direction (Karapetyan et al., 2013). Moreover, coincidence systems are used in different research The pulse level was adjusted to ﬁve different values be- tween 0 and 10 V (minimum and maximum input voltage al- lowed) and the conversion time was measured for each of them. The results are shown in Fig. 7. As we can see, the higher the pulse height, the larger the conversion time. In this work, only minimum and maximum conversion times were needed. www.geosci-instrum-method-data-syst.net/5/437/2016/ www.geosci-instrum-method-data-syst.net/5/437/2016/ Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 S. Ayuso et al.: A coincidence detection system based on real-time software 441 Table 1. Processing time of some tasks. In bold, the task to know the status of a GPIO. Executed tasks Iteration Total Average number time time per (s) iteration (µs) Setting value of one GPIO 107 34 3.4 µs Reading only one GPIO 107 42 4.2µs Reading and writing one GPIO 107 87 8.7 µs Reading and writing two GPIOs 107 162 16.2 µs Discriminator Detector 1 ADC Gate SAS Data Detector 2 ADC Gate Data & PC Discriminator Figure 8. Hardware coincidence detector block diagram. The AND gate is the core of the circuit; its output becomes active when both inputs are activated. Here, the SAS only acquires and records data. It does not work as a coincidence system. The PC is only used to launch and stop the SAS software. S. Ayuso et al.: A coincidence detection system based on real-time software Table 1. Processing time of some tasks. In bold, the task to know the status of a GPIO. Executed tasks Iteration Total Average number time time per (s) iteration (µs) Setting value of one GPIO 107 34 3.4 µs Reading only one GPIO 107 42 4.2µs Reading and writing one GPIO 107 87 8.7 µs Reading and writing two GPIOs 107 162 16.2 µs Discriminator Detector 1 Detector 2 & Discriminator Figure 8. Hardware coinc gate is the core of the cir inputs are activated. Here It does not work as a coi launch and stop the SAS S. Ayuso et al.: A coincidence detection system based on real-time software 441 Discriminator Detector 1 ADC Gate SAS Data Detector 2 ADC Gate Data & PC Discriminator Figure 8. Hardware coincidence detector block diagram. The AND gate is the core of the circuit; its output becomes active when both inputs are activated. Here, the SAS only acquires and records data. It does not work as a coincidence system. The PC is only used to launch and stop the SAS software. Discriminator Detector 1 ADC Gate SAS Data Detector 2 ADC Gate Data & PC Discriminator Figure 8. Hardware coincidence detector block diagram. The AND gate is the core of the circuit; its output becomes active when both inputs are activated. Here, the SAS only acquires and records data. 3.1 Hardware coincidence A hardware coincidence circuit is an electronic device with one output and two (or more) inputs. The output is acti- vated only when all input signals are received within a cer- tain time window. Figure 8 shows a typical coincidence cir- cuit, where the output of the AND gate triggers the ADCs’ conversion process. This circuit is appropriate for detecting coincidence because of its high speed of operation, since the AND gate switching time is only a few nanoseconds (Texas Instruments, 2010). The process to determine coincidence between two pulses is as follows: the ADC starts the conversion after the lead- ing edge of the input pulse surpasses the LLD setting. If both ADCs receive pulses in coincidence, they will start the con- version at the same time. After ﬁnishing the conversion pro- cess, each ADC activates its respective DR signals, which are detected by the SAS. This will decide whether or not coinci- dence has happened, depending on the time elapsed between both DR signals. www.geosci-instrum-method-data-syst.net/5/437/2016/ It does not work as a coincidence system. The PC is only used to launch and stop the SAS software. areas such as medical applications, quantum physics or op- tics (Joost and Salomon, 2015). Relativistic particles, such as high-energy muons, require less than a few nanoseconds to go through two scintillator layers separated by 1 m (Remmen and McCreary, 2012). A coincidence in this case is therefore deﬁned by pulses from both scintillators detected within a time window of a few nanoseconds. dence processing. This is the basic working principle of our new software coincidence system. As a matter of fact, the to- tal muon ﬂux crossing unit horizontal area from above, at sea level, is approximately one muon per minute per cm2 and re- mains fairly constant over time (Grieder, 2010). Thus, one of our MD1 muon scintillators (30 × 30 cm) would detect about 15 muons s−1 (900 muons min−1). Given that our telescope is located at 708 m a.s.l. (above sea level), it will detect less than 18 muons per second (Ramesh et al., 2011), that is, on average, 1 muon every 55.6 ms, and, this period is at least 3 orders of magnitude above the software processing time (25.2 µs in the worst case, as will be seen further on). This is the basis for demonstrating the feasibility of a software- based coincidence system for low-rate applications. 3.2 Software coincidence We deﬁne software coincidence as the ability to detect coin- cidence by means of a program running in a CPU-based sys- tem. A priori, software-based real-time coincidence seems unfeasible because the CPU has to be shared with the under- lying operating system. If this is a general purpose operat- ing system, and therefore it has no real-time capabilities, it is impossible to establish deterministically whether the ac- quisition activities will be executed on time. Therefore, the average access time to the hardware has to be taken into ac- count when our software is running. Commercially available embedded development platforms like that used in this work require a time in the range of microseconds to read and com- pare two GPIOs (see Sect. 4.2.1 for more detail). This time is several orders of magnitude longer than the time required by a relativistic muon to cross through two stacked detec- tors. However, if the particle ﬂux is steady and low enough (like that of muon ﬂux), the average time elapsed between the detection of two incident particles will be well above the software processing time, thus allowing for software coinci- As seen in Fig. 7, conversion time depends on the pulse amplitude, so the difference between both conversion times (both DR enables) can be up to 8.8 µs (16–7.2 µs). In order to solve the coincidence problem, the software pools both DR lines continuously. When a DR goes active, the software waits enough time to be sure that the conversion of the sec- ond ADC has ﬁnished. Then, it checks the state of the second ADC DR line (DR2). If it has been activated, the software concludes that there is coincidence and the data from both ADC are recorded. After that, the software resets both ADCs sending a DA and the system is again ready for a new event. Otherwise, if DR2 is not active, the software considers that there is no coincidence and sends a DA (reset) to both ADCs without recording the data. The SAS always sends the DA to both ADC simultaneously (they are both connected at the same circuit wire) to ensure that they are always reset, and Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 S. Ayuso et al.: A coincidence detection system based on real-time software Probability of detecting as coinci- dent two different muons in a period of δt = 25 µs as a function of muon ﬂux (lower axis) or scintillator area (upper axis) at sea level. (a) ADC2 ADC1 Muon 1 16 μs 7.2 μs Reset Muon 2 4.2 μs 8.8 μs DR2 check DR1 check t (μs) (a) t (μs) ADC2 ADC1 Muon 1 16 μs 7.2 μs Reset Muon 2 (b) 21.4 μs 4.2 μs 4.2 μs 4.2 μs DR2 check DR2 check DR1 check DR1 check (b) Figure 10. Poisson distribution. Probability of detecting as coinci- dent two different muons in a period of δt = 25 µs as a function of muon ﬂux (lower axis) or scintillator area (upper axis) at sea level. DR1 check: ADC1 Data Ready checking instant. DR2 check: ADC2 Data Ready checking instant In red, time from ADC end of conversion to DA (reset) activation. Figure 9. Different muons considered like a single particle (coinci- dent) because of ADC conversion time (blue and green) and soft- ware checking time (4.2 µs between DR1 and DR2). In red, the time from the ADC end of conversion to DA activation. DR1 check: ADC1 Data Ready checking instant. DR2 check: ADC2 Data Ready checking instant. (a) Muon 1 generates the maximum height pulse in ADC2 and takes the maximum conversion time, whereas muon 2 generates the minimum height pulse in ADC1 and takes the min- imum conversion time. Both ADCs ﬁnish conversion at the same time and the SAS considers a single muon. (b) Muon 1: maximum conversion time in ADC1. Muon 2: minimum conversion time in ADC2. In the worst case, taking into account the checking time and the waiting time to ensure the ADC end of conversion, muon 2 can arrive up to 21.4 µs later than muon 1, and the SAS considers both muons as a single muon. secutive muons that would be erroneously counted as coinci- dent is P = 1.03 × 10−7. Figure 10 shows the Poisson probability for k = 2 muons and a time window δt = 25 µs according to scintillator area or muon count rate at sea level. As we can see, using scintil- lators with areas up to 1 m2, the probability of taking as coin- cident two different muons is negligible. Thus, this software coincidence technique can be applied accepting a minimal number of errors. 4 SAS design and implementation The design and implementation of the SAS prototype can be split into two well-differentiated parts: hardware and soft- ware. S. Ayuso et al.: A coincidence detection system based on real-time software In order to reduce the number of wrong co- incidences as much as possible, the acquisition chains must be adjusted (discrimination levels of ADC, LLD and ULD) in such a way as the particle detected is in the muon en- ergy range, avoiding noise and other particles which would increase the total ﬂux. they start the waiting for a new input pulse at the same time, as suggested by Medina (1987). As mentioned above, the use of software coincidence is limited by the probability of false coincidence we are willing to accept. For low count rates such as those of muon ground- based detectors, our prototype can work with most available scintillators (areas up to 3 m2 with probability of false coin- cidence = 1.1 × 10−4). Obviously, the received pulses may correspond to differ- ent muon arrivals up to 8.8 µs apart and the software could declare them coincident (Fig. 9a). Moreover, taking into ac- count the pooling time (4.2 µs between each DR; see Table 1 and Sect. 4.2.2 below), particles up to 21.4 µs apart (Fig. 9b) could be considered coincident. Actually, in order to guaran- tee that both ADC conversions have ﬁnished, our software waits 25.2 µs (see Sect. 4.2.2). However, in our muon tele- scope (MD1) it is highly unlikely to have two or more muon arrivals in 25.2 µs because of its steady ﬂux with an average rate of one muon every 55.6 ms. As the arrival of these par- ticles has a random and independent behaviour, it follows a Poisson distribution, and the probability of detecting k con- secutive muons in a δt time window is given by S. Ayuso et al.: A coincidence detection system based on real-time software 442 A coincidence detection system based on real-time software Figure 10. Poisson distribution. Probability of detecting as coinci- dent two different muons in a period of δt = 25 µs as a function of muon ﬂux (lower axis) or scintillator area (upper axis) at sea level. 442 S. Ayuso et al.: t (μs) ADC2 ADC1 Muon 1 16 μs 7.2 μs Reset Muon 2 (b) 21.4 μs 4.2 μs 4.2 μs 4.2 μs DR2 check DR2 check DR1 check DR1 check (a) ADC2 ADC1 Muon 1 16 μs 7.2 μs Reset Muon 2 4.2 μs 8.8 μs DR2 check DR1 check t (μs) DR1 check: ADC1 Data Ready checking instant. DR2 check: ADC2 Data Ready checking instant In red, time from ADC end of conversion to DA (reset) activation. Figure 9. Different muons considered like a single particle (coinci- dent) because of ADC conversion time (blue and green) and soft- ware checking time (4.2 µs between DR1 and DR2). In red, the time from the ADC end of conversion to DA activation. DR1 check: ADC1 Data Ready checking instant. DR2 check: ADC2 Data Ready checking instant. (a) Muon 1 generates the maximum height pulse in ADC2 and takes the maximum conversion time, whereas muon 2 generates the minimum height pulse in ADC1 and takes the min- imum conversion time. Both ADCs ﬁnish conversion at the same time and the SAS considers a single muon. (b) Muon 1: maximum conversion time in ADC1. Muon 2: minimum conversion time in ADC2. In the worst case, taking into account the checking time and the waiting time to ensure the ADC end of conversion, muon 2 can arrive up to 21.4 µs later than muon 1, and the SAS considers both muons as a single muon. t (μs) ADC2 ADC1 Muon 1 16 μs 7.2 μs Reset Muon 2 (b) 21.4 μs 4.2 μs 4.2 μs 4.2 μs DR2 check DR2 check DR1 check DR1 check (a) ADC2 ADC1 Muon 1 16 μs 7.2 μs Reset Muon 2 4.2 μs 8.8 μs DR2 check DR1 check t (μs) DR1 check: ADC1 Data Ready checking instant. DR2 check: ADC2 Data Ready checking instant In red, time from ADC end of conversion to DA (reset) activation. Figure 9. Different muons considered like a single particle (coinci- dent) because of ADC conversion time (blue and green) and soft- ware checking time (4.2 µs between DR1 and DR2). www.geosci-instrum-method-data-syst.net/5/437/2016/ www.geosci-instrum-method-data-syst.net/5/437/2016/ S. Ayuso et al.: A coincidence detection system based on real-time software Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 4.1 Hardware The hardware implementation involved, ﬁrstly, the selection of the processing platform, secondly, the design and building of the interface card and, ﬁnally, the box assembly. P(k,δt) = (λδt)k · e−λδt k! , (2) (2) where λ is the mean number of muons per second. where λ is the mean number of muons per second. Considering λ = 18 muons s−1, k = 2 muons and a time window of δt = 25.2 µs, the probability of having two con- S. Ayuso et al.: A coincidence detection system based on real-time software In red, the time from the ADC end of conversion to DA activation. DR1 check: ADC1 Data Ready checking instant. DR2 check: ADC2 Data Ready checking instant. (a) Muon 1 generates the maximum height pulse in ADC2 and takes the maximum conversion time, whereas muon 2 generates the minimum height pulse in ADC1 and takes the min- imum conversion time. Both ADCs ﬁnish conversion at the same time and the SAS considers a single muon. (b) Muon 1: maximum conversion time in ADC1. Muon 2: minimum conversion time in ADC2. In the worst case, taking into account the checking time and the waiting time to ensure the ADC end of conversion, muon 2 can arrive up to 21.4 µs later than muon 1, and the SAS considers both muons as a single muon. Figure 10. Poisson distribution. Probability of detecting as dent two different muons in a period of δt = 25 µs as a fun muon ﬂux (lower axis) or scintillator area (upper axis) at se secutive muons that would be erroneously counted as dent is P = 1.03 × 10−7. Figure 10 shows the Poisson probability for k = 2 and a time window δt = 25 µs according to scintillat or muon count rate at sea level. As we can see, using lators with areas up to 1 m2, the probability of taking a cident two different muons is negligible. Thus, this so coincidence technique can be applied accepting a m number of errors. In order to reduce the number of wro incidences as much as possible, the acquisition chain be adjusted (discrimination levels of ADC, LLD and t (μs) ADC2 ADC1 Muon 1 16 μs 7.2 μs Reset Muon 2 (b) 21.4 μs 4.2 μs 4.2 μs 4.2 μs DR2 check DR2 check DR1 check DR1 check (a) ADC2 ADC1 Muon 1 16 μs 7.2 μs Reset Muon 2 4.2 μs 8.8 μs DR2 check DR1 check t (μs) DR1 check: ADC1 Data Ready checking instant. DR2 check: ADC2 Data Ready checking instant In red, time from ADC end of conversion to DA (reset) activation. Figure 9. Different muons considered like a single particle (coinci- dent) because of ADC conversion time (blue and green) and soft- ware checking time (4.2 µs between DR1 and DR2). In red, the Figure 10. Poisson distribution. 4.1.2 Interface card and box – Applying software-based coincidence detection. – Storing the data on a micro-SD card with a time tag (hour, minute, second and millisecond) and number of registered data per minute. An interface card and a box have been designed and imple- mented (Fig. 11) taking into account the technical speciﬁca- tions of BBB and its processor manufacturers (Cooley, 2014; Texas Instruments, 2013, 2014). The interface is based on the 74LVC245 tri-state transceiver (IDT, 1999), which pro- vides electrical isolation between the BBB processor and ex- ternal devices (ADCs), 3.3 to 5 V voltage level conversion and buffered signals. 4.1.1 Hardware platform Figure 11. Left panel: interface with two DB25 connectors. Right panel: interface mounted on the process platform (Beaglebone Black) headers. In order to minimize the time employed in design and imple- mentation tasks, we have taken full advantage of the avail- able commercial platform performances. Nowadays, dozens of card-sized embedded processor systems can be purchased with different input and output possibilities. Beaglebone Black (BBB) was chosen for the following reasons: Figure 11. Left panel: interface with two DB25 connectors. Right panel: interface mounted on the process platform (Beaglebone Black) headers. – a great number of GPIO and connection possibilities. We need 37 GPIOs in this work (Table 2); – high processing power; – Enabling the transceivers of the interface after booting the system. – low power consumption; – Communicating with the ADCs using their protocol. – includes a micro-SD card slot. It is used to store all pro- cessed data. – Converting ADC binary data to their decimal value. S. Ayuso et al.: A coincidence detection system based on real-time software 443 Table 2. GPIOs required by the processor card to control the ADC. We need 18 lines to read data and control one ADC. Another line is needed to enable and disable the interface buffers, which has been added to protect the processor card. Name Acro. No. of lines Description Data D 13 Binary data value Data Ready DR 1 Active when conversion is complete Invalid INV 1 Conversion error Overﬂow OVF 1 Value exceeds ULD settings Enable Data ED 1 Gate the 13-bit data onto the output lines. Data Accepted DA 1 Acknowledgment of data acceptance. Reset the ADC. TOTAL lines 1 ADC 18 Line to enable interface buffers 1 TOTAL to control 2 ADCs 37 18 + 18 + 1 4.2.1 Processing time After the software development, it was necessary to know the time required to accomplish several tasks, such as read- ing or writing a bit in a GPIO. As we have seen in Sect. 3.2, to establish the duration of the coincidence time window (in which we consider two pulses as coincident) is a critical de- cision. Since it must be as short as possible, the software that veriﬁes DR signals must also be as fast as possible. For this reason, after writing code, the execution times of the differ- ent routines were veriﬁed, revised and optimized to achieve the best results. www.geosci-instrum-method-data-syst.net/5/437/2016/ www.geosci-instrum-method-data-syst.net/5/437/2016/ Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 www.geosci-instrum-method-data-syst.net/5/437/2016/ Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 4.2 SAS software The BBB used in this work (revision B) was delivered with the Angstrom distribution of the Linux operating system. Our software has been developed in C++ and it is compiled in the BBB itself. It performs the following tasks. – GPIO conﬁguration. To access any external device through GPIO, it must be conﬁgured by means of a de- vice structure system called “Device Tree”. It has its own language to describe which devices should be made available (Power.OrgTM, 2011). – GPIO conﬁguration. To access any external device through GPIO, it must be conﬁgured by means of a de- vice structure system called “Device Tree”. It has its own language to describe which devices should be made available (Power.OrgTM, 2011). In order to measure the time spent on each routine, the software was adapted to make 10 million iterations of a sin- gle task. Thus, the average time of every task was estimated from the total time to run all the iterations (Table 1). In this Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 5 SAS experimental validation To validate the SAS reliability and proper functioning, sev- eral data acquisition experiments with the MD1 muon tele- scope (see Sect. 2.1) were performed. Both, hardware and software coincidence conﬁgurations, were simultaneously tested and their results were compared. To make comparisons between both types of coincidence detection systems, we acquired data during 1 day with the experimental set-up shown in Fig. 13. Obviously, the data registered by both software and hardware coincidence chains should be identical. Figure 14 shows the corresponding his- tograms produced, which are nearly identical, showing only a minor difference in the total amount of data acquired by both systems (0.05 %). Figure 13 shows the experimental set-up operating in hard- ware (blue background block) and software coincidence (red background block) simultaneously. In hardware coincidence, the SAS is used to store data, so it does not work as a coin- cidence detector module, and that is the reason why its soft- ware has been slightly simpliﬁed; it waits for the activation of both DR signals to read and store data, and then resets the ADCs. y Although this difference may be considered negligible, further tests were performed in order to ﬁnd out the origin of this discrepancy. Sometimes, the ADC conversion process produces errors and conversion is aborted (see Sect. 2.4). In these cases, the DR signal is not generated and the INV sig- nal activated, which causes data not to be registered. An ad hoc code was written to register the INV signal and several samples were taken and analysed. As can be seen in Fig. 15, an error causes the INV activation in the hardware coinci- dence chain. However, the software coincidence prototype stores the correct value because its ADCs have not produced conversion errors. In normal operation, these hardware co- incidence data would not be registered and, as a result, the total amount of hardware coincidence data would be lower than the one registered with software coincidence. That is the origin of the small difference between the histograms cor- responding to hardware and software coincidence shown in The software coincidence block shows how this conﬁgu- ration is signiﬁcantly simpler than the one based on hard- ware coincidence, saving three modules: two SCAs and one coincidence detector module (yellow modules in Fig. 13). Moreover, the SAS has the capability of storing and trans- ferring data to a PC, avoiding the use of an interface module. 4.2.2 Software coincidence detection In the worst case, after detecting the ﬁrst ADC end of conversion (b), the SAS must wait to check the DR signal another two times (every time takes 8.4 µs) in order to always ensure the second ADC end of conversion. www.geosci-instrum-method-data-syst.net/5/437/2016/ www.geosci-instrum-method-data-syst.net/5/437/2016/ S. Ayuso et al.: A coincidence detection system based on real-time software S. Ayuso et al.: A coincidence detection system based on real-time software 444 coincidence detection system based on real-time software t (μs) t (μs) DR1 check DR2 check ADC2 ADC1 Muon 16 μs 7.2 μs Reset (a) ADC2 ADC1 Muon 16 μs 7.2 μs Reset (b) 4.2 μs 4.2 μs 4.2 μs 4.2 μs 4.2 μs 4.6 μs 4.2 μs DR1 check DR2 check DR1 check DR1 check DR2 check DR1 check DR2 check 8.8 μs DR2 check 4.2 μs 4.2 μs 4.2 μs 25.2 μs Reset DR1 check: ADC1 Data Ready checking instant. DR2 check: ADC2 Data Ready checking instant In red, time from ADC end of conversion to DA (reset) activation. t (μs) DR1 check DR2 check ADC2 ADC1 Muon 16 μs 7.2 μs Reset (a) 4.2 μs 4.2 μs 4.6 μs 4.2 μs DR1 check DR2 check work, the most relevant task timings are those to get the cur- rent status of a single GPIO (4.2 µs) and to set the value of a single GPIO (3.4 µs). (a) 5 SAS experimental validation From an economical point of view, the total cost of those four NIM modules is well above EUR 6000 (only one SCA mod- ule costs more than EUR 1600), and the SAS implementation components have a cost of less than EUR 150. So, we can say that the SAS, working in software coincidence, reduces the costs of the laboratory equipment replaced by a factor of 40. Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 4.2.2 Software coincidence detection The incident particle causes simultaneous pulses in the ADC inputs and, therefore, both ADCs start the conversion simul- taneously. Bearing in mind that the software checks DR1 and DR2 sequentially in this order, if DR1 is active and DR2 is inactive in the ﬁrst checking (Fig. 12a), the minimum wait- ing time to guarantee ADC2 end conversion (DR2 active) is 4.2 µs, which means another checking of both DR (8.4 µs). Otherwise, if DR2 is active and DR1 is inactive in the ﬁrst checking (Fig. 12b), the minimum waiting time to ADC1 end conversion is 8.8 µs; therefore, the software must wait to check both DRs twice (16.8 µs). t (μs) ADC2 ADC1 Muon 16 μs 7.2 μs Reset (b) 4.2 μs 4.2 μs 4.2 μs DR1 check DR1 check DR2 check DR1 check DR2 check 8.8 μs DR2 check 4.2 μs 4.2 μs 4.2 μs 25.2 μs Reset (b) Consequently, in order to guarantee that both ADC con- versions have ﬁnished, the software pools the state of both ADC DR lines three times after each reset, which takes it 25.2 µs. The software considers their state only the third time; if one or both are inactive, it sends the DA signal to reset both ADCs and start the cycle again (there is no coincidence). Otherwise, if both DR lines are active, a coincidence has been detected and the data are then stored before resetting both ADCs. This process is repeated over and over again. DR1 check: ADC1 Data Ready checking instant. DR2 check: ADC2 Data Ready checking instant In red, time from ADC end of conversion to DA (reset) activation. Figure 12. Coincidence evaluation. Maximum and minimum conversion times and waiting time to ensure ADC conversion. (a) ADC1 minimum conversion time and ADC2 maximum conver- sion time. (b) ADC2 minimum conversion time and ADC1 max- imum conversion time. In the worst case, after detecting the ﬁrst ADC end of conversion (b), the SAS must wait to check the DR signal another two times (every time takes 8.4 µs) in order to always ensure the second ADC end of conversion. Figure 12. Coincidence evaluation. Maximum and minimum conversion times and waiting time to ensure ADC conversion. (a) ADC1 minimum conversion time and ADC2 maximum conver- sion time. (b) ADC2 minimum conversion time and ADC1 max- imum conversion time. www.geosci-instrum-method-data-syst.net/5/437/2016/ Ayuso et al.: A coincidence detection system based on real-time software In the second one, software coincidence is applied to ensure the pulses collected by the PMTs every time correspond to the same passing muon through a small scintillator area. 6 Applications The software-based coincidence system presented in this work is an effective low-cost replacement for conventional hardware coincidence, valid for low-rate experimental parti- cle detection systems (up to 500 muons s−1 or up to 3 m−2 scintillator area at sea level, using our prototype, with proba- Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 Ayuso et al.: A coincidence detection system based on real-time software S. Ayuso et al.: A coincidence detection system based on real-time software 445 PMT1 PREAMP 1 AMP 1 ADC A Coicidence detector SCA 1 PMT2 PREAMP 2 AMP 2 SCA 2 ADC B ADC 1 ADC 2 SAS 1 SAS 2 GATE Hardware coincidence Software coincidence PREAMP: Preamplifier. AMP: Amplifier. SCA: Single Channel Analyzer. PMT: Photomultiplier. AMP 2 PREAMP 2 PREAMP: Preamplifier. AMP: Amplifier. SCA: Single Channel Analyzer. PMT: Photomultiplier. Figure 13. Schematic set-up for the hardware coincidence and software coincidence results comparison. The same analogue signals detected by PMT1 and PMT2 are introduced into both hardware coincidence and software coincidence chains. Working in hardware coincidence, SAS 1 only stores data from both chains. Working in software coincidence, SAS 2 detects coincidence and stores data. We can see in yellow the unnecessary modules when SAS is working in software coincidence. Figure 14. Comparative tests between data acquired with hardware (left panel) and software (right panel) coincidence by the muon tele- scope (MD1). Black and red lines correspond to the histogram of upper and lower detectors, respectively. Figure 14. Comparative tests between data acquired with hardware (left panel) and software (right panel) coincidence by the muon tele- scope (MD1). Black and red lines correspond to the histogram of upper and lower detectors, respectively. bility of false coincidence = 1.1 × 10−4). In this section two examples of speciﬁc scientiﬁc applications are provided. In the ﬁrst one, using software coincidence counting greatly re- duces the chance of a signal being caused by an event other than the passage of an energetic muon (Ramesh et al., 2011). In the second one, software coincidence is applied to ensure the pulses collected by the PMTs every time correspond to the same passing muon through a small scintillator area. Fig. 14. Therefore, seeing that the rest of the data are similar in time and amplitude values, we conclude that under the ex- perimental conditions used in this work, both kinds of coin- cidence detection systems (hardware and software) produce equivalent results. bility of false coincidence = 1.1 × 10−4). In this section two examples of speciﬁc scientiﬁc applications are provided. In the ﬁrst one, using software coincidence counting greatly re- duces the chance of a signal being caused by an event other than the passage of an energetic muon (Ramesh et al., 2011). www.geosci-instrum-method-data-syst.net/5/437/2016/ www.geosci-instrum-method-data-syst.net/5/437/2016/ Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 S. Ayuso et al.: A coincidence detection system based on real-time software Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 S. Ayuso et al.: A coincidence detection system based on real-time software 446 Figure 16. From top to bottom, muon count rate (black line) and smoothed count rate (red line), neutron count rate (black line) and smoothed count rate (red line), solar wind density, solar wind temperature, solar wind speed, interplanetary magnetic ﬁeld com- ponents and magnetic ﬁeld intensity. “Complex ejecta” refers to a complex solar wind structure composed of different interacting structures like shocks, ICMEs and interaction regions. The vertical purple lines mark the interplanetary shock positions and the ejecta’s limits. Hardware coincidence Software coincidence ADC A ADC B INV A INV B dt dt ADC 1 ADC 2 274 199 0 0 10 10 275 198 593 209 0 0 268 268 597 209 377 538 0 0 128 128 379 539 475 8191 0 1 159 160 478 270 217 285 0 0 2 1 218 285 350 301 0 0 2 2 352 301 125 277 0 0 91 91 125 277 286 222 0 0 15 15 287 221 327 250 0 0 13 13 328 249 346 227 0 0 24 24 347 226 335 596 0 0 4 5 337 598 383 276 0 0 61 60 385 276 ADC A & ADC B: Data from both ADCs in hardware coincidence. Range: 0 1023 ADC 1 & ADC 2: Data from both ADCs in software coincidence. Range: 0–1023 INV A & INV B: Invalid signal. 1 = active (conversion process error). dt: time elapsed from previous event in ms. – ADC A & ADC B: Data from both ADCs in hardware coincidence. Range: 0 1023 ADC 1 & ADC 2: Data from both ADCs in software coincidence. Range: 0–1023 INV A & INV B: Invalid signal. 1 = active (conversion process error). dt: time elapsed from previous event in ms. – ADC A & ADC B: Data from both ADCs in hardware coincidence. Range: 0 1023 ADC 1 & ADC 2: Data from both ADCs in software coincidence. Range: 0–1023 INV A & INV B: Invalid signal. 1 = active (conversion process error). dt: time elapsed from previous event in ms. – Figure 15. Data analysis. We can see the same data acquired in hardware coincidence and software coincidence columns, with an insigniﬁcant difference between values, which is due to ADC con- version. Sometimes, a conversion error is produced in an ADC, the invalid line is activated and the ADC data are out of range. S. Ayuso et al.: A coincidence detection system based on real-time software That is what happens in the fourth row. In yellow, the invalid i2 acti- vated (1) and the ADC2 data value (out of range = 8191). These are not valid data. Although the hardware has detected the coincidence, these data are not registered in the normal acquisition process be- cause the Data Ready signal is not activated. In this fragment there would be one less data in hardware coincidence. This situation is in- herent to ADC’s operation, and it has nothing to do with hardware or software coincidence. Figure 16. From top to bottom, muon count rate (black line) and smoothed count rate (red line), neutron count rate (black line) and smoothed count rate (red line), solar wind density, solar wind temperature, solar wind speed, interplanetary magnetic ﬁeld com- ponents and magnetic ﬁeld intensity. “Complex ejecta” refers to a complex solar wind structure composed of different interacting structures like shocks, ICMEs and interaction regions. The vertical purple lines mark the interplanetary shock positions and the ejecta’s limits. www.geosci-instrum-method-data-syst.net/5/437/2016/ www.geosci-instrum-method-data-syst.net/5/437/2016/ S. Ayuso et al.: A coincidence detection system based on real-time software S. Ayuso et al.: A coincidence detection system based on real-time software Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 6.1 Monitoring solar activity with ground-based cosmic ray counters The muon telescope used in this application (MD1) is in- stalled in the facilities of the Castilla-La Mancha Neutron Monitor (CaLMa) (Medina et al., 2013; Blanco et al., 2015; García-Población et al., 2014). The MD1 and the neutron monitor are located in the same room and their measurements can be directly compared. Neutrons and muons observed at ground level are secondary particles produced by collisions between cosmic rays and atmospheric atoms. The cosmic ray (protons) energy threshold to produce neutrons detected by CaLMa is above 7 GeV because of the geomagnetic location of this neutron monitor, while the energy threshold of pri- mary cosmic rays rises up to higher than 10 GeV for muon production (Duldig, 2000). Transient interplanetary distur- bances associated with solar activity may cause decreases in both the neutron and muon count rates observed on the Earth’s surface, in an event known as Forbush decrease (For- bush, 1938). In order to observe these cosmic ray ﬂux vari- ations, the effect atmospheric pressure variations must be removed from the data using a correction procedure (see e.g. Paschalis et al., 2013, and references therein). on 21 December 2014. The count rate in CaLMa decreased by 6 % with respect to the previous neutron count rate (72.62 Hz on average). This decrease was also observed by the muon telescope, working in software coincidence, as a reduction in the steady muon count rate of about 3 % (7.66 Hz on average). As can be observed in Fig. 16, a sharp decrease is observed in CaLMa after an interplane- tary shock passage that marks the arrival of complex in- terplanetary ejecta (21 December 2014, 18:00 UTC). These complex ejecta seem to be composed of two consecutive in- terplanetary coronal mass ejections (ICMEs) and comprise a second interplanetary shock probably related to a com- pression region created by a fast solar wind stream follow- ing the ejecta. The ﬁrst ICME is listed in the Richardson and Cane ICME list (http://www.srl.caltech.edu/ACE/ASC/ DATA/level3/icmetable2.htm) with limits between 22 De- Figure 16 shows pressure-corrected muon and neutron count rates and plasma and interplanetary magnetic ﬁeld measurements during a Forbush decrease detected by CaLMa www.geosci-instrum-method-data-syst.net/5/437/2016/ Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 S. Ayuso et al.: A coincidence detection system based on real-time software 447 Figure 17. 6.1 Monitoring solar activity with ground-based cosmic ray counters Software coincidence with four PMTs (MD2), three of them placed in the sides of a 100 cm × 100 cm × 5 cm scintillator. The fourth one is set with a 6 cm × 2 cm bismuth germanate scintillator (BGO). Coincident particle tracks pass through a relatively small region of the large scintillator, just under the BGO. When moving the BGO through the big scintillator surface, the histograms shift depending on the distance and the angle formed by the PMT axis and the line between the PMT and scintillator impact place. (a) Conﬁguration sketch with the BGO in the centre of the big scintillator, (b) result histograms of the upper-left conﬁguration and (c) result histograms with different distances and angles. Figure 17. Software coincidence with four PMTs (MD2), three of them placed in the sides of a 100 cm × 100 cm × 5 cm scintillator. The fourth one is set with a 6 cm × 2 cm bismuth germanate scintillator (BGO). Coincident particle tracks pass through a relatively small region of the large scintillator, just under the BGO. When moving the BGO through the big scintillator surface, the histograms shift depending on the distance and the angle formed by the PMT axis and the line between the PMT and scintillator impact place. (a) Conﬁguration sketch with the BGO in the centre of the big scintillator, (b) result histograms of the upper-left conﬁguration and (c) result histograms with different distances and angles. sensitive muon detector (MD2; see Sect. 2.1). The experi- mental set-up uses four PMTs operating in software coinci- dence. Three of them were placed attached to the sides of a plastic scintillator. The fourth PMT was placed inside an opaque box, gathering the light emitted by a small BGO scin- tillator (see Fig. 17a). The BGO can be moved horizontally in order to select only muon trajectories crossing a certain spot over the surface of the plastic scintillator. cember 2014 04:00 and 17:00 UTC, including a smooth mag- netic ﬁeld rotation and low and stable solar wind temper- ature, as can be expected when a well-developed magnetic cloud is observed in the solar wind (ﬁrst shadowed region in Fig. 16). A second rotation in magnetic ﬁeld components is observed between 22 December 1014 17:00 UTC and 23 De- cember 2014 (second shadowed region in Fig. 6.2 Position-sensitive muon detector In this experiment we used our software-based coincidence system to acquire data from a prototype of a position- 6.1 Monitoring solar activity with ground-based cosmic ray counters 16), suggest- ing the presence of a second ICME; however, solar wind properties show less clear signatures (Hidalgo et al., 2013). The signal generated by each PMT was ampliﬁed and in- jected into an ADC to carry out its conversion. The four ADCs were connected to our prototype in order to detect coincidence and to record the pulse heights (see the block diagram in Fig. 18). The BGO was located in different po- sitions on the big scintillator surface and corresponding data were acquired and registered. The good agreement between the muon and neutron data, presented in Fig. 16, validates the software-based coinci- dence system used to acquire the muon data. Both of them show a clear response to the passage of interplanetary distur- bances. The difference in their count rate decreases observed by both instruments in shape (faster, sharper and deeper de- crease in CaLMa) and in magnitude is likely related to the different energy of the primary cosmic ray producing the secondary neutrons and muons observed at ground level, as could be expected when the primary cosmic ray energy threshold for CaLMa (neutrons mainly) is about 7 GeV and, for the muon telescope, about 10 GeV. Figure 17b shows the pulse-height distribution registered by the three lateral PMTs (labelled 1, 2 and 3 in the ﬁgure) when the BGO is located over the centre of the plastic scin- tillator. In this case, the three PMTs observed identical dis- tributions. Figure 17c shows the pulse-height distribution corre- sponding to PMT 1 obtained for three different locations of the BGO. As expected, the distribution is shifted towards larger pulse heights when the BGO is located closer to the PMT. The combined pulse height information from PMTs 1, Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 7 Conclusions Blanco, J. J., Medina, J., García-Población, O., Gomez-Herrero, R., García-Tejedor, I., Ayuso, S., and Catalán, E. J.: CaLMa neutron monitor: current status, ﬁrst observations and fu- ture improvements, J. Phys., 632, 012052, doi:10.1088/1742- 6596/632/1/012052, 2015. A software-coincidence acquisition system (SAS) capable of detecting coincidence by using software and based on a low- cost development platform has been implemented and tested. It works autonomously (i.e. without a dedicated computer), recording data on a micro-SD card and transferring them to a PC through USB or Ethernet connections. In order to evalu- ate the SAS operation in software coincidence in comparison with that of hardware coincidence, several tests have been carried out, acquiring and recording data from both coinci- dence methods simultaneously. The results make it evident that software coincidence is as effective as hardware coinci- dence with a low ﬂux of particles like that of a cosmic ray ground-based muon telescope (scintillator areas up to 3 m2). Brancaccio, F., Silva Dias, M., and Toledo, F.: Development of an analysis methodology applied to 4πβ −γ software coincidence data acquisition system, International Nuclear Atlantic Confer- ence – INAC, 27 September–2 October 2009, Río de Janeiro, Brazil, 2009. CANBERRA Industries: Inc. Model 8075, Analog-to-Digital Con- verter, Operator’s manual, Canberra Industries Inc. Meriden, Connecticut, USA, November 1983. Cecchini, S. and Spurio, M.: Atmospheric muons: experimen- tal aspects, Geosci. Instrum. Method. Data Syst., 1, 185–196, doi:10.5194/gi-1-185-2012, 2012. Furthermore, our software coincidence system has been tested in two different experimental set-ups for cosmic ray muon detection: a two-element muon telescope, requiring single coincidence, and a position-sensitive muon detector requiring quadruple coincidence. The results were entirely satisfactory. The ﬁrst device clearly observed a cosmic ray Forbush decrease, conﬁrmed using neutron monitor data and well correlated with the passage of an interplanetary distur- bance. The second device was able to record different PMT pulse levels, depending on the location of the incident muon tracks. Chilingarian, A., Angelov, C., Arakelyan, K., Arsov, T., Avakyan, K., Chilingarian, S., Hovhannisyan, A., Hovsepyan, G., Hrzina, D., Hovhannisyan, T., Maricic, D., Nishev, A., Tchorbadjieff, A., Kalapov, I., Karapetyan, T., Kozliner, L., Mailyan, B., Reymers, A., Romstajn, I., Rosa, D., Stamenov, J., Tserunyan, S., and Yeghikyan, A.: New Particle Detector Network for Solar Physics and Space Weather research, Procedings of the 31st ICRC, Lodz, 2009a. S. Ayuso et al.: A coincidence detection system based on real-time software 448 PMT 1 PREAMP 1 AMP 1 PMT 2 PREAMP 2 AMP 2 SAS Analogue signal PMT 3 PREAMP 3 AMP 3 PMT 4 PREAMP 4 AMP 4 ADC 1 ADC 2 ADC 3 ADC 4 Digital data PREAMP: Preamplifier. AMP: Amplifier. Figure 18. NIM modules and SAS interconnection block diagram working in software coincidence with four detection chains. The signal from each PMT is ampliﬁed by a preampliﬁer and an am- pliﬁer in order to get the appropriate ADC input level. SAS detects coincidence and registers data. PMT 1 PREAMP 1 AMP 1 PMT 2 PREAMP 2 AMP 2 SAS Analogue signal PMT 3 PREAMP 3 AMP 3 PMT 4 PREAMP 4 AMP 4 ADC 1 ADC 2 ADC 3 ADC 4 Digital data PREAMP: Preamplifier. AMP: Amplifier. This system provides a reliable and low-cost replacement for hardware-based coincidence system modules over 40 times its value. www.geosci-instrum-method-data-syst.net/5/437/2016/ Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 8 Data availability All experimental data used have been deposited in a reliable public repository (doi:10.5281/zenodo.154682). Public data used in Fig. 16: http://www.nmdb.eu/nest/ search.php and http://cdaweb.sci.gsfc.nasa.gov/istp_public/ (satellite: ACE. Options: AC_H0_SWE, AC_H1_MFI). AMP: Amplifier. AMP: Amplifier. Figure 18. NIM modules and SAS interconnection block diagram working in software coincidence with four detection chains. The signal from each PMT is ampliﬁed by a preampliﬁer and an am- pliﬁer in order to get the appropriate ADC input level. SAS detects coincidence and registers data. Acknowledgements. This work has been partially supported by the University of Alcalá through project CCG2014/EXP-013 and by the Ministerio de Ciencia y Tecnología through project ESP2013- 48346-C2-1-R. Acknowledgements. This work has been partially supported by the University of Alcalá through project CCG2014/EXP-013 and by the Ministerio de Ciencia y Tecnología through project ESP2013- 48346-C2-1-R. We would like to thank José Salvador Pérez Bachiller, who helped us in the SAS box mechanical design, machining and assembly. 2 and 3 can be used to reconstruct the location of the particle track. Obviously, this practical application could be carried out with hardware coincidence, but we take advantage of the eas- ier adjustment, simpler connection and lower cost of our soft- ware coincidence. The ﬁnal conﬁguration of this application is now under development. Edited by: L. Eppelbaum Reviewed by: two anonymous referees Edited by: L. Eppelbaum Reviewed by: two anonymous referees Edited by: L. Eppelbaum Reviewed by: two anonymous referees Reviewed by: two anonymous referees Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 7 Conclusions Chilingarian, A., Hovsepyan, G., Arakelyan, K., Chilingaryan, S., Danielyan, V., Avakyan, K., Yeghikyan, A., Reymers, A., and Tserunyan, S.: Space Environmental Viewing and Analysis Net- work (SEVAN), Earth Moon Planet, 104, 195–210, 2009b. Coley, G.: Beaglebone Black system reference manual, Rev B. Ref. BBONEBLK_SRM, Beagleboard.org Foundation, Ock- land Twp, Pennsylvania, USA, https://github.com/CircuitCo/ Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 S. Ayuso et al.: A coincidence detection system based on real-time software 449 BeagleBone-Black/blob/rev_b/BBB_SRM.pdf?raw=true (last access: 17 September 2016), 20 January 2014. Müller-Mellin, R., Kunow, H., Fleixxner, V., Pehlke, E., Rode, E., Röschmann, N., Scharmberg, C., Sierks, H., Rusznyak, P., Mckenna-Lawlor, S., Elendt, I., Sequeiros, J., Meziat, D., Sanchez, S., Medina, J., Del Peral, L., Witte, M., Marsden, R., and Henrion, J.: COSTEP-Comprehensive Suprathermal and En- ergetic Particle Analyzer, Sol. Phys., 162, 483–504, 1995. Del Peral, L., Medina, J., Sanchez, S., Bronchalo, E., Rodriguez- Pacheco, J., Sequeiros, J., and Meziat, D.: Detector system for low-energy cosmic ions study, Nucl. Instrum. Meth. Phys. Res. A, 354, 539–546, 1995. Duldig, M. L.: Muon observations, Space Sci. Rev., 93, 207–226, 2000. Neutron Monitor Database: http://www.nmdb.eu/nest/search.php, last access: 23 September 2016. Forbush, S. E.: On world-wide changes in cosmic ray intensity, Phys. Rev., 54, 975–988, 1938. Paschalis, P., Mavromichalaki, H., Yanke, V., Belov, V., Eroshenco, E., Gerontidou, M., and Koutroumpi, I.: Online application for the barometric coefﬁcient calculation of the NMDB stations, New Astron., 19, 10–18, 2013. García-Población, O., Blanco, J. J., Gómez-Herrero, R., Steigies, C. T., Medina, J., Tejedor, I. G., and Sánchez, S.: Embedded Data Acquisition System for Neutron Monitors, J. Instrum., 9, T08002, doi:10.1088/1748-0221/9/08/T08002, 2014. Philips: Philips Electronic Tube Handbook, part 6: devices for nuclear equipment, 53AVP data sheet, https://frank.pocnet. net/sheets/030/5/53AVP.pdf (last access: 17 September 2016), March 1959. TM ™ Goddard Space Flight Center, Space Physics Data Facility: Coordi- nated Data Analysis Web, http://cdaweb.sci.gsfc.nasa.gov/istp_ public/, last access: 23 September 2016. Power OrgTM: Standard for embedded power architecture™plat- form requirements (ePAPR), https://www.power.org/download_ ﬁle.php?ﬁle=7920 (last access: 17 September 2016), April 2011. Grieder, P. K. F.: Extensive Air Showers, Springer-Verlag, Berlin, Heidelberg, 2010. Hasebe, N., Ezawa, Y., and Yoshii, H.: Charge waveform of a new two-dimensional position-sensitive silicon detector, Jpn. J. Appl. Phys., 27, 816–820, 1988. Ramesh, N., Hawron, M., Martin, C., and Bachri, A.: Flux variation of cosmic muons, J. Arkansas Acad. Sci., 65, 1–6, 2011. Remmen, G. and McCreary, E.: Measurement of the Speed and Energy Distribution of Cosmic Ray Muons, J. Undergrad. Res. Phys., 25, MS138, 2012. Havelka, M., Auerbach, P., and Sochorová, J.: Software coincidence counting, Appl. Radiat. Isot., 56, 265–268, 2002. Texas Instruments: Quadruple 2-input positive-and gate, Data sheet, Ref. SCAS283Q, http://www.ti.com/lit/ds/symlink/sn74lvc08a. pdf (last access: 17 September 2016), Rev. October 2010. ® TM Hidalgo, M. A., Nieves-Chinchilla, T., and Blanco, J. J.: On the Flux-Rope Topology of Ejecta Observed in the Period 1997– 2006, Sol. Phys., 284, 151–166, 2013. www.geosci-instrum-method-data-syst.net/5/437/2016/ www.geosci-instrum-method-data-syst.net/5/437/2016/ S. Ayuso et al.: A coincidence detection system based on real-time software Texas Instruments: AM335x ARM® CortexTM-A8 Microproces- sors (MPUs) Technical reference manual, Ref. SPRUH73H, October 2011, http://phytec.com/wiki/images/7/72/AM335x_ techincal_reference_manual.pdf (last access: 17 Septem- ber 2016), Rev. April 2013. IDT – Integrated Device Technology: Inc. IDT74LVC245A 3.3V CMOS octal bus transceiver with 3-state Output and 5 V tolerant, Datasheet, http://pdf.datasheetcatalog.com/datasheet/ idt/74LVC245A_DS_91896.pdf (last access: 17 Septem- ber 2016), April 1999. Joost, R. and Salomon, R.: CDL, a precise, low-cost coincidence detector latch, Electronics, 4, 1018–1032, 2015. Texas Instruments: Beaglebone Black schematic sheet, Ref. 450-5500-001, https://beagleboard.org/static/beaglebone/ BEAGLEBONE_SCHEM_A3.pdf (last access: 17 Septem- ber 2016), January 2014. Karapetyan, G., Ganeva, M., and Hippler, R.: Directional sensitivity of MuSTAnG muon telescope, J. Space Weather Space Clim., 3, A16, doi:10.1051/swsc/2013040, 2013. US NIM Committee: Standard NIM instrumentation system, DOE/ER-0457T, Rev. 5, http://www.osti.gov/scitech/servlets/ purl/7120327 (last access: 17 September 2016), USA, 1978. Medina, J., Sequeiros, J., and Henrion, J.: HP-NIM data acquisition system, Nucl. Instrum. Meth. A, 257, 321–324, 1987. Zenodo: https://zenodo.org/record/154682#.V-Uh703r2e9, doi:10.5281/zenodo.154682, last access: 23 September 2016. Medina, J., Blanco, J. J., García, O., Gómez-Herrero, R., Catalán, E. J., García I., Hidalgo, M. A., Meziat, D., Prieto, M., Rodríguez- Pacheco, J., and Sanchez, S.: Castilla-La Mancha neutron moni- tor, Nucl. Instrum. Meth. A, 727, 97–103, 2013. Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016 www.geosci-instrum-method-data-syst.net/5/437/2016/ Geosci. Instrum. Method. Data Syst., 5, 437–449, 2016
https://openalex.org/W3043543029	http://real.mtak.hu/151427/1/1788-6120-article-p1243.pdf	Hungarian	null	A diabeteses neuropathia és egyéb szövődmények előfordulása a Debreceni Egyetem Diabeteses Neuropathia Centrumában	Orvosi hetilap	2,020	cc-by	6,454	Debreceni Egyetem, Általános Orvostudományi Kar, Anyagcsere Betegségek Nem Önálló Tanszék, Belgyógyászati Intézet, Debrecen Bevezetés: Világszerte jelentősen növekszik a cukorbetegség előfordulása. A distalis típusú szenzomotoros polyneuro pathia (DSPN) a leggyakrabban előforduló és a legkorábban kimutatható microvascularis szövődmény, mely változa tos klinikai megjelenése és sokszor atípusos tünetei miatt gyakran csak a cukorbetegség előrehaladott stádiumában kerül felismerésre. Célkitűzés és módszer: Munkánk során 431 beteg adatait dolgoztuk fel, akiket 2011 és 2018 között a Debreceni Egye tem Diabeteses Neuropathia Centrumában vizsgáltunk Neurometer® segítségével, és összefüggéseket kerestünk a különböző szív-ér rendszeri és microvascularis szövődmények (retinopathia, microalbuminuria), a laboratóriumi paraméterek és a DSPN súlyossága között. Eredmények: A betegek átlagéletkora 63,4 év, 62%-uk nő volt, 92%-uk 2-es típusú cukorbetegségben szenvedett. A cukorbetegség fennállásának átlagos ideje 13,7 év volt. Cardiovascularis betegség a betegek 42%-ánál volt ismert. A microvascularis szövődmények közül a retinopathia előfordulása 12% volt, perzisztáló microalbuminuria a betegek 16%-ánál volt igazolva. A vizsgált betegek 19%-ánál a típusos DSPN-panaszok ellenére sem tudtunk idegi károsodást kimutatni; 49%-ban enyhe fokú, 19%-ban közepes fokú és 13%-ban súlyos fokú neuropathia volt kimutatható Neu rometer® segítségével. A cukorbetegséggel összefüggő idegi károsodás kifejezettebb volt diabeteses retinopathia (p<0,001) és perzisztáló microalbuminuria esetén (p<0,001), e microvascularis szövődmények előfordulása összefüg gést mutatott a DSPN súlyosságával. A cardiovascularis szövődmények megjelenése nem mutatott korrelációt a peri fériás idegkárosodás mértékével, és nem találtunk összefüggést a DSPN súlyossági foka és a cardiovascularis betegsé gek előfordulása között. Következtetés: Eredményeink alapján a diabeteses neuropathia progressziója előre jelezheti az egyéb microvascularis szövődmények megjelenését 2-es típusú cukorbetegségben, adataink azonban nem mutattak összefüggést a cardio vascularis betegségek kialakulásával. A Neurometer® segítségével végzett perifériás idegrendszeri vizsgálat alkalmas a DSPN követésére és a neuropathia súlyosságának megállapítására. A cardiovascularis kockázatot az autonóm ideg rendszeri funkciót vizsgáló Ewing-féle reflextesztek segítségével jobban meg tudjuk ítélni cukorbetegeinknél. Orv Hetil. 2020; 161(30): 1243–1251. Kulcsszavak: cukorbetegség, diabeteses neuropathia, cardiovascularis szövődmények, microvascularis szövődmén EREDETI KÖZLEMÉNY EREDETI KÖZLEMÉNY A diabeteses neuropathia és egyéb szövődmények előfordulása a Debreceni Egyetem Diabeteses Neuropathia Centrumában Sztanek Ferenc dr. ■ Balogh Bernadett dr. Molnár Ágnes dr. ■ Zöld Eszter dr. ■ Tóth Nóra dr. Jakab András Áron dr. ■ Paragh György dr. Sztanek Ferenc dr. ■ Balogh Bernadett dr. Molnár Ágnes dr. ■ Zöld Eszter dr. ■ Tóth Nóra dr. Jakab András Áron dr. ■ Paragh György dr. Debreceni Egyetem, Általános Orvostudományi Kar, Anyagcsere Betegségek Nem Önálló Tanszék, Belgyógyászati Intézet, Debrecen Debreceni Egyetem, Általános Orvostudományi Kar, Anyagcsere Betegségek Nem Önálló Tanszék, Belgyógyászati Intézet, Debrecen Rövidítések tag és vékony szenzoros idegrostok károsodása fordul elő DSPN-ben a tapintás-, az egyensúly- és hőérzés zava rát okozva. Az időben történő felismerés, a specifikus terápia és a megelőző lábápolás jelentősen csökkenti az érzéskiesésből adódó lábsérülések és a diabeteses lábfe kélyek kialakulásának kockázatát. Továbbá az autonóm neuropathia felismerése és kezelése csökkenti a cardio vascularis kockázatot, és javítja az életminőséget [6]. Ko rábban igazolódott, hogy a DSPN a cardiovascularis ese mény bekövetkezésének független prediktora [7], valamint szoros összefüggést mutat az egyéb microvas cularis szövődményekkel, a diabeteses retinopathia és a microalbuminuria fennállásával [8]. ATP = adenozil-trifoszfát; BNO = betegségek nemzetközi osztályozása; CV = cardiovascularis; CVD = (cardiovascular di sease) cardiovascularis betegség; DNS = dezoxiribonukleinsav; DSPN = (distal sensorimotor neuropathy) distalis típusú szen zomotoros polyneuropathia; eGFR = (estimated glomerular filtration rate) becsült glomerulusfiltrációs ráta; HbA1c = he moglobin-A1c; HDL = (high-density lipoprotein) magas sűrű ségű lipoprotein; SD = standard deviáció; T2DM = (type 2 diabetes mellitus) 2-es típusú cukorbetegség; USA = (United States of America) Amerikai Egyesült Államok A diabetes mellitus előfordulása világszerte jelentősen emelkedik, 2018-ban a 2-es típusú cukorbetegségben (T2DM) szenvedő betegek arányát a felnőtt lakosság 8,8%-ára, számát mintegy 500 millióra becsülték; a pre valencia a fejlett és a fejlődő országokban egyaránt emel kedik [1]. A betegek körülbelül 77%-a 20–64 éves, a munkaképes korosztályba tartozik, tehát jelentős a gaz dasági és társadalmi jelentősége egyaránt. Az előrejelzé sek szerint 2045-re az idős betegpopuláció aránya is megduplázódik [2]. Magyarországon a cukorbetegség prevalenciája 6,4% körüli, amely az utóbbi években stag náló tendenciával, az életkor előrehaladtával azonban fokozatosan növekszik [3]. A betegek több mint 90%-a T2DM-ben szenved. [ ] A DSPN leggyakoribb endogén metabolikus oka a cu korbetegség, kialakulásának pontos mechanizmusa azon ban jelenleg sem ismert. Kialakulását két fő tényező idézi elő: az idegszövet vérellátásáért felelős vasa nervorum károsodása és a sejtanyagcsere kóros változásainak hatá sa. Az érfali károsodások többféle mechanizmus révén jöhetnek létre. Az intracelluláris glükóztartalom megnö vekedése fokozza a mitokondriumok szuperoxid-anion jainak termelését, felgyorsítja a glikolízis folyamatát, nö veli a szabad gyökök képződését, továbbá felerősíti a gyulladásos folyamatokat [9]. A hyperglykaemia fokozza a fehérjék nem enzimatikus glikolizációját, aminek kö vetkezménye az előrehaladott glikációs végtermékek fel szaporodása, az endothelium diszfunkciója, fokozódik a vasoconstrictor anyagok (például endotelin, angioten zin) felszabadulása, a vasodilatatiót kiváltó faktorok (pél dául prosztaciklin, bradikinin) aránya csökken [10]. Kü lönböző kóros szignálútvonalak aktivációja fokozza a proinflammatorikus citokinek termelődését és az apop tózis indukcióját. (Beérkezett: 2020. március 20.; elfogadva: 2020. április 6.) (Beérkezett: 2020. március 20.; elfogadva: 2020. április 6.) 2020 ■ 161. évfolyam, 30. szám Sztanek F, Balogh B, Molnár Á, Zöld E, Tóth N, Jakab AÁ, Paragh Gy. [Diabetic neuropathy and other diabetic complications at the Diabetic Neuropathy Center of the University of Debrecen]. Orv Hetil. 2020; 161(30): 1243– 1251. Diabetic neuropathy and other diabetic complications at the Diabetic Neuropathy Center of the University of Debrecen Introduction: The prevalence of diabetes mellitus is significantly increasing worldwide. Distal sensorimotor neuropa thy (DSPN) is the most common and the earliest detectable microvascular complication. Due to its diverse clinical appearance and atypical symptoms, DSPN is often recognized in an advanced stage. pp yp y p g g Aim and method: In our study, the data of 431 patients who were examined using the Neurometer® between 2011 and 2018 at the Diabetic Neuropathy Center of the University of Debrecen were processed and the correlations between cardiovascular and microvascular complications, laboratory parameters and the severity of DSPN were in vestigated. 2020 ■ 161. évfolyam, 30. szám ■ 1243–1251. 1243 DOI: 10.1556/650.2020.31799 ■ © Szerző(k) Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC EREDETI KÖZLEMÉNY Results: The average age of patients was 63.4 years, 62% of them were women, and 92% had type 2 diabetes mellitus. The average duration of diabetes was 13.7 years. Cardiovascular disease (CVD) was diagnosed in 42% of the patients. The incidence of retinopathy was 12%, persistent microalbuminuria was 16%. Despite DSNP complaints, neuronal damage could not be detected in 19%; in the examined patients 49% had mild, 19% moderate and 13% severe neu ropathy. Diabetes-related neurological damage was more serious in the presence of both diabetic retinopathy (p<0.001) and microalbuminuria (p<0.001). The incidence of these microvascular complications and the severity of DSPN showed a significant positive correlation (p<0.001). There was no correlation between the severity of periph eral neuropathy and the development of CVD, and we did not find any correlations between the severity of DSPN and CVD. Conclusion: Based on our investigation, correlation between the progression of diabetic neuropathy and cardiovascu lar complications was not found, although the progression of diabetic neuropathy indicated the development of other microvascular diseases. Peripheral neurological examination using the Neurometer® is appropriate for control ling the DSPN status and the establishment of the severity of neuropathy determines the quality of life in diabetic patients. Among these patients, the risk of CVD can be assessed by Ewing’s test for autonomic nervous system func tion. Keywords: diabetes mellitus, diabetic neuropathy, cardiovascular complications, microvasular complications Sztanek F, Balogh B, Molnár Á, Zöld E, Tóth N, Jakab AÁ, Paragh Gy. [Diabetic neuropathy and other diabetic complications at the Diabetic Neuropathy Center of the University of Debrecen]. Orv Hetil. 2020; 161(30): 1243– 1251. Rövidítések Az endoneuralis vérátáramlás csökke A distalis típusú szenzomotoros polyneuropathia (DSPN) prevalenciája Európában T2DM esetében 18– 35% között mozog [4]. A DSPN változatos klinikai megjelenése és sokszor nem típusos tünetei miatt sok esetben nem valósulhat meg a korai diagnózis, ami a megfelelő oki és tüneti kezelés elkezdése szempontjából kiemelkedő fontosságú lenne [5]. A leggyakrabban a vas 1244 ORVOSI HETILAP Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC EREDETI KÖZLEMÉNY nése, az idegszövet integritásának megbomlása és a javí tó (repair) mechanizmusok is károsodnak [11]. A reaktívoxigén-gyökök termelődése fokozódik, a szuper oxidion és a hidrogén-peroxid közvetlenül károsítják a fehérjéket és a mitokondriális DNS-t is, valamint az ATP termelése is gátlás alá kerül. A mitokondriális károsodás következménye a csökkent neurotróf anyagok termelő dése is [12]. A reaktív gyökök, többek között a szuper oxid-anion és peroxinitrit termelődése központi szerepet játszik az oxidatív stressz fokozódásában, a nitrogén-mo noxid termelődése csökken, károsodik az endothel funk ciója és az idegszövet véráramlása [13]. A tartósan magas vércukorszint következtében a neuronokban a glikolízis folyamata telítődik, és alternatív anyagcsere-útvonalak, többek között a poliolútvonal aktiválódik, melynek so rán szorbitol és a belőle keletkező fruktóz felhalmozó dik. Mivel a sejtmembrán átjárhatatlan a szorbitol szá mára, nő az ozmotikus nyomás, aminek következtében végső soron az axonális transzport és a strukturális fehér jék is károsodhatnak [14]. A hexózamin-útvonal is alter natív glükózlebontási folyamatnak felel meg, ennek során végül N-acetil-glükózamin keletkezik, mely transz kripciós faktorok oldalláncaihoz kapcsolódva citokinter melődést és apoptózist indukál [15]. szükséges a diabeteses neuropathia diagnózisának felállí tásához. A magyarországi diabeteses neuropathia centru mokban a Neurometer® (Neurotron Inc., Baltimore, MD, USA, 2002) segítségével kvantitatívan tudjuk vizs gálni az áramérzet-küszöbérték kóros változásait. A vizs gálat során a nervus medianust és a nervus peroneus su perficialist ellenőrizzük különböző áramerősségekkel (0,01–0,99 mA) többször ismételve, három különböző frekvencián. 2000 Hz-en a vastag mielinizált rostokat, 250 Hz-en a vékony mielinizált rostokat, még 5 Hz-en a vékony mielinizálatlan rostokat ingereljük, majd a beteg áramérzet-küszöbértéke alapján határozzuk meg az idegi károsodás mértékét. Minél magasabb a páciens áram érzet-küszöbértéke, annál súlyosabb neuropathiát való színűsíthetünk. A vizsgálat nemcsak a DSPN szűrésére, hanem a betegség követésére is alkalmazható [16]. A di agnózis felállításához együtt kell értékelnünk a betegek klinikai tüneteit, szénhidrátháztartását és az eszközös vizsgálatok során kapott eredményeket. A cardiovascula ris autonóm neuropathia kimutatása a Ewing-féle cardio vascularis reflextesztek elvégzésével történik. A para szimpatikus funkciót a mélybelégzés-teszttel, a Valsalva- hányadossal és a felállás okozta szívfrekvencia-változás segítségével vizsgáljuk. Rövidítések A szimpatikus idegrendszer mű ködését a felállás során mért szisztolés vérnyomáscsökke néssel, illetve a kézszorítási teszt során mért diasztolés vérnyomás-emelkedés megállapításával határozhatjuk meg. A cardiovascularis autonóm neuropathia jelenléte T2DM-ben a szív- és érrendszeri betegségek előfordulá sának kockázati tényezője, ezért korai diagnosztikája je lentős szerepet játszhat a fő cardiovascularis események, mint a szívizominfarktus, a stroke és a szívelégtelenség megelőzése szempontjából [17]. A diabeteses neuropathia a legtöbbször jellegzetes kli nikai képpel rendelkezik, azonban atípusos tünetek ese tén nehezített lehet a diagnózis felállítása. A DSPN-re jellemző panaszok a szimmetrikus, gyakran az alsó és fel ső végtagokat érintő, harisnya-, illetve kesztyűujj formá jában fennálló bizsergés vagy tűszúrásszerű fájdalom. A betegek jellegzetesen éjszaka és nyugalomban a tünetek súlyosbodását panaszolják. Az alsó végtagi reflexek reny hesége vagy kiesése is szembetűnő lehet. Az idegrend szeri és társuló microangiopathiás károsodások követ kezménye a diabetesesláb-szindróma kialakulása, mely a DSPN legrettegettebb szövődménye: a beteg nem érzé keli a lábán keletkező sérüléseket, mély fekélyek alakul hatnak ki rossz gyógyulási hajlammal és az amputáció veszélyével. A DSPN következtében a láb szerkezete is megváltozhat, ún. Charcot-féle neuropathiás osteoarth ropathia alakulhat ki [16]. Munkánk során a Diabeteses Neuropathia Centru munkban vizsgálaton megjelenő cukorbetegek adatait gyűjtöttük össze, és DSPN jelenlétében kerestük a mic ro- és macrovascularis szövődményeket, a kóros laborpa raméterek és az egyéb kockázati tényezők összefüggése it. Vizsgálatunkkal hangsúlyozni kívánjuk a DSPN korai felismerésének és kezelésének fontosságát cukorbetege ink körében. A cukorbetegek szűrése DSPN irányában a legtöbb ször a háziorvosi rendelőkben, diabetes-szakrendelése ken és belgyógyászati osztályokon zajlik, részletes kivizs gálás azonban Magyarországon a regionális diabeteses neuropathia centrumokban történik [16]. A vibrációér zést a Rydel–Seiffer-féle, 128 Hz-es kalibrált hangvilla segítségével vizsgálhatjuk, a nyomásérzés vizsgálatára a 10 grammos Semmes–Weinstein-féle mikrofilamentum- tesztet alkalmazzuk, így a vastag mielinizált idegrostok állapota ítélhető meg. Lehetőségünk van Neurotips®- vizsgálatra, melynek során steril műanyag tűvel enyhe nyomást gyakorolunk a beteg talpára, s egészséges eset ben fájdalomérzetet váltunk ki. A kóros sudomotoros funkciót a Neuropad® segítségével tudjuk igazolni. Az elektroneurográfiás vizsgálat a leginkább differenciáldi agnosztikai szempontból jelentős, és a legtöbbször nem Betegek és módszer 0 1 Retinopathia jelenléte (1) / hiánya (0) 260 280 300 320 340 360 380 400 420 440 460 480 500 n = 367 n = 46 p<0,0001 A Neurometer®-rel mért áramérzet-küszöbérték átlaga ORVOSI HETILAP 0 1 Retinopathia jelenléte (1) / hiánya (0) 260 280 300 320 340 360 n = 367 n = 46 A Neurometer®-rel mért á 0 1 Microalbuminuria jelenléte (1) / hiánya (0) 260 280 300 320 340 360 380 400 420 440 460 480 500 n = 349 n = 64 p<0,0001 A Neurometer®-rel mért áramérzet-küszöbérték átlaga 0 1 Szív- és érrendszeri betegség jelenléte (1) / hiánya (0) 270 280 290 300 310 320 330 340 350 n = 241 p = 0,43 n = 172 A Neurometer®-rel mért áramérzet-küszöbérték átlaga 1. ábra A diabeteses neuropathia súlyossága (az áramérzet-küszöbérték alapján) a retinopathiás szövődmény jelenléte esetén 2. ábra A diabeteses neuropathia súlyossága (az áramérzet-küszöbérték alapján) a microalbuminuria jelenléte esetén 3. ábra A diabeteses neuropathia súlyossága (az áramérzet-küszöbérték alapján) az összevont CVD jelenléte esetén CVD = cardiovascularis betegség 0 1 Retinopathia jelenléte (1) / hiánya (0) 1. ábra A diabeteses neuropathia súlyossága (az áramérzet-küszöbérték alapján) a retinopathiás szövődmény jelenléte esetén 1. ábra A diabeteses neuropathia súlyossága (az áramérzet-küszöbérték alapján) a retinopathiás szövődmény jelenléte esetén 0 1 Microalbuminuria jelenléte (1) / hiánya (0) 260 280 300 320 340 360 380 400 420 440 460 480 500 n = 349 n = 64 p<0,0001 A Neurometer®-rel mért áramérzet-küszöbérték átlaga 2. ábra A diabeteses neuropathia súlyossága (az áramérzet-küszöbérték Betegek és módszer Vizsgálatunkba olyan, korábban igazolt cukorbetegeket választottunk be, akik 2011 és 2018 között a Debreceni Egyetem Diabeteses Neuropathia Centrumában neuro pathiás szűrővizsgálaton estek át, és akiknek a dokumen tációjában (a MedSolution rendszer adatbázisa és a Neu ropathia Centrum regisztere alapján) rendelkezésünkre álltak információk a macro- és microvascularis szövőd mények jelenlétéről vagy hiányáról, az egyéb vizsgált ri zikófaktorokkal (dohányzás, alkoholfogyasztás, hyperto nia) és laborparaméterekkel együtt. Kizártuk azon betegeket, akiknek igazoltan más etiológiájú, nem diabe teses neuropathiájuk volt, vagy az előbb említett adatok nem álltak rendelkezésünkre. Kizáró tényezőink közé 1245 2020 ■ 161. évfolyam, 30. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC EREDETI KÖZLEMÉNY ÖZLEMÉNY 0 1 Retinopathia jelenléte (1) / hiánya (0) 260 280 300 320 340 360 380 400 420 440 460 480 500 n = 367 n = 46 p<0,0001 A Neurometer®-rel mért áramérzet-küszöbérték átlaga 0 1 Microalbuminuria jelenléte (1) / hiánya (0) 260 280 300 320 340 360 380 400 420 440 460 480 500 n = 349 n = 64 p<0,0001 A Neurometer®-rel mért áramérzet-küszöbérték átlaga 280 290 300 310 320 330 340 350 n = 241 p = 0,43 n = 172 eter®-rel mért áramérzet-küszöbérték átlaga 1. ábra A diabeteses neuropathia súlyossága (az áramérzet-küszöbérték alapján) a retinopathiás szövődmény jelenléte esetén 2. ábra A diabeteses neuropathia súlyossága (az áramérzet-küszöbérték alapján) a microalbuminuria jelenléte esetén tartozott a kóros alkoholfogyasztás, az alkohol indukálta májkárosodás és az egyéb eredetű hepatopathia, melye ket az anamnézis és a májfunkciós értékek alapján álla pítottunk meg. A Neurometer® segítségével meghatá rozott áramérzet-küszöbértékek mindkét lábon és mindhárom neuronalis rosttípusra vonatkozó átlagát használtuk fel a statisztikai számítások során. Vizsgála tunk során SAS for Windows 6.12 (SAS Institute Inc., Cary, NC, USA) számítógépes programot használtunk statisztikai analízis céljából. Az adatok normáleloszlását, ezáltal az összehasonlíthatóságukat Kolmogorov–Szmir nov-teszttel ellenőriztük. A normális eloszlást mutató paramétereknél az adatokat átlagérték ± szórás (SD) for mában, nem normális eloszlás esetén medián és alsó-fel ső kvartilisek megadásával ábrázoltuk. A különböző cso portok összehasonlítása során normális eloszlás esetén kétmintás t-próbát (Student-féle t-teszt), a nem normális eloszlást mutató paramétereknél pedig Mann–Whitney- próbát alkalmaztunk; szignifikánsnak tekintettük a p<0,05 értéket. A nem normális eloszlású változók 10-es alapú logaritmusát véve végeztünk lineáris regressziós analízist. Minőségi változók esetén khi-négyzet-szignifi kanciatesztet alkalmaztunk. Eredmények Vizsgálatunkba 431 beteget vontunk be, akiknek antro pometriai adatait és laboratóriumi eredményeit az 1. táb lázatban foglaltuk össze. A betegek több mint 90%-a 2020 ■161 évfolyam 30 szám 12 1. táblázat A diabeteses neuropathiás betegek antropometriai és laborpara méterei Paraméterek Átlag ± SD/Medián (kvartilisek) Betegszám (fő) 431 T1DM/T2DM (fő) 36/395 Férfi/nő (fő) 164/267 Életkor (év) 63,39 ± 10,99 Diabetes fennállása (év) 13,74 ± 9,81 Neurometer®-átlag (mA) 304,02 (213,83–337,17) sTSH (mIU/l) 2,35 (1,07–2,80) D-vitamin (nmol/l) 64,20 ± 28,66 Hemoglobin (g/l) 133,65 (125,00–144,00) HbA1c (%) 7,44 (6,3–8,3) eGFR (ml/perc/1,73 m2) 73,84 ± 19,41 Triglicerid (mmol/l) 2,06 (1,10–2,40) Koleszterin (mmol/l) 4,97 (3,90–5,70) HDL-koleszterin (mmol/l) 1,33 (1,00–1,60) LDL-koleszterin (mmol/l) 2,85 (2,00–3,60) eGFR = becsült glomerulusfiltrációs ráta; HbA1c = hemoglobin-A1c; HDL = magas sűrűségű lipoprotein; LDL = alacsony sűrűségű lipopro tein; SD = standard deviáció; sTSH = szenzitív thyreoideastimuláló hormon; T1DM = 1-es típusú cukorbetegség; T2DM = 2-es típusú cukorbetegség 1. táblázat A diabeteses neuropathiás betegek antropometriai és laborpara méterei 0 1 Microalbuminuria jelenléte (1) / hiánya (0) 2. ábra A diabeteses neuropathia súlyossága (az áramérzet-küszöbérték alapján) a microalbuminuria jelenléte esetén 0 1 Szív- és érrendszeri betegség jelenléte (1) / hiánya (0) 270 280 290 300 310 320 330 340 350 n = 241 p = 0,43 n = 172 A Neurometer®-rel mért áramérzet-küszöbérték átlaga 3 áb A di b hi úl á ( á é kü öbé ék 0 1 Szív- és érrendszeri betegség jelenléte (1) / hiánya (0) eGFR = becsült glomerulusfiltrációs ráta; HbA1c = hemoglobin-A1c; HDL = magas sűrűségű lipoprotein; LDL = alacsony sűrűségű lipopro tein; SD = standard deviáció; sTSH = szenzitív thyreoideastimuláló hormon; T1DM = 1-es típusú cukorbetegség; T2DM = 2-es típusú cukorbetegség 1246 ORVOSI HETILAP 2020 ■ 161. évfolyam, 30. szám Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC EREDETI KÖZLEMÉNY 2. Eredmények táblázat A CV-betegségben szenvedő és azoktól mentes alcsoportok összehasonlítása Változók Nem igazolt CVD jelenléte Igazolt CVD jelenléte p-érték Életkor (év) 64,12 ± 7,35 65,26 ± 6,98 0,23 Neurometer®-átlag (mA) 266,17 (214,00–332,83) 263,00 (212,92–342,08) 0,60 Diabetes fennállása (év) 12,00 ± 4 12,00 ± 4,5 0,57 sTSH (mIU/l) 1,72 (1,13–2,96) 1,57 (0,97–2,60) 0,14 D-vitamin (nmol/l) 57,60 ± 13,20 58,20 ± 12,40 0,94 Hemoglobin (g/l) 136,00 (127,00–145,00) 132,50 (123,00–143,00) 0,13 HbA1c (%) 7,20 (6,30-8,20) 7,10 (6,35–8,35) 0,52 eGFR (ml/perc/1,73 m2) 83,50 ± 6,30 76,00 ± 7,20 0,03 Triglicerid (mmol/l) 1,40 (1,00–2,30) 1,80 (1,30–2,70) 0,001 Koleszterin (mmol/l) 4,65 (4,00–5,70) 4,60 (3,80–5,70) 0,85 HDL-koleszterin (mmol/l) 1,30 (1,10–1,60) 1,20 (0,90–1,50) 0,003 LDL-koleszterin (mmol/l) 2,70 (2,00–3,60) 2,74 (2,00–3,60) 0,65 CV = cardiovascularis; CVD = cardiovascularis betegség; eGFR = becsült glomerulusfiltrációs ráta; HbA1c = hemoglobin-A1c; HDL = magas sűrű ségű lipoprotein; LDL = alacsony sűrűségű lipoprotein; sTSH = szenzitív thyreoideastimuláló hormon 2. táblázat A CV-betegségben szenvedő és azoktól mentes alcsoportok összehasonlítása CV = cardiovascularis; CVD = cardiovascularis betegség; eGFR = becsült glomerulusfiltrációs ráta; HbA1c = hemoglobin-A1c; HDL = magas sűrű ségű lipoprotein; LDL = alacsony sűrűségű lipoprotein; sTSH = szenzitív thyreoideastimuláló hormon CV = cardiovascularis; CVD = cardiovascularis betegség; eGFR = becsült glomerulusfiltrációs ráta; HbA1c = hemoglobin-A1c; HDL = magas sűrű ségű lipoprotein; LDL = alacsony sűrűségű lipoprotein; sTSH = szenzitív thyreoideastimuláló hormon szignifikáns különbség a CVD-szövődménytől mentes betegeinkkel összehasonlítva (3. ábra). (n = 395) T2DM-ben szenvedett. A vizsgált kohorsz 62%-át nők alkották. A betegek átlagéletkora 63,4 év, a betegek átlagos HbA1c-értéke 7,4% volt. A rendelkezé sünkre álló dokumentáció alapján igazolt macrovascula ris szövődmény 183 betegnél, vagyis a kohorsz 42%-ánál volt jelen. Microvascularis szövődményt (diabeteses reti nopathia, microalbuminuria) a betegek 28%-ánál talál tunk, ami a vizsgálatban 119 főt jelentett. A Neurometer®- vizsgálattal alátámasztott diabeteses neuropathiás páciensek száma 350 volt, ami a kohorsz 81%-át tette ki. A betegek 19%-ánál (81 fő) a típusos neuropathiás tüne tek ellenére nem tudtunk elváltozást kimutatni a Neurometer®-vizsgálat alapján, ők feltehetően vékony rost-neuropathiában szenvedtek. Összehasonlítottuk a CVD-szövődményekkel rendel kező cukorbetegeket a szövődményektől mentes bete gekkel, az eredményeket a 2. táblázatban foglaltuk ös� sze. A CVD-szövődménnyel rendelkező alcsoportban a szérumtriglicerid-szint szignifikánsan magasabbnak, a HDL-koleszterin és az eGFR-érték szignifikánsan ala csonyabbnak bizonyult. Lineáris regressziós analízist alkalmazva hasonlítottuk össze a különböző változók és a Neurometer® segítségé vel meghatározott áramérzet-küszöbérték átlagával jel lemzett neuropathiás károsodás súlyosságát. A HbA1c pozitív korrelációt mutatott a DSPN súlyosságával (r = 0,1981, p<0,001; 4. ábra). Az 1. Eredmények ábrán a Neurometer® segítségével meghatáro zott áramérzet-küszöbértéket, azaz a fennálló neuropat hia súlyosságát hasonlítottuk a diabeteses retinopathia fennállásához. Ezen az ábrán látható, hogy a retinopathi ás betegeknél szignifikánsan magasabb volt az áramér zet-küszöbérték átlagértéke (p<0,0001), tehát súlyosabb neuropathia véleményezhető. A különböző CV-rizikófaktorok és microvascularis szövődmények összehasonlításához a betegek tünetei és a Neurometer® segítségével meghatározott áramérzet- küszöbérték szerinti DSPN súlyossága alapján a betege ket négy különböző csoportba soroltuk. Az első cso portban Neurometer®-rel nem volt kimutatható az áramérzet-küszöbérték kóros változása (vékonyrost-ne uropathia), a második csoportba az enyhe fokú, a harma dikba a mérsékelten súlyos és a negyedikbe a súlyos fokú DSPN-betegek kerültek. A kategorizálás után az adato kat khi-négyzet-szignifikanciatesztekkel vizsgáltuk. Az első diagramon a férfi-nő megoszlást ábrázoltuk; férfido minancia figyelhető meg a neuropathia súlyosbodásával (p<0,05; 5. ábra). Vizsgálatunk során a neuropathia súlyossága és a mic roalbuminuria közötti összefüggést is elemeztük: a reti nopathiás esetekhez hasonlóan perzisztáló microalbumi nuria esetén szignifikánsan magasabb áramérzet-küszöb értéket (p<0,0001), azaz előrehaladottabb neuropathiás idegkárosodást találtunk (2. ábra). A cardiovascularis betegségeket mint összevont vég pontot kezeltük vizsgálatunk során, idetartozik a kór előzményben szereplő stabil angina pectoris, instabil an gina pectoris, akut myocardialis infarctus, ischaemiás szívbetegség, igazolt szisztolés vagy diasztolés szívelég telenség, előrehaladott balkamra-hypertrophia, átmeneti agyi ischaemia, stroke, perifériás alsó végtagi érszűkület és hasiaorta-aneurysma. Az összevont CVD esetében nem igazolódott az áramérzet-küszöbérték tekintetében Az inzulinnal kezelt cukorbetegek száma szignifikán san emelkedett a neuropathia súlyosságának függvé nyében (p<0,01; 6. ábra). A CV-rizikófaktorok közül a dohányzás, az alkoholfo gyasztás nem mutatott szignifikáns eltérést a DSPN súlyossága alapján történt összehasonlítások során. A microvascularis szövődmények szempontjából mind a 1247 2020 ■ 161. évfolyam, 30. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC EREDETI KÖZLEMÉNY 4. ábra A Neurometer® segítségével meghatározott áramérzet-küszöbérték átlagával jellemzett neuropathiás károsodás súlyossága és a HbA1c összefüggése HbA1c = hemoglobin-A1c 0,6 0,7 0,8 0,9 1,0 1,1 1,2 logHba1c 1,8 2,0 2,2 2,4 2,6 2,8 3,0 3,2 r e t e m o r u e N g ol logHba1c:logNeurometer: r = 0,1981; p = 0,0005; r2 = 0,0393 0,6 0,7 0,8 0,9 1,0 1,1 1,2 logHba1c 1,8 2,0 2,2 2,4 2,6 2,8 3,0 3,2 r e t e m o r u e N g ol logHba1c:logNeurometer: r = 0,1981; p = 0,0005; r2 = 0,0393 4. ábra A Neurometer® segítségével meghatározott áramérzet-küszöbérték átlagával jellemzett neuropathiás károsodás súlyossága és a HbA1c összefüggése HbA1c = hemoglobin-A1c 4. Eredmények ábra 0% 20% 40% 60% 80% 100% Nincs Enyhe Mérsékelt Súlyos 62 121 47 26 19 90 34 32 Az inzulinnal kezeltek aránya A diabeteses neuropathia súlyossága Nem Igen p = 0,0015 6. ábra Az inzulinnal kezeltek aránya a DSPN súlyosságának függvé nyében DSPN = distalis típusú szenzomotoros polyneuropathia 0% 20% 40% 60% 80% 100% Nincs Enyhe Mérsékelt Súlyos 62 121 47 26 19 90 34 32 Az inzulinnal kezeltek aránya A diabeteses neuropathia súlyossága Nem Igen p = 0,0015 6. ábra Az inzulinnal kezeltek aránya a DSPN súlyosságának függvé nyében DSPN = distalis típusú szenzomotoros polyneuropathia 0% 20% 40% 60% 80% 100% Nincs Enyhe Mérsékelt Súlyos 62 121 47 26 19 90 34 32 Az inzulinnal kezeltek aránya A diabeteses neuropathia súlyossága 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Nincs Enyhe Mérsékelt Súlyos 28 70 34 32 53 141 47 26 Férﬁ/nő arány A diabeteses neuropathia súlyossága Férﬁ Nő p = 0,02 5. ábra A férfi/nő arány a DSPN súlyosságával összehasonlítva DSPN = distalis típusú szenzomotoros polyneuropathia ressziós analízist végezve azt találtuk, hogy mind férfiak (r = 0,21, p<0,05; nincs ábrázolva), mind nők (r = 0,26, p<0,001; nincs ábrázolva) esetében szignifikáns pozitív összefüggést mutat a cukorbetegség tartama a neuropa thia súlyosságával. Vizsgálatunkban alcsoportelemzés során a HDL-szint szignifikánsan magasabbnak bizo nyult a nőbetegek körében (HDLnő: 1,42 ± 0,44 mmol/l; HDLffi: 1,19 ± 0,35 mmol/l, p<0,0001; nincs ábrázolva). microalbuminuria, mind a retinopathia előfordulási ará nya nőtt a DSPN súlyossági fokával (mindkét összeha sonlításra vonatkoztatva: p<0,0001; 7/a és 7/b ábra). p , ; ) Az összevont CVD-szövődmény, valamint külön érté kelve a hypertoniával a diabeteses neuropathia súlyossága nem mutatott szignifikáns összefüggést (8/a és 8/b ábra). A diabeteses neuropathia súlyosságát a diabetes fenn állásának időtartamával összehasonlítva, és lineáris reg 1248 2020 ■ 161. évfolyam, 30. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC EREDETI KÖZLEMÉNY 7. ábra A microvascularis szövődmények (microalbuminuria [a]; retinopathia [b]) kapcsolata a DSPN súlyosságával DSP di li í ú l hi a) b) 0% 50% 100% 78 187 62 36 3 24 19 22 A microalbuminuria aránya A diabeteses neuropathia súlyossága 0% 50% 100% 75 194 69 42 6 17 12 16 A retinopathia aránya A diabeteses neuropathia súlyossága Nincs Enyhe Mérsékelt Súlyos Nincs Enyhe Mérsékelt Súlyos Nem Igen p<0,0001 Nem Igen p = 0,00029 a) 0% 50% 100% 78 187 62 36 3 24 19 22 A microalbuminuria aránya A diabeteses neuropathia súlyossága Nincs Enyhe Mérsékelt Súlyos Nem Igen p<0,0001 b) 0% 50% 100% 75 194 69 42 6 17 12 16 A retinopathia aránya Nincs Enyhe Mérsékelt Súlyos 8. ábra A cardiovascularis szövődmények (a) és a hypertonia (b) jelenlétének összefüggései a DSPN súlyosságával DSPN = distalis típusú szenzomotoros polyneuropathia Nincs Enyhe Mérsékelt Súlyos Nincs Enyhe Mérsékelt Súlyos a) b) 0% 20% 40% 60% 80% 100% 23 43 19 18 58 168 62 40 Hypertonia A diabeteses neuropathia súlyossága Nem Igen p = 0,26 0% 20% 40% 60% 80% 100% 53 118 49 28 28 93 32 30 Cardiovascularis betegség A diabeteses neuropathia súlyossága Nem Igen p = 0,20 Nincs Enyhe Mérsékelt Súlyos b) 0% 20% 40% 60% 80% 100% 23 43 19 18 58 168 62 40 Hypertonia A diabeteses neuropathia súlyossága Nem Igen p = 0,26 Nincs Enyhe Mérsékelt Súlyos a) 0% 20% 40% 60% 80% 100% 53 118 49 28 28 93 32 30 Cardiovascularis betegség A diabeteses neuropathia súlyossága Nem Igen p = 0,20 b) A cardiovascularis szövődmények (a) és a hypertonia (b) jelenlétének összefüggései a DSPN súlyosságával 8. ábra A cardiovascularis szövődmények (a) és a hypertonia (b) jelenlétének összefüggései a DSPN súlyosságával DSPN = distalis típusú szenzomotoros polyneuropathia korlátai állhatnak. A szív- és érrendszeri betegségeket nem tudtuk külön elemezni keresztmetszeti és obszervá ciós jellegű adatelemzésünk során, mivel a rendelkezésre álló, sokszor hiányos kórelőzményi adatok és a változa tos, BNO szerinti kódolás alapján megfelelő esetszámú, egységes alcsoportokat nem tudtunk alkotni. A microvas cularis szövődmények (diabeteses retinopathia és nephro pathia) vizsgálata szintén a kórelőzményi adatok alapján történt; az eltérő vizsgálati módszerek főleg a diabeteses nephropathia esetén jelenthettek limitációt, mivel sok szor nem volt elemezhető, hogy az albuminuria jelenléte (kóros microalbuminuria-eredmény a laborokban vagy kóros albumin/kreatinin hányados) egyszeri vagy kons tans tényező-e. Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC Mind a microvascularis, mint a CVD-szö vődmények és a diabeteses neuropathia kapcsolatának elemzésére további prospektív vizsgálatok, akár több ma gyarországi régiót magukban foglaló, multicentrikus ada tok elemzése bővítheti ismereteinket. Munkánk során nem vizsgáltuk a diabeteses autonóm neuropathia össze függését az egyéb szövődményekkel; a Ewing-féle cardio vascularis reflextesztek eredményével való összehasonlítás további információkat nyújthat a macrovascularis szövőd mények és a DSPN súlyosságának kapcsolatáról. Kereszt metszeti jellegéből adódóan mortalitási adatok elemzésé re vizsgálatunk nem terjedt ki. Irodalom [1] Zheng Y, Ley SH, Hu FB. Global aetiology and epidemiology of type 2 diabetes mellitus and its complications. Nat Rev Endo crinol. 2018; 14: 88–98. Eredményeink alapján a súlyosabb fokú neuropathiá ban szenvedő betegek körében férfidominancia volt megfigyelhető, ami már korábban is ismert volt a szak irodalomban. A férfiakban a cukorbetegség korábbi stá diumában és súlyosabb formában jelentkezik ez a micro vascularis idegi szövődmény [22]. Egyrészt a férfiak nagyobb átlagmagassága és a perifériás idegrostok hossza összefüggésben állhat a súlyosabb fokú neuropathiás ká rosodással. Másrészt a metabolikus faktorok közül a cu korbetegségre jellemző dyslipidaemia is befolyásolhatja a neuropathia mértékét. Experimentális egérkísérletek alapján a magasabb HDL-koleszterin-szintet protektív faktorként azonosították a nőstény egyedek esetén. A HDL-hez kötött enzimek (például paraoxonáz, mie loperoxidáz) antioxidáns szerepet töltenek be az oxidatív stressz károsító hatásával szemben, így késleltetve a DSPN megjelenését [23]. Vizsgálatunkban alcsoport elemzés során a HDL-szint szignifikánsan magasabbnak bizonyult a női betegek körében, ami alátámaszthatja ezt az experimentális megfigyelést. [2] International Diabetes Federation. IDF Diabetes Atlas, 9th edi tion. IDF, Brussels, 2019. Available from: https://www.diabete satlas.org. [3] Kempler P, Putz Zs, Kiss Z, et al. Prevalence and financial burden of type 2 diabetes mellitus in Hungary between 2001–2014 – results of the analysis of the National Health Insurance Fund database. [A 2-es típusú diabetes előfordulása és költségterheinek alakulása Magyarországon 2001–2014 között – az Országos Egészségbiztosítási Pénztár adatbázis-elemzésének eredményei.] Diabet Hung. 2016; 24: 177–188. [Hungarian] [4] Tamayo T, Rosenbauer J, Wild SH, et al. Diabetes in Europe: an update. Diabetes Res Clin Pract. 2014; 103: 206–217. [5] Winkler G, Kempler P. Pathomechanism of diabetic neuropathy: background of the pathogenesis-oriented therapy. [A neuro pathia diabetica patomechanizmusa: az oki kezelés elméleti hát tere.] Orv Hetil. 2010; 151: 971–981. [Hungarian] [6] Pop-Busui R, Boulton AJ, Feldman EL, et al. Diabetic neuropa thy: a position statement by the American Diabetes Association. Diabetes Care 2017; 40: 136–154. [7] Brownrigg JR, Lusignan S, McGovern A, et al. Peripheral neu ropathy and the risk of cardiovascular events in type 2 diabetes mellitus. Heart 2014; 100: 1837–1843. Az inzulinnal kezelt betegek körében súlyosabb fokú neuropathia volt megfigyelhető. Irodalmi adatok alapján a hosszabb betegségtartam és az elégtelen szénhidrát- anyagcsere együttesen állhat a súlyosabb neuropathiás károsodás hátterében az inzulinkezelésben részesülő cu korbetegek esetén. Az inzulinkezelés feltehetően legfel jebb neutrális hatású a neuropathia progressziójára. To vábbá az inzulin jól ismert gyulladáscsökkentő hatása akár protektív tényező is lehetne a progresszió lassítására [24]. Irodalom [8] Kärvestedt L, Mårtensson E, Grill V, et al. Peripheral sensory neuropathy associates with micro- or macroangiopathy: results from a population-based study of type 2 diabetic patients in Swe den. Diabetes Care 2009; 32: 317–322. [9] Brownlee M. Biochemistry and molecular cell biology of dia betic complications. Nature 2001; 414: 813–820. [10] Cameron NE, Eaton SE, Cotter MA, et al. Vascular factors and metabolic interactions in the pathogenesis of diabetic neuropa thy. Diabetologia 2001; 44: 1973–1988. [11] Callaghan BC, Cheng HT, Stables CL, et al. Diabetic neuropa thy: clinical manifestations and current treatments. Lancet Neu rol. 2012; 11: 521–534. [12] Hosseini A, Abdollahi M. Diabetic neuropathy and oxidative stress therapeutic perspectives. Oxid Med Cell Longev. 2013; 2013: 168039. Megbeszélés Vizsgálatunk során a microvascularis és CVD-szövődmé nyek, illetve egyéb rizikófaktorok előfordulását és az ezekkel kapcsolatos összefüggéseket elemeztük a Debre ceni Egyetem Neuropathia Centrumában gondozott be tegek adatai alapján. A cukorbetegség legkorábban és legjobban szűrhető szövődménye a DSPN, azonban en nek ellenére jelentősen aluldiagnosztizált állapot. A mic rovascularis szövődmények (retinopathia, microalbumin uria) esetén súlyosabb neuropathiás károsodást találtunk, valamint a microvascularis szövődmények gyakrabban fordulnak elő a DSPN súlyosságának függvényében, ahogy ezt a nemzetközi irodalomban korábban már leír ták [18]. Az általunk kimutatott eltérések egyenes arányú összefüggésben álltak az áramérzet-küszöbérték változá sával, tehát a Neurometer®-rel történő neuropathiás ká rosodás meghatározása és annak követése alkalmas lehet a többi microvascularis szövődmény előrejelzésére is. Ko rábbi irodalmi adatok is arra utalnak, hogy a nephropa thia és a retinopathia kialakulása szorosan összefügg sú lyosabb DSPN-tüneteket mutató egyének esetén [19, 20]. Adataink elemzése során az összevontan kezelt CVD-szövődmények nem mutattak összefüggést a DSPN súlyosságával. Ennek hátterében feltehetően vizsgálatunk 1249 2020 ■ 161. évfolyam, 30. szám ORVOSI HETILAP Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC EREDETI KÖZLEMÉNY Számos tényezőből adódhat, hogy a dohányzással kapcsolatban nem találtunk szignifikáns összefüggést a diabeteses neuropathia súlyosságával. A dohányzás okoz ta károsodás multifaktoriális: nemcsak idegkárosodást, de angiopathiát is okoz, valamint a dohányosok hypoxia tűrő képessége is fokozottabb, ami befolyásolhatja az áramérzet-küszöbérték meghatározását [20]. Az alko hollal kapcsolatosan a korreláció hiányát a gondos bevá lasztási kritériumok befolyásolhatták. Az alkoholfogyasz tásról a betegek anamnézise alapján tájékozódtunk, a májenzimértékek alapján az egyértelmű alkoholos máj károsodást és az esetleges alkoholos eredetű neuropathi ás eseteket kizártuk. A magas vérnyomás nemzetközi összehasonlításban sem mutatott összefüggést a DSPN- nel 2-es típusú cukorbetegek esetén [21]. Anyagi támogatás: Munkánk a Magyar Diabetes Társa ság 2019. évi klinikai kutatási pályázatának támogatásá val készülhetett el. A publikáció elkészítését az EFOP- 3.6.2-16-2017-00009. számú projekt támogatta. Anyagi támogatás: Munkánk a Magyar Diabetes Társa ság 2019. évi klinikai kutatási pályázatának támogatásá val készülhetett el. A publikáció elkészítését az EFOP- 3.6.2-16-2017-00009. számú projekt támogatta. Szerzői munkamegosztás: B. B. és Sz. F. a szakirodalmi adatok feldolgozásában, adatok gyűjtésében és értékelé sében, T. N., Z. E. és J. A. Á. a kézirat szerkesztésében, M. Á. és P. Gy. a végső változat ellenőrzésében műkö dött közre. A cikk végleges változatát valamennyi szerző elolvasta és jóváhagyta. Szerzői munkamegosztás: B. B. és Sz. F. a szakirodalmi adatok feldolgozásában, adatok gyűjtésében és értékelé sében, T. N., Z. E. és J. A. Á. a kézirat szerkesztésében, M. Á. és P. Gy. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Megbeszélés a végső változat ellenőrzésében műkö dött közre. A cikk végleges változatát valamennyi szerző elolvasta és jóváhagyta. 2020 ■ 161. évfolyam, 30. szám Részvételi szándék 2020. augusztus 20-ig naponta 14–16 óra között Koltayné Bartha Magdánál jelezhető. Telefon: 06-20/960 5854 – e-mail: baratikor.saar@gmail.com Az Orvostalálkozók eddig döntően a résztvevők áldozatos adományaiból valósultak meg. Kérjük, hogy lehetősége szerint ebben az évben is támogassa rendezvényünket a Szár Községért Baráti Kör részére (UniCredit Bank 10918001-00000036-60180000) átutalható vagy a helyszínen kapható csekken befizetett összeggel, Romhányi Orvostalálkozó megjegyzéssel. Következtetés 98., 4032 e-mail: sztanekf@yahoo.com) (Sztanek Ferenc dr., Debrecen, Nagyerdei krt. 98., 4032 e-mail: sztanekf@yahoo.com) [20] Tesfaye S, Chaturvedi N, Eaton SE, et al. Vascular risk factors and diabetic neuropathy. N Engl J Med. 2005; 352: 341–350. A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) Következtetés [13] Sztanek F, M Molnár Á, Balogh Z. The role of oxidative stress in the development of diabetic neuropathy. [Az oxidatív stressz szerepe a diabeteses neuropathia kialakulásában.] Orv Hetil. 2016; 157: 1939–1946. [Hungarian] Összefoglalásképpen azt mondhatjuk, hogy a diabeteses neuropathia szűrő jellegű vizsgálata kiemelt fontosságú a cukorbetegek körében. A diabeteses neuropathia centru mokban Neurometer® segítségével a DSPN progresszió ját követhetjük, és ezzel előre jelezhetjük egyéb micro vascularis szövődmények megjelenését is. A DSPN cardiovascularis betegségekkel való összefüggésének elemzésére további vizsgálatok szükségesek. [14] Schemmel KE, Padiyara RS, D’Souza JJ. Aldose reductase in hibitors in the treatment of diabetic peripheral neuropathy: a re view. J Diabetes Compl. 2010; 24: 354–360. [15] Du XL, Edelstein D, Rossetti L, et al. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosa mine pathway and induces plasminogen activator inhibitor-1 1250 2020 ■ 161. évfolyam, 30. szám ORVOSI HETILAP ORVOSI HETILAP Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC EREDETI KÖZLEMÉNY expression by increasing Sp1 glycosylation. Proc Natl Acad Sci USA 2000; 97: 12222–12226. [21] Grisold A, Callaghan BC, Feldman EL. Mediators of diabetic neuropathy: is hyperglycemia the only culprit? Curr Opin Endo crinol Diabetes Obes. 2017; 24: 103–111. [16] Putz Zs, Hermányi Zs, Tóth N, et al. Testing distal sensory neu ropathy in diabetes care. [A distalis típusú sensoros neuropathia diagnosztikája a diabetológiai gyakorlatban.] Diabet Hung. 2008; 16: 157–164. [Hungarian] [22] Aaberg ML, Burch DM, Hud ZR, et al. Gender differences in the onset of diabetic neuropathy. J Diab Compl. 2008; 22: 83– 87. [17] Kempler P, Tesfaye S, Chaturvedi N, et al. Autonomic neuropa thy is associated with increased cardiovascular risk factors: the EURODIAB IDDM complications study. Diabet Med. 2002; 19: 900–909. [23] O’Brien PD, Hur J, Robell NJ, et al. Gender-specific differences in diabetic neuropathy in BTBR ob/ob mice. J Diab Compl. 2016; 30: 30–37. [24] Sun Q, Li J, Gao F. New insights into insulin: the anti-inflamma tory effect and its clinical relevance. World J Diabetes 2014; 5: 89–96. [18] Papanas N, Ziegler D. Risk factors and comorbidities in diabetic neuropathy: an update 2015. Rev Diabet Stud. 2015; 12: 48–62. [19] Dyck PJ, Kratz KM, Karnes JL, et al. The prevalence by staged severity of various types of diabetic neuropathy, retinopathy, and nephropathy in a population-based cohort: the Rochester Dia betic Neuropathy Study. Neurology 1993; 43: 817–824. [Cor rection: Neurology 1993; 43: 2345.] (Sztanek Ferenc dr., Debrecen, Nagyerdei krt. Fontos információk Részvételi szándék 2020. augusztus 20-ig naponta 14–16 óra között Koltayné Bartha Magdánál jelezhető. Telefon: 06-20/960 5854 – e-mail: baratikor.saar@gmail.com Program: A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) Program: 900 Szentmise 1000 Üdvözlések Németh Norbert, polgármester Prof. Dr. Kellermayer Miklós: „Húsz év” 1030 Márfi Gyula (nyugalmazott veszprémi érsek): „Gondolatok Európa jövőjéről” 1100 Avanesian Alex (dramaturg, szerkesztő, rendező): „Kőország” 1130 Lehoczky László (rendező, szerkesztő): „A szeretet a legnagyobb! – gondolatok a média hivatásáról" 1400 Prof. Dr. Kellermayer Miklós, Prof. Dr. Sándor Zoltán: „A molekuláktól a komplex rendszerekig” 1430 Prof. Dr. Cziráki Attila: „A kardiológia új dimenziói” 1500 Prof. Dr. Pár Alajos: „Haladás a vírushepatitiszek prevenciójában és gyógyításában” 1530 Prof. Dr. Than Péter: „Innovatív endoprotetika” A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) g 900 Szentmise 1000 Üdvözlések Németh Norbert, polgármester Prof. Dr. Kellermayer Miklós: „Húsz év” 1030 Márfi Gyula (nyugalmazott veszprémi érsek): „Gondolatok Európa jövőjéről” 1100 Avanesian Alex (dramaturg, szerkesztő, rendező): „Kőország” 1130 Lehoczky László (rendező, szerkesztő): „A szeretet a legnagyobb! – gondolatok a média hivatásáról" 1400 Prof. Dr. Kellermayer Miklós, Prof. Dr. Sándor Zoltán: „A molekuláktól a komplex rendszerekig” 1430 Prof. Dr. Cziráki Attila: „A kardiológia új dimenziói” 1500 Prof. Dr. Pár Alajos: „Haladás a vírushepatitiszek prevenciójában és gyógyításában” 1530 Prof. Dr. Than Péter: „Innovatív endoprotetika” A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) 900 Szentmise 1000 Üdvözlések Németh Norbert, polgármester Prof. Dr. Kellermayer Miklós: „Húsz év” 1030 Márfi Gyula (nyugalmazott veszprémi érsek): „Gondolatok Európa jövőjéről” 1100 Avanesian Alex (dramaturg, szerkesztő, rendező): „Kőország” 1130 Lehoczky László (rendező, szerkesztő): „A szeretet a legnagyobb! – gondolatok a média hivatásáról" 1400 Prof. Dr. Kellermayer Miklós, Prof. Dr. Sándor Zoltán: „A molekuláktól a komplex rendszerekig” 1430 Prof. Dr. Cziráki Attila: „A kardiológia új dimenziói” 1500 Prof. Dr. Pár Alajos: „Haladás a vírushepatitiszek prevenciójában és gyógyításában” 1530 Prof. Dr. Program: Than Péter: „Innovatív endoprotetika” 900 Szentmise 1030 Márfi Gyula (nyugalmazott veszprémi érsek): „Gondolatok Európa jövőjéről” 1100 Avanesian Alex (dramaturg, szerkesztő, rendező): „Kőország” 1500 Prof. Dr. Pár Alajos: „Haladás a vírushepatitiszek prevenciójában és gyógyításában” 1530 Prof. Dr. Than Péter: „Innovatív endoprotetika” 1530 Prof. Dr. Than Péter: „Innovatív endoprotetika” A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) 0 ■ 161. évfolyam, 30. szám 1251 ORVOSI HETILAP Unauthenticated \| Downloaded 10/10/22 12:31 PM UTC
https://openalex.org/W4293879621	https://www.duo.uio.no/bitstream/10852/100668/1/1-s2.0-S002236972200378X-main.pdf	English	null	In-situ electron loss spectroscopy reveals surface dehydrogenation of hydrated ceria nanoparticles at elevated temperatures	Journal of physics and chemistry of solids	2,022	cc-by	5,943	In-situ electron loss spectroscopy reveals surface dehydrogenation of hydrated ceria nanoparticles at elevated temperatures sen a,, Xinwei Sun b, Ingvild Thue Jensen a, Øystein Prytz c, Truls Norby b Annett Thøgersen a,, Xinwei Sun b, Ingvild Thue Jensen a, Øystein Prytz c, Tr a SINTEF, Materials Physics, Forskningsveien 1, Oslo, NO-0373, Norway b University of Oslo, Department of Chemistry, Centre for Materials Science and Nanotechnology, FERMIO, Gaustadall´een 21, Oslo, NO-0349, Norway c University of Oslo, Department of Physics, Centre for Materials Science and Nanotechnology, Box 1048 Blindern, Oslo, NO-0316, Norway A R T I C L E I N F O Keywords: ETEM EELS Ceria Microscopy Nanopowder Hydrogen Keywords: ETEM EELS Ceria Microscopy Nanopowder Hydrogen Ceria (CeO2) exhibits high reversible oxygen storage capacity at intermediate temperatures (500–800 ◦C) related to an extraordinary and not fully understood reduction of its surfaces. We have investigated pristine and alcohol- dispersed commercially available ceria nanoparticles by in-situ scanning transmission electron microscopy with electron energy loss spectroscopy (STEM-EELS) to examine the dynamic changes during the initial redox reaction process of ceria nanoparticles in an ultra-high vacuum atmosphere using an in-situ heating holder. High spatially resolved EELS was used to estimate the amounts of Ce3+ and Ce4+ in the nanoparticles as a function of tem perature, based on the white-line ratios M5/M4 of the EELS spectra. The results show a nm-range thick surface layer rich in Ce3+ on pristine particles prior to heating. During heating, this oxidises to Ce4+. Heating in high vacuum should normally not lead to oxidation, but the observed results can be understood if the surface layer has an oxyhydroxide composition such as CeOOH, which by heating in the vacuum dehydrogenates and hence oxidises to CeO2, a process that requires diffusion of hydrogen only. This process occurred for all samples, but was more pronounced for the particles that were previously dispersed in ethanol. Thermogravimetric analysis (TGA) by heating the pristine powder in dry atmosphere yielded a considerable weight loss confirming the content of hydroxide and probably water in and on the CeO2 particles. The results suggest that CeO2 surfaces are reduced to a layer of oxyhydroxide by hydrogen-containing molecules like water vapour or alcohols. Contents lists available at ScienceDirect Contents lists available at ScienceDirect * Corresponding author. E-mail address: annett.thogersen@sintef.no (A. Thøgersen). 1. Introduction been made at high temperatures. Previous TEM investigations of the surface of ceria have shown a thin layer of Ce3+ [4,6–8]. Studies have also shown that investigating the particles under high vacuum produces more Ce3+ on the surface than if investigating the particles in an oxygen atmosphere [9,10]. Baalousha et al. [11] and Wu et al. [11] found that ceria nanoparticles have more Ce3+ than larger particles and bulk ceria. It is important to map the compositional variations on the nanoparticle surface in-situ at low temperatures, as reduced ceria nanoparticles may be unstable and small changes may have a big influence in the catalytic functionality of the particles [4]. We have therefore investigated the composition of pristine ceria nanoparticles as well as the composition of ceria dispersed in alcohols prior to analysis, which is the most common way of preparing and dispersing nanoparticles for TEM analysis. Ceria (CeO2) nanoparticles find use in applications such as oxygen sensors, solid oxide fuel cells, and three-way catalysts to remove NOx and CO from combustion exhausts [1–3], owing to the oxide ion con ductivity, electronic structure, and variable stoichiometry. Ceria particles exhibit high and reversible oxygen storage capacities by changing the valence state of Ce between +4 and +3 in the surface at intermediate temperatures (500–800 ◦C). The reduced state involves oxygen vacancies and electrons (representing Ce3+) introducing oxide ion and electronic conductivity in the surface [4,5]. The separation of the Ce3+ and Ce4+ components can be carried out by peak fitting the electron energy loss spectra (EELS) M4,5 peak using transmission elec tron microscopy (TEM), as well as measuring the white line intensity ratio [4,5]. For pure ceria, the reduction from +4 to +3 was found to occur by heating above 730 ◦C, in a hydrogen rich atmosphere [4]. Most studies on the temperature evolution of ceria particles have therefore * Corresponding author. E-mail address: annett.th Available online 27 August 2022 0022-3697/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.jpcs.2022.110955 Received 27 April 2022; Received in revised form 6 August 2022; Accepted 8 August 2022 Journal of Physics and Chemistry of Solids 170 (2022) 110955 Journal of Physics and Chemistry of Solids 170 (2022) 110955 2. Methodology Ceria nanoparticles from Sigma-Aldrich Co. were used for the ex Ceria nanoparticles from Sigma-Aldrich Co. were used for the ex 0022-3697/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). lished by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Journal of Physics and Chemistry of Solids 170 (2022) 110955 A. Thøgersen et al. periments (Cerium (IV) Oxide nanopowder, Pcode: 1001924131, <25 nm particle size). For the in-situ heating measurements, a Protochips Fusion holder was used. TEM samples were prepared by dispersing dry ceria powder or a drop of ceria mixed with either isopropanol or ethanol on a Protochips in-situ heating chip. The samples were analysed by a probe corrected and monochromated FEI Titan G2 60–300 microscope operated at 300 kV, using high resolution scanning TEM (STEM), elec tron energy loss spectroscopy (EELS) with a Gatan imaging filter and detector, and Super-X energy dispersive spectroscopy (EDS). In order to reduce beam damage, electron beam current of less than 0.1 nA was used. In some of our experiments we also performed a double analysis of each particle in the heating test to see if there were any changes in our results after the spectral image EELS acquisition. No damage was observed in our extra tests. The EELS spectra were acquired using dual EELS spectrum image (SI), in order to avoid referencing issues. This means that we acquire a low loss EELS spectrum with the zero-loss peak together with the core-loss EELS spectrum of the Ce-M4,5 peak, and using the zero-loss peak as the referencing peak. The post acquisition data processing was performed using Digital Micrograph from Gatan Inc. and CasaXPS for peak fitting analysis. More details on the peak fitting is given in the Results chapter. corresponding well to the CeO2 structure. A 1:2 compositional rela tionship between Ce and O was also confirmed by EDS. i The content of Ce3+ in the samples may be affected by the treatment of the ceria nanoparticles prior to the analysis. We have therefore investigated ceria treated in three different ways common to the TEM- sample preparation process namely, as-received (pristine) ceria pow der and ceria dispersed in ethanol or isopropanol. EELS spectra in Fig. 2 shows spectra taken from bulk and surface of our caria nanopowder, with a clear Ce3+ and Ce4+ signature. 2. Methodology Even though there may be minor Ce4+ and Ce3+ present in the spectra as well, these spectra shows the signature shapes we are analyzing in this paper. The spectra are composed of the two peaks, due to spin-orbit splitting, (white lines) M4,5. These lines are caused by excitations from the M4,5 (3d) to the unoccupied states in the N(4f) band, reflecting changes in the occupancy of the 4f band and thereby also the oxidation state [4]. The EELS Ce-M4,5 peaks have been peak-fitted with Gaussian-Lorentzian peaks corre sponding to Ce-M5 and Ce-M4, where the Ce-M5 and Ce-M4 peaks of Ce3+ are located at energy losses of 880.1 eV and 898 eV respectively, with an energy separation of about 17.9 ± 0.2 eV. For the Ce4+ component, the Ce-M5 peak is located at 881.4 eV and Ce-M4 peak at 899.6 eV, with an energy separation of about 18.2 ± 0.2 eV. Intensity ratios of the M5 to M4 white lines of Ce3+ was found to be 1.3 (IM5/IM4 = 1.3.), while for Ce4+ the intensity difference is IM5/IM4 = 0.8. A variation in-between these two numbers can be related to a change in oxidation state. In-situ heating was performed with temperature steps of 0.05 ◦C per second, for then to wait about 10 min (before taking EELS spectra) to avoid drift due to heating. EELS spectra were acquired with either a 0.25 eV/ch dispersion (collection semi-angle 12 mrad and convergence semi-angle 21 mrad), or with a 0.1 eV/ch dispersion (collection semi- angle 100 mrad and convergence semi-angle 30 mrad), with some var iations in spectrum image pixel size. We have used the zero-loss peak (from dual EELS) for initial energy referencing. However, due to some misalignment of the drift tube or the image filter, we also experienced a shift in the peaks between samples dispersed in ethanol and isopropanol. In order to more easily compare the peaks we aligned the 3+ peaks for the samples at room temperature. The analysis has been carried out for eight different particles, and the results of some of them are presented in the following sections. Thermogravimetric analysis (TGA) was made with a Netzsch 449 F1 Jupiter® thermal analyser (Netzsch GmbH, Germany) on the as-received CeO2 nanopowder, uniaxially pressed at 3 MPa into a pellet. The sample was first dried at 26 ◦C in N2 gas for 3 h. 2. Methodology It was further dried and degassed during stepwise heating to 400 ◦C at 3 K/min, and then held at 400 ◦C for 6 h to ensure complete removal of water and hydrogen and any organic residue from the surface. The weight increase was then measured by flowing wet (pH2O = 0.026 atm) N2 over the sample at 400, 125 and 26 ◦C during cooling. The relative increase in weight and corresponding uptake of water is calculated on the basis that the content of adsorbed water and other gases in dry N2 at 400 ◦C was zero. Fig. 2. EELS spectra of the Ce-M4,5 peak, in an area with mainly Ce3+ and Ce4+ showing the different spectral signatures. 3.4. Temperature evolution of the ceria samples The composition of Ce3+ in the four samples as a function of tem perature is shown in Fig. 7, for the whole particle (A) and for the surface (B). The error bars are estimated uncertainty/errors in the calculated oxidation state (y-axis, ± 0.1), and calculated composition from EELS data (y-axis, ± 4%), found by varying the fitting parameters and mea surements of the spectra. All samples start with a relatively high amount of Ce3+ in the whole particle, which decreases with increasing temper ature (Fig. 7A). The sample dispersed in isopropanol shows a small initial increase in the amount of Ce3+, with a slight increase at 200 ◦C, before it decreases further. 3.3. CeO2-powder in isopropanol solution p p EELS spectra of the pristine ceria particle is shown in Fig. 3A and B, from the whole particle and from the surface layer respectively. The percentages of Ce3+ at the corresponding temperature, as well as the estimated oxidation state based on the white line ratio are presented in Table 1. The pristine particle contains 53% Ce3+ in the whole particle at room temperature (estimated oxidation state of +3.3), and 80% Ce3+ at the surface, as shown in Fig. 3B. During heating in high vacuum, the Ce- M4,5 peak moves to higher energy loss, and the white line intensity ratio shifts to a clear Ce4+ signature. At 400 ◦C, the particle contains only 13% Ce3+, and we can observe 31% Ce3+ at the surface layer. Energy loss mapping of the integrated EELS signal of the Ce4+ and Ce3+ peaks pre sented in Fig. 4 shows that at room temperature the particle contains a mix of both Ce3+ and Ce4+. During heating, most of the Ce3+ in the bulk particle transform into Ce4+, while the transformation at the surface is not as quick, resulting in a layer of Ce3+ at the surface. This evolution profile was also found for the three other samples we have examined. EELS spectra of ceria dispersed in isopropanol prior to analysis are shown in Fig. 3E and F. Ce3+ is observed at both the whole nanoparticle and the surface at room temperature, with 64% and 100% Ce3+, respectively. Increasing the temperature shows a significant reduction in Ce3+ content of the whole particle, resulting in more Ce4+. However, no significant decrease in Ce3+ is observed at the surface. The corre sponding energy filtered image are shown in Fig. 6. The images show that at room temperature, Ce3+ is pronounced at the surface. During heating, Ce3+ is reduced overall in the particle. 3. Results The shape and sizes of the ceria nanoparticles studied in this work are shown in the STEM images in Fig. 1 A and E, with the corresponding electron diffraction images in B and D. The diffraction pattern in Fig. 1B is consistent with the CeO2 crystal structure with a space group of Fm- 3m (225) in the [110] zone axis. Diffraction of several particles in (E) shows ring patterns resulting from many crystal orientations, all Fig. 1. A) HAADF STEM image of a CeO2 nanoparticle, B) corresponding electron diffraction pattern, C) high resolution HAADF STEM image, D) electron diffraction pattern from E), and E) STEM image of many CeO2 particles. Fig. 1. A) HAADF STEM image of a CeO2 nanoparticle, B) corresponding electron diffraction pattern, C) high resolution HAADF STEM image, D) electron diffraction pattern from E), and E) STEM image of many CeO2 particles. Fig. 2. EELS spectra of the Ce-M4,5 peak, in an area with mainly Ce3+ and Ce4+ showing the different spectral signatures. Fig. 2. EELS spectra of the Ce-M4,5 peak, in an area with mainly Ce3+ and Ce4+ showing the different spectral signatures. 2 A. Thøgersen et al. A. Thøgersen et al. Journal of Physics and Chemistry of Solids 170 (2022) 110955 3.1. Pristine CeO2-powder particle at the different temperatures. The result reveals that the Ce3+ is mainly found at the surface of the particle. There is also a clear decrease in Ce3+ at 400 ◦C. We examined four pristine particles at 25 ◦C, 200 ◦C, and 400 ◦C, where most of the changes in composition were found to occur. Only the spectra and images of one of the particles will be presented, while the data of the other three nanoparticles are plotted in Section 3.4. Table 1 Table 1 Amount of Ce3+ present in pristine ceria, ceria dispersed in ethanol, and ceria dispersed in isopropanol, with the mean oxidation state (ox. state) calculated based on the white line ratio. Table 1 Amount of Ce3+ present in pristine ceria, ceria dispersed in ethanol, and ceria dispersed in isopropanol, with the mean oxidation state (ox. state) calculated based on the white line ratio. ou t o Ce p ese t p st e ce a, ce a d spe sed et a o , a d ce a d spe sed sop opa o , t t e ea o dat o state (o . state) ca cu ated ased o the white line ratio. Particle Surface Pristine Ethanol Isopropanol Pristine Ethanol Isopropanol Temp. Ce3+ ox. Ce3+ ox. Ce3+ ox. Ce3+ ox. Ce3+ ox. Ce3+ ox. (◦C) (%) state (%) state (%) state (%) state (%) state (%) state 25 53 +3.3 71 +3.3 64 +3.3 80 +3.0 54 +3.4 100 +3.0 100 58 +3.5 61 +3.4 59 +3.6 100 +3.0 200 26 +3.6 13 +3.8 74 +3.1 24 +3.4 26 +3.7 100 +3.1 400 13 +3.8 8 +3.7 35 +3.8 31 +3.6 16 +3.9 100 +3.0 500 6 +3.7 11 +3.7 600 13 +3.8 29 +3.7 700 16 +3.7 29 +3.6 Fig. 4. STEM image of a pristine ceria particle, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at 25 ◦C, 200 ◦C, and 400 ◦C. Particle Surface Pristine Ethanol Isopropanol Pristine Ethanol Isopropanol Temp. Ce3+ ox. Ce3+ ox. Ce3+ ox. Ce3+ ox. Ce3+ ox. Ce3+ ox. (◦C) (%) state (%) state (%) state (%) state (%) state (%) state 25 53 +3.3 71 +3.3 64 +3.3 80 +3.0 54 +3.4 100 +3.0 100 58 +3.5 61 +3.4 59 +3.6 100 +3.0 200 26 +3.6 13 +3.8 74 +3.1 24 +3.4 26 +3.7 100 +3.1 400 13 +3.8 8 +3.7 35 +3.8 31 +3.6 16 +3.9 100 +3.0 500 6 +3.7 11 +3.7 600 13 +3.8 29 +3.7 700 16 +3.7 29 +3.6 Fig. 4. STEM image of a pristine ceria particle, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at 25 ◦C, 200 ◦C, and 400 ◦C. Fig. 4. STEM image of a pristine ceria particle, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at 25 ◦C, 200 ◦C, and 400 ◦C. 3.2. CeO2-powder in ethanol solution EELS spectra of ceria particles dispersed in ethanol prior to analysis are shown in Fig. 3C and D. At room temperature, the particle contains 71% Ce3+ (estimated oxidation state +3.3). The white line intensity ratio also shows a clear Ce3+ signature. During heating in high vacuum, we observe a clear shift in the Ce-M4,5 peak to higher energy loss, as well as a shift in the white line intensity ratio to a pronounced Ce4+ signature. At 700 ◦C, the particle contains only 16% of Ce3+. This can also be observed in the spectra from the surface (in Fig. 3D) where Ce4+ dom inates at higher temperatures. Fig. 5 shows energy filtered images of the The calculated oxidation state based on the white line ratio as a function of Ce3+ content is shown in Fig. 8, using data from all samples investigated. A linear thread-line is fitted to the data points and shows a near-linear evolution of the oxidation state, with a R2 value of about 0.86 (a measurement of how good the line fits the data points, where 1 is the best). However, some of the data points are not connected to the ig. 3. EELS spectra of the Ce-M4,5 peaks, both from the whole particle (A,C,E) and the surface (B,D,F), of pristine ceria particle (A and B), ceria first dispersed in thanol (C and D), and ceria first dispersed in isopropanol (E and F), at 25 ◦C–700 ◦C. The component Ce4+ is shown in blue, Ce3+is shown in red, and the black is the xperimental spectra. Fig. 3. EELS spectra of the Ce-M4,5 peaks, both from the whole particle (A,C,E) and the surface (B,D,F), of pristine ceria particle (A and B), ceria first dispersed in ethanol (C and D), and ceria first dispersed in isopropanol (E and F), at 25 ◦C–700 ◦C. The component Ce4+ is shown in blue, Ce3+is shown in red, and the black is the experimental spectra. Fig. 3. EELS spectra of the Ce-M4,5 peaks, both from the whole particle (A,C,E) and the surface (B,D,F), of pristine ceria particle (A and B), ceria first dispersed in ethanol (C and D), and ceria first dispersed in isopropanol (E and F), at 25 ◦C–700 ◦C. The component Ce4+ is shown in blue, Ce3+is shown in red, and the black is the experimental spectra. 3 Journal of Physics and Chemistry of Solids 170 (2022) 110955 A. Thøgersen et al. Table 1 age of a pristine ceria particle, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at 25 ◦C, 200 ◦C, and 400 ◦C. Fig. 4. STEM image of a pristine ceria particle, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at 25 ◦C, 200 Fig. 5. STEM image of a ceria particle dispersed in ethanol, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at room temperature 25 ◦C, 100 ◦C, 200 ◦C, 400 ◦C, 500 ◦C, 600 ◦C, and 700 ◦C. Fig. 5. STEM image of a ceria particle dispersed in ethanol, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at room temperature 25 ◦C, 100 ◦C, 200 ◦C, 400 ◦C, 500 ◦C, 600 ◦C, and 700 ◦C. A. Thøgersen et al. Journal of Physics and Chemistry of Solids 170 (2022) 110955 Fig. 6. A) STEM image of a ceria particle dispersed in isopropanol, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at room temperature (25◦), 100 ◦C, 200 ◦C, and 400 ◦C. icle dispersed in isopropanol, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at room temperature (25◦), 100 ◦C Fig. 6. A) STEM image of a ceria particle dispersed in isopropanol, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) 200 ◦C, and 400 ◦C. Fig. 6. A) STEM image of a ceria particle dispersed in isopropanol, and EELS-mapping of the Ce3+ (red) and Ce4+ (blue) peaks at room temperature (25◦), 100 ◦C, 200 ◦C, and 400 ◦C. Fig. 7. Plot showing the fraction of Ce3+ present in A) the entire ceria particle, and B) ceria particle surface, with increasing temperature. thread-line. From our experience, a combination of the oxidation state based on the white line ratio together with the fitted peaks is necessary to correctly determine the compositional profile of the samples Fig. 7. Plot showing the fraction of Ce3+ present in A) the entire ceria particle, and B) ceria particle surface, with increasing temperature. Fig. 8. A graph showing the average oxidation state found from using the white line ratio of the EELS Ce-M4,5 spectra of the entire particle, as a function of the fraction of Ce3+ present in the sample found by peak fitting the EELS spectra of the different particles. Fig. 9. Table 1 Time-dependent mass change of CeO2 under dry and wet (pH2O = 0 026 atm) N (left y axis) in response to temperature change (right y axis) Fig. 8. A graph showing the average oxidation state found from using the white line ratio of the EELS Ce-M4,5 spectra of the entire particle, as a function of the fraction of Ce3+ present in the sample found by peak fitting the EELS spectra of the different particles. 3.5. Thermogravimetry (TG) This shows that H in CeO2 is indeed present as H+ compensated by Ce3+ in what may be termed CeOOH-like structures in a surface layer. If the TG results showing a loss of water from the pristine oxide of 0.092 mol H2O/mol CeO2 is interpreted as loss of H2, this corresponds to as much as 0.83 mol H2/mol CeO2, or 1.66 H/CeO2. It is hence likely that pristine CeO2 nanoparticles have a mixture of H inside or on the surface, e.g. like regions or a layer of CeOOH, and H2O adsorbed on the surface. The TG results for the temperature range 26–400 ◦C during heating in dry N2 and cooling in wet N2 are shown in Fig. 9. During heating under dry conditions, a significant amount of water is desorbed from the oxide upon heating to 125–200 ◦C, normally taken to reflect physisorbed water [12]. Further heating to 400 ◦C represents the removal of chem isorbed water, possibly also any water in the structure. In total, the water lost, supposedly present in and on the pristine CeO2, amounts to around 0.092 mol H2O per mol CeO2. The amount of water adsorbed in the bulk of CeO2 may be assumed to be negligible due to its unfavourable hydration thermodynamics. Therefore, the mass change may reflect adsorbed water. There may also be water or hydrogen left inside the crystallites from their synthesis. The following water uptakes in wet N2 at 400, 125 and 26 ◦C correspond to approx. 0.003, 0.03 and 0.12 mol H2O per mol CeO2, respectively, in rough agreement with a previous report on CeO2 powder with a specific surface area of 55 m2/g [13]. The existence of a layer of CeOOH on CeO2 surfaces explains many observations, behaviours, and properties of CeO2, e.g. redox and cata lytic behaviour as well as protons found on the surface, but not bulk. The reasons why the surface phase has not been observed earlier may stem from the fact that it has the same oxygen content and a similar structure as the host CeO2, and is very thin, requiring the use of TEM and the understanding gained by in-situ heating experiments. We observed different evolution of the oxidation process for the different particles. Such differences have also been observed in the redox activity level [4,16]. Crozier et al. 4. Discussion EELS results show that both pristine ceria as well as ceria dispersed in alcohols prior to analysis start with high amounts of Ce3+ at the surface of the particles. When heating in vacuum, this partly transforms into Ce4+, where it is stable up to 700 ◦C. Johnston-Peck et al. [14] studied dose-rate-dependent damage of cerium dioxide by STEM-EELS. They found that when ceria is exposed to high beam damage, oxygen va cancies will form, and ceria will be reduced from +4 to +3 oxidation state. Since the particles in our study mainly experience a shift from +3 to +4 oxidation state, this process cannot be due to beam damage. The amount of Ce3+ and the evolution process during heating in the three types of samples are not significantly different, only a small increase in Ce3+ was found in the samples dispersed in ethanol. However, the amount of Ce3+ in the pristine samples is surprising. Heating in high vacuum should normally not lead to further oxidation. If the reoxidation to Ce4+ was due to annihilation of oxygen vacancies, it would require uptake of oxygen (which is not available in high vacuum) and lower temperatures (due to the negative entropy change of gas uptake). However, the results can be rationalized if the surface layer is an oxy hydroxide (based on the known stability of rare earth (III) oxy hydroxides) that we for simplicity represent as CeOOH, not oxide like Ce2O3 (both being Ce3+). Dehydrogenation of a hydroxide yields hydrogen release, which is natural in the TEM vacuum, and it increases with temperature due to the positive entropy change of gas release. Thereby, when heating the particles in high vacuum, the surface layer dehydrogenates (oxidises) to CeO2, a process that requires diffusion of protons only: 3.5. Thermogravimetry (TG) [4] reported that some particles were found to be inactive at room temperature and that heating till 700 ◦C in hydrogen resulted in no significant changes. Wang et al. [16] found that CeO2–ZrO2 solid solution nanoparticles with a large variation in con centrations through the particle were more active than homogeneous ones. They identified types of ceria nanoparticle behaviour depending on their redox activity level. In our case, with samples dispersed in al cohols, the evolution of the oxidation process may be dependent on the thickness of the CeOOH layer at the surface. The surface of ceria dispersed in isopropanol showed predominately Ce3+ with very little changes after heating. For particles dispersed in ethanol we observed both Ce3+ and Ce4+ at the surface, with a clear reduction upon heating. The ethanol sample has overall more Ce3+ in the whole particle at room temperature compare to the isopropanol sample, and it experiences a more abrupt decrease in Ce3+ content. The isopropanol sample on the other hand has still a large amount of Ce3+ left in the particle at 200 ◦C, located mostly at the surface. To our knowledge, such results have not been reported, and we recommend future studies on a larger set of particles to fully understand the difference between ceria dispersed in ethanol vs isopropanol. 5. Conclusion We have found that during low temperature heating of ceria nano particles in vacuum, the oxidation state changes from +3 to +4 in a surface layer. There are small differences in the oxidation process be tween different particles, but at 400 ◦C and higher, they all have about the same compositional structure with mainly Ce4+. This process occurs for both pristine sample and samples dispersed in alcohols. However, the Ce3+ content is much higher in the latter. Heating in vacuum should normally not lead to further oxidation, and our observations indicate that a layer of oxyhydroxide like CeOOH dehydrogenates to CeO2, a process that only requires diffusion of H. This means that much of the Ce3+ we see on the surface is not a reduced oxide such as Ce2O3 but a reduced oxyhydroxide like CeOOH, i.e. the Ce3+ is not compensated by oxygen vacancies but by protons. These results are supported by TG results showing that the pristine CeO2 powder contains a considerable amount of water and possibly hydrogen, probably partly adsorbed from ambient humidity and partly left from the synthesis of the powder. The use of in-situ TEM and EELS to map the evolution process of Ce3+ to Ce4+ in ceria nanoparticles has hence provided new insight to understand their properties, utilizing that the M4,5 white line ratio gives good indication on the oxidation state of the particles. CeOOH⟶CeO2 + 1 2H2(g) The formation of the CeOOH layer is expected to happen under ambient conditions according to a reducing hydrogenation from water vapour: CeO2 + 1 2H2O(g)⟶CeOOH + 1 4O2(g) Wang et al. [15] found the presence of hydroxyl groups (O–H) after water exposure of ceria surfaces. They suggest that these groups as well as hydrocarbons may also be absorbed from ambient environments. This will have a large effect on the wettability of the surface of ceria. The formation of a layer and not a complete hydrogenation of bulk CeO2 suggests that the formation of a surface of CeOOH and the interface between CeO2 and CeOOH, along with the associated charge separation and space charge layers, lowers the energy compared to a single CeO2 surface. The growth of the layer then stops as soon as the surface attains bulk properties and/or the two space charge layers in the CeOOH layer meet. Fig. 8. A graph showing the average oxidation state found from using the white line ratio of the EELS Ce-M4,5 spectra of the entire particle, as a function of the fraction of Ce3+ present in the sample found by peak fitting the EELS spectra of the different particles. Fig. 9. Time-dependent mass change of CeO2 under dry and wet (pH2O = 0.026 atm) N2 (left y-axis) in response to temperature change (right y-axis). Fig. 7. Plot showing the fraction of Ce3+ present in A) the entire ceria particle, and B) ceria particle surface, with increasing temperature. thread-line. From our experience, a combination of the oxidation state based on the white line ratio together with the fitted peaks is necessary to correctly determine the compositional profile of the samples. thread-line. From our experience, a combination of the oxidation state based on the white line ratio together with the fitted peaks is necessary to correctly determine the compositional profile of the samples. Fig. 9. Time-dependent mass change of CeO2 under dry and wet (pH2O = 0.026 atm) N2 (left y-axis) in response to temperature change (right y-axis). 5 A. Thøgersen et al. Journal of Physics and Chemistry of Solids 170 (2022) 110955 Data availability p y y p , Mater. 20 (16) (2010) 2664–2674, https://doi.org/10.1002/adfm.201000279. URL, https://onlinelibrary.wiley.com/doi/abs/10.1002/adfm.201000279. Data will be made available on reasonable request. [9] A.C. Johnston-Peck, W.-C.D. Yang, J.P. Winterstein, R. Sharma, A.A. Herzing, In situ oxidation and reduction of cerium dioxide nanoparticles studied by scanning transmission electron microscopy, Micron 115 (2018) 54–63, https://doi.org/ 10.1016/j.micron.2018.08.008. URL, https://www.sciencedirect.com/science/ article/pii/S0968432818302361. A. Thøgersen et al. Journal of Physics and Chemistry of Solids 170 (2022) 110955 Xinwei Sun: Data curation; Formal analysis; Investigation; Method ology (TG + samples), Writing part of manuscript and help with edit. ultramic.2008.05.015. URL, http://www.sciencedirect.com/science/article/pii/ S0304399108001782. [5] S. Turner, S. Lazar, B. Freitag, R. Egoavil, J. Verbeeck, S. Put, Y. Strauven, G. Van Tendeloo, High resolution mapping of surface reduction in ceria nanoparticles, Nanoscale 3 (2011) 3385–3390, https://doi.org/10.1039/C1NR10510H. URL. Ingvild Thue Jensen: Formal analysis; Investigation; Øystein Prytz: Formal analysis; Funding acquisition; Project administration; Øystein Prytz: Formal analysis; Funding acquisition; Project administration; [6] U.M. Graham, M.T. Tseng, J.B. Jasinski, R.A. Yokel, J.M. Unrine, B.H. Davis, A. K. Dozier, S.S. Hardas, R. Sultana, E.A. Grulke, D.A. Butterfield, Invivo processing of ceria nanoparticles inside liver: impact on free-radical scavenging activity and oxidative stress, arXiv:https://chemistry-europe.onlinelibrary.wiley.com/doi/pd f/10.1002/cplu.201402080, Chem Plus Chem. 79 (8) (2014) 1083–1088, https:// doi.org/10.1002/cplu.201402080. URL, https://chemistry-europe.onlinelibrary. wiley.com/doi/abs/10.1002/cplu.201402080. [6] U.M. Graham, M.T. Tseng, J.B. Jasinski, R.A. Yokel, J.M. Unrine, B.H. Davis, A. K. Dozier, S.S. Hardas, R. Sultana, E.A. Grulke, D.A. Butterfield, Invivo processing of ceria nanoparticles inside liver: impact on free-radical scavenging activity and oxidative stress, arXiv:https://chemistry-europe.onlinelibrary.wiley.com/doi/pd Truls Norby: Formal analysis; Funding acquisition; Supervision; Writing part of manuscript and help with edit. f/10.1002/cplu.201402080, Chem Plus Chem. 79 (8) (2014) 1083–1088, https:// doi.org/10.1002/cplu.201402080. URL, https://chemistry-europe.onlinelibrary. wiley.com/doi/abs/10.1002/cplu.201402080. Declaration of competing interest [7] B. Goris, S. Turner, S. Bals, G. Van Tendeloo, Three-dimensional valency mapping in ceria nanocrystals, ACS Nano 8 (10) (2014) 10878–10884, pMID: 25286190. arXiv:10.1021/nn5047053. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. [8] J.M. Perkins, S. Fearn, S.N. Cook, R. Srinivasan, C.M. Rouleau, H.M. Christen, G. D. West, R.J.H. Morris, H.L. Fraser, S.J. Skinner, J.A. Kilner, D.W. McComb, Anomalous oxidation states in multilayers for fuel cell applications, arXiv:htt ps://onlinelibrary.wiley.com/doi/pdf/10.1002/adfm.201000279, Adv. Funct. Mater. 20 (16) (2010) 2664–2674, https://doi.org/10.1002/adfm.201000279. URL, https://onlinelibrary.wiley.com/doi/abs/10.1002/adfm.201000279. Acknowledgement [10] F. Zhang, P. Wang, J. Koberstein, S. Khalid, S.-W. Chan, Cerium oxidation state in ceria nanoparticles studied with x-ray photoelectron spectroscopy and absorption near edge spectroscopy, Surf. Sci. 563 (1) (2004) 74–82, https://doi.org/10.1016/ j.susc.2004.05.138. URL, https://www.sciencedirect.com/science/article/pii/ S003960280400812X. Acknowledgement to Kathrin Michel, at Institute of Physical Chem istry, Justus Liebig University Giessen, for her contributions to this project. The Research Council of Norway (RCN) is acknowledged for the support to The Norwegian Center for Transmission Electron Microscopy [197405], and for support through projects “MoZEES” (257653) and “SUPROX” (280868).” [11] J. I, J.R. L, M. Baalousha, P. Le Coustumer, Characterisation of structural and surface speciation of representative commercially available cerium oxide nanoparticles, Environ. Chem. 7 (2010) 377–385. [12] S. Raz, K. Sasaki, J. Maier, I. Riess, Characterization of adsorbed water layers on y2o3-doped zro2, Solid State Ionics 143 (2) (2001) 181–204, https://doi.org/ 10.1016/S0167-2738(01)00826-8. URL, https://www.sciencedirect.com/science/ article/pii/S0167273801008268. Author statement This is the individual author’s contributions to the paper: Annett Thøgersen: Data curation; Formal analysis; Funding acquisi tion; Investigation; Methodology (TEM/EELS work); Project adminis tration; Main writer of manuscript/Review and editing. 6 References [1] J. Kaˇspar, P. Fornasiero, M. Graziani, Use of ceo2-based oxides in the three-way catalysis, Catal. Today 50 (2) (1999) 285–298, https://doi.org/10.1016/S0920- 5861(98)00510-0. URL, http://www.sciencedirect.com/science/article/pii/ S0920586198005100. p [13] E. Mamontov, Observation of fragile-to-strong liquid transition in surface water in ceo2, J. Chem. Phys. 123 (17) (2005), 171101 arXiv:10.1063/1.2125729. [14] A.C. Johnston-Peck, J.S. DuChene, A.D. Roberts, W.D. Wei, A.A. Herzing, Dose- rate-dependent damage of cerium dioxide in the scanning transmission electron microscope, Ultramicroscopy 170 (2016) 1–9, https://doi.org/10.1016/j. ultramic.2016.07.002. URL, https://www.sciencedirect.com/science/article/pii/ S0304399116300936. [2] A. Alinezhadchamazketi, A.A. Khodadadi, Y. Mortazavi, A. Nemati, Catalytic evaluation of promoted ceo2-zro2 by transition, alkali, and alkaline-earth metal oxides for diesel soot oxidation, J. Environ. Sci. 25 (12) (2013) 2498–2506, https://doi.org/10.1016/S1001-0742(12)60334-9. URL, http://www.sciencedirec t.com/science/article/pii/S1001074212603349. [15] Y. Wang, Q. Zhou, L. Kang, L. Yang, H. Wu, Z. Zhou, C. Xiao, J. Guo, F. Yang, S. Zhang, G. Li, Y. Jin, Oxide-water interaction and wetting property of ceria surfaces tuned by high-temperature thermal aging, Appl. Surf. Sci. 554 (2021), 149658, https://doi.org/10.1016/j.apsusc.2021.149658. URL, https://www.sci encedirect.com/science/article/pii/S0169433221007340. [ ] i h d i h d i i f [3] A. Trovarelli, C. de Leitenburg, M. Boaro, G. Dolcetti, The utilization of ceria in industrial catalysis, Catal. Today 50 (2) (1999) 353–367, https://doi.org/10.1016/ S0920-5861(98)00515-X. URL, http://www.sciencedirect.com/science/article/ pii/S092058619800515X. [16] R. Wang, P.A. Crozier, R. Sharma, J.B. Adams, Measuring the redox activity of individual catalytic nanoparticles in cerium-based oxides, Nano Lett. 8 (3) (2008) 962–967, pMID: 18251517. arXiv:10.1021/nl073135c. [4] P.A. Crozier, R. Wang, R. Sharma, In situ environmental tem studies of dynamic changes in cerium-based oxides nanoparticles during redox processes, Ultramicroscopy 108 (11) (2008) 1432–1440, https://doi.org/10.1016/j.
https://openalex.org/W1642527378	https://www.frontiersin.org/articles/10.3389/fevo.2015.00022/pdf	English	null	The spatial information content of the honey bee waggle dance	Frontiers in human neuroscience	2,015	cc-by	6,451	ORIGINAL RESEARCH published: 18 March 2015 doi: 10.3389/fevo.2015.00022 Edited by: Edited by: Madeleine Beekman, The University of Sydney, Australia Reviewed by: Timothy Martin Schaerf, The University of Sydney, Australia Martin Middendorf, University of Leipzig, Germany Correspondence: Roger Schürch, Clinical Trials Unit, University of Bern, Finkenhubelweg 11, 3012 Bern, Switzerland roger.schuerch@ctu.unibe.ch Reviewed by: Timothy Martin Schaerf, The University of Sydney, Australia Martin Middendorf, University of Leipzig, Germany Reviewed by: Timothy Martin Schaerf, The University of Sydney, Australia Martin Middendorf, University of Leipzig, Germany Keywords: honey bee, waggle dance, information theory, spatial information, Apis mellifera The spatial information content of the honey bee waggle dance Roger Schürch 1, 2 and Francis L. W. Ratnieks 1 1 Laboratory of Apiculture and Social Insects, School of Life Sciences, University of Sussex, Brighton, UK, 2 Clinical Trials Unit, University of Bern, Bern, Switzerland In 1954, Haldane and Spurway published a paper in which they discussed the information content of the honey bee waggle dance with regard to the ideas of Norbert Wiener, who had recently developed a formal theory of information. We return to this concept by reanalyzing the information content in both vector components (direction, distance) of the waggle dance using recent empirical data from a study that investigated the accuracy of the dance. Our results show that the direction component conveys 2.9 bits and the distance component 4.5 bits of information, which agrees to some extent with Haldane and Spurway’s estimates that were based on data gathered by von Frisch. Of course, these are small amounts of information compared to what can be conveyed, given enough time, by human language, or compared to what is routinely transferred via the internet. Nevertheless, small amounts of information can be very valuable if it is the right information. The receivers of this information, the nestmate bees, know how to react adaptively so that the value of the information is not negated by its low information content. 1. Introduction Correspondence: Roger Schürch, Clinical Trials Unit, University of Bern, Finkenhubelweg 11, 3012 Bern, Switzerland roger schuerch@ctu unibe ch Correspondence: Roger Schürch, Clinical Trials Unit, University of Bern, Finkenhubelweg 11, 3012 Bern, Switzerland roger schuerch@ctu unibe ch In 1954, Haldane and Spurway (1954) published a paper in the scientiﬁc journal Insectes Sociaux with the title “A statistical analysis of communication in Apis mellifera and a comparison with com- munication in other animals.” Haldane and Spurway (1954), using the data set of Karl von Frisch, looked at the waggle dance communication using an information theory approach, at least in terms of the direction communicated by a dancing bee (von Frisch, 1946, 1967). Of course, von Frisch’s primary target was to understand the dance language, not to obtain a precise calibration to study where the bees had foraged: he chose to work only with good dancers (Chittka and Dornhaus, 1999), which seems to underestimate systematically the error present in the dances (Schürch and Couvillon, 2013; Schürch et al., 2013) and therefore bias the data. Specialty section: This article was submitted to Behavioral and Evolutionary Ecology, a section of the journal Frontiers in Ecology and Evolution Received: 05 December 2014 Accepted: 24 February 2015 Published: 18 March 2015 Specialty section: This article was submitted to Behavioral and Evolutionary Ecology, a section of the journal Frontiers in Ecology and Evolution Specialty section: This article was submitted to Behavioral and Evolutionary Ecology, a section of the journal Frontiers in Ecology and Evolution Many years have now passed since the original paper, and we have a better understanding of what—and how well—the bees indicate with their dance (Couvillon, 2012; Couvillon et al., 2012; Schürch et al., 2013). Therefore, we were interested to revisit the ideas and theories put forward by Haldane and Spurway (1954) by analyzing them with our own calibration data from Schürch et al. (2013). Received: 05 December 2014 Accepted: 24 February 2015 Published: 18 March 2015 2. Information and Communication When a bee performs a waggle dance, she transfers information to her unoccupied nestmates about the location of a resource, for example food or a new nest site she has visited (von Frisch, 1946, 1967). In information theory, information is deﬁned as a reduc- tion in uncertainty (Shannon, 1948). Hence, from an information theory viewpoint, a dancing bee will reduce in her dance follow- ers the uncertainty of where she, the dancer, has been collecting her food, or where she has found a suitable nest site. Informa- tion theory aﬀords us a way of quantifying the initial uncertainty and the uncertainty remaining after a dance has occurred, which therefore allows for a quantiﬁcation of the information. I = Hbefore −Hafter = log 10 −log 2 = 2.3 bit. (3) (3) From the deﬁnition of entropy, it also follows that the informa- tion, I, depends on the set of values of the a priori probabili- ties, pi. If we observe the dances of marked bees that have been trained to feeders at known distances, we cannot gain informa- tion about foraging distance by observing waggle run durations. The received information is not an absolute quantity (Norwich, 1994). The uncertainty in information theory is usually termed entropy and commonly represented by the symbol H (Shannon, 1948; Norwich, 1994). If we ﬁrst consider a random event with x discrete outcomes and the probability of the ith possible outcome has probability pi, H is deﬁned as Regarding the waggle dance communication, we are now only left with one thing to consider. The foraging distance, as well as the directional component, are not communicated discretely but on a continuous scale. Let us suppose that the probability density function p(x) represents the probability of a bee having foraged at distance x. Let x be measured in meters, and 1x is the constant length between discrete partitions of the continuous foraging dis- tance. Then p(x)1x = p(500)(1) is a reasonable approximation to the probability that the foraging distance of a returned forager selected at random from the population will be found to have foraged between 500 and 500 + 1 m. We can also choose a con- venient upper limit for our considerations; 14 km is maximum foraging distance in bees reported in the literature and seems a reasonable choice (Eckert, 1933). Citation: Schürch R and Ratnieks FLW (2015) The spatial information content of the honey bee waggle dance. Front. Ecol. Evol. 3:22. doi: 10.3389/fevo.2015.00022 Here we ﬁrst present the components of information theory that are necessary to understand the information content of events with continuous outcomes, such as foraging distance and direction. We then apply the theory thus presented to the data from our previous calibration experiment (Schürch et al., 2013). Our estimates should give a more accurate depiction of the information March 2015 \| Volume 3 \| Article 22 Frontiers in Ecology and Evolution \| www.frontiersin.org 1 Information in the waggle dance Schürch and Ratnieks content of the dance and therefore will have implications for research on the evolution of the waggle dance communication. Norwich (1994) gives an example related to language trans- mission over a noisy channel, for example a radio. We are asked to consider 10 possible numerals that are equally probable to be transmitted, so that entropy equals log 10. The spoken words for “ﬁve” and “nine” can be confused. So, even after one has heard the spoken numeral, there may be some uncertainty about whether “ﬁve” or “nine” was uttered at the sender’s end. If after the trans- mission the probabilities of confusing “ﬁve” with “nine” and vice versa are equal, the entropy has been reduced from log 10 to log 2, but not to log 1 = 0. The information in this case is given by the uncertainty before hearing the word minus the uncertainty remaining after hearing the word, that is 2. Information and Communication We could fragment the proba- bility density function into a number of narrow rectangles with 1x = 1 m and calculate the entropy H: H = − x X i = 1 pi log(pi). (1) (1) (1) For example, in the case of waggle dance communication, the outcomes could represent honey bee foraging distances in hun- dreds of meters, that is we look at the probabilities that a returned forager has foraged at 0–100 (i = 1), 100–200 (i = 2), 200–300 (i = 3), . . . meters, and pi is the probability that a forager has foraged in the ith interval. In words, the uncertainty H is the sum of the weighted logarithms of the probabilities of the outcomes (Norwich, 1994). Choosing the base of the logarithm in the cal- culation of H is arbitrary. In computer science, the choice falls on base 2 because it reﬂects the on/oﬀstate of electronic switches, and the unit of H is the familiar bit. Following Norwich (1994) we use base e and natural units here, but we then convert our calculations to bits: H = − 14,000 X i = 1 p(x = xi)log p(x = xi) (4) bits = natural units ln2 . (2) (4) (2) If information is the reduction in entropy, then, when H is reduced completely because we observe one of the discrete x outcomes, then information, I, is given by I = H. Note that if we would change our unit of measurement of bee for- aging distances to cm, the uncertainty expressed by the entropy H would increase. If, on the other hand, we would take coarser mea- surements, say if we measured foraging distance in km, H would decrease. Similarly, we would see a change in the information conveyed by waggle dances. If we changed our unit of measure- ment to cm, we could receive a higher quantity of information, and with smaller and smaller units of measurement (1x →0), we could in the end obtain an inﬁnite quantity of information (Norwich, 1994). This is a question of resolution. Note that if we would change our unit of measurement of bee for- aging distances to cm, the uncertainty expressed by the entropy H would increase. If, on the other hand, we would take coarser mea- surements, say if we measured foraging distance in km, H would decrease. 2. Information and Communication In (B), smaller bins have been chosen, and the entropies increase compared to (A), but the information does not change other than that the measurement of I is more accurate. deviation σ (dark gray bins). The difference between our prior uncertainty and the uncertainty that remains after observing the dance is the information communicated by the dance. The uncertainties, the entropies H, depend on the size of the bins, but the information I does not. In (B), smaller bins have been chosen, and the entropies increase compared to (A), but the information does not change other than that the measurement of I is more accurate. deviation σ (dark gray bins). The difference between our prior uncertainty and the uncertainty that remains after observing the dance is the information communicated by the dance. The uncertainties, the entropies H, depend on the size of the bins, but the information I does not. In (B), smaller bins have been chosen, and the entropies increase compared to (A), but the information does not change other than that the measurement of I is more accurate. deviation σ (dark gray bins). The difference between our prior uncertainty and the uncertainty that remains after observing the dance is the information communicated by the dance. The uncertainties, the entropies H, depend on the size of the bins, but the information I does not. In (B), smaller bins have been chosen, and the entropies increase compared to (A), but the information does not change other than that the measurement of I is more accurate. FIGURE 1 \| Information of the dance as the reduction in uncertainty in the dance. (A) If we know that a bee has foraged between 0 and 3 km, but not where, our prior knowledge attributes equal probabilities to the possibilities that the bee has visited any of 30 × 100 m bins (light gray bins). Knowing how the dance translates into distance, after observing the dance our uncertainty is reduced to a narrower area around the estimated distance µ, which is dependent on the standard human observers (Seeley and Visscher, 1988; Towne and Gould, 1988; Tautz and Sandeman, 2003). It is unlikely that the accu- racy of the measurement is as small as 1 m. 2. Information and Communication Therefore, there is little point to measuring H with 1x = 1 m other than conve- nience, and therefore, we do not obtain an inﬁnite quantity of information from a measurement of foraging distances through the waggle dance. entropy tends to inﬁnity. As we also have seen, the information I = Hbefore−Hafter will quickly approach our desired value, as we choose ﬁner and ﬁner resolutions. We therefore think it suﬃcient to consider a discrete approximation of our random variables, for distance, for example on the meter scale because that is con- venient. To measure the information contained in both vector components, we use a numerical approach. In our calibration experiment, we used normal errors to model the waggle run duration–distance calibration, and for the angles, we used the von Mises distribution. We will need to be able to calculate the probability densities for both vector components to calculate the entropy after observing a dance (Hafter) using the respective probability distribution. To calculate Hafter for the dis- tance, we calculate the probabilities pi at each discrete step i using the built-in function dnorm in R (R Core Team, 2013) using our empirical estimates of how accurately we could measure dis- tance (Schürch et al., 2013; Couvillon et al., 2014a,b). To calculate Hafter for the direction component of the vector, we used the dvonmises function from the circular package (Agostinelli and Lund, 2013) in R for all discrete steps i. Once we have pi, we can then calculate the information easily, which we will do here for distance and direction separately. The programs to estimate the respective information contents can be found in the electronic Supplementary Material. Luckily for us, while the diﬀerential entropy Hafter tends toward inﬁnity, for our practical considerations, it does not play a role. Since the terms that tend toward inﬁnity in both Hbefore and Hafter are the same, they cancel each other out when the diﬀerence is formed. As our prior expectations of where a bee has been, that is Hbefore is also increasing as our measuring intervals decrease, I = Hbefore −Hafter approaches a constant as measuring inter- vals get smaller and smaller. For the purpose of measuring the information content empirically, it should therefore suﬃce to use discrete approximations for estimating Hbefore and Hafter. Using the information theory outlined above, we chose to pursue the procedure outlined below. 2. Information and Communication Similarly, we would see a change in the information conveyed by waggle dances. If we changed our unit of measure- ment to cm, we could receive a higher quantity of information, and with smaller and smaller units of measurement (1x →0), we could in the end obtain an inﬁnite quantity of information (Norwich, 1994). This is a question of resolution. In our example of honey bee foraging distances measured in hundreds of meters, we would exactly know whether a bee has foraged between 200 and 300 m, or 300 and 400 m. Of course, interference may prevent the dance followers from exactly deter- mining the duration of a dance’s waggle run that reﬂects the foraging distance, or dancing bees may be constrained in how accurately they can dance (Tanner and Visscher, 2010; Couvil- lon et al., 2012; Preece and Beekman, 2014). For a dance follower, or for human observers, some residual uncertainty may remain, and hence the number of possible intervals at which a bee could have foraged after we have observed her dance is greater than 1, and I < H (Figure 1). Of course, we do not gain an unlimited amount of informa- tion from a dance, because 1x is not getting smaller without boundaries. The accuracy with which the foraging distance of a bee can be estimated from observing a dance is limited (Schürch et al., 2013), and this seems to be true also for nestmates, not only Frontiers in Ecology and Evolution \| www.frontiersin.org March 2015 \| Volume 3 \| Article 22 2 Schürch and Ratnieks Information in the waggle dance FIGURE 1 \| Information of the dance as the reduction in uncertainty in the dance. (A) If we know that a bee has foraged between 0 and 3 km, but not where, our prior knowledge attributes equal probabilities to the possibilities that the bee has visited any of 30 × 100 m bins (light gray bins). Knowing how the dance translates into distance, after observing the dance our uncertainty is reduced to a narrower area around the estimated distance µ, which is dependent on the standard deviation σ (dark gray bins). The difference between our prior uncertainty and the uncertainty that remains after observing the dance is the information communicated by the dance. The uncertainties, the entropies H, depend on the size of the bins, but the information I does not. 2. Information and Communication When observing foragers that have returned, but before having observed their dances, we assume a ﬂat prior knowledge: in terms of the direction the for- ager has ﬂown, we give equal probability to any possible angle on the interval [0, 2π). Similarly, we assume that the probability that a bee has ﬂown any distance in the potential foraging range from [0, 14 km) is equal to any other distance. That is, for both vector components, we assume uniform random distributions for our pi before we observe the dance. How wide should our intervals for the pi be? Should we measure distance in meters, or tens or hundreds of meters? In reality, the measurement would be a con- tinuous one. If we were interested in the uncertainty before or after observing a dance exclusively, we would run into problems if we choose inﬁnitesimally ﬁne resolutions (1x →0) for our mea- surements. As outlined above, for continuous random variables, Frontiers in Ecology and Evolution \| www.frontiersin.org 3. Distance As the information gained when observing the waggle dance depends on the prior information available, much in our calcula- tions depends on the choice of the functions for these prior expec- tations. If we observe a bee that we know is a returning forager, for example because she is trophallaxing, what is our knowledge? In a previous paper on the waggle run duration–distance calibration, March 2015 \| Volume 3 \| Article 22 Frontiers in Ecology and Evolution \| www.frontiersin.org 3 Schürch and Ratnieks Information in the waggle dance TABLE 1 \| Ten randomly selected dances from Couvillon et al. (2014a). TABLE 1 \| Ten randomly selected dances from Couvillon et al. (2014a). Mean SD 1896.5 152.6 2222.1 156.6 2263.7 162.5 1133.7 129.0 544.6 128.5 3204.2 176.2 923.3 133.4 657.2 136.3 1279.7 145.5 676.8 131.6 we have argued that we only really know that the bee has not been further than 14 km (Eckert, 1933; Schürch et al., 2013), and we attributed equal probabilities to all distances between 0 and 14 km as our prior knowledge. We follow this argument here as well. However, we would like to caution the reader that we will probably get an estimate of the maximum information that can be transferred by the dance. An experienced forager (or human dance decoder) who has followed many dances on a given day already could “gain” less knowledge, because, for example, she already knows that average foraging distances are much shorter than the 14 km maximum foraging range. Calculating Hbefore is straight-forward. If we measure foraging distance in meters, the probability pi that a bee has visited any given ith distance is 1/14,000 for each of the is, and the entropy is Hbefore = − 14,000 X i = 1 1 14,000 log 1 14,000 = 9.55. (5) (5) The number of steps i will depend on how we divide the cir- cle. For example, if we divide the circle into 16 slices, our step size will be π/8. Through trial and error, we found that a step size of π/512 is suﬃcient for an accurate measurement of the information content. 5. The Total Information in the Dance I = Hbefore −Hafter = 9.55 −6.41 = 3.14 natural units (8) The beneﬁt of our recent approach to map the dance as a cloud of probabilities instead of a single point (Schürch et al., 2013; Cou- villon et al., 2014a; Garbuzov et al., 2014) will now also allow us for the ﬁrst time to calculate the combined spatial information directly (see Figure 2). We can overlay a ﬁnite landscape with a grid, and based on the simulated dances used in Couvillon et al. (2014a), we can calculate the probability that each of the squares or 4.53 bits. or 4.53 bits. 3. Distance From the posterior samples of this model (N = 1000 per dance) we then calculated the mean and the standard deviation for a given dance. Table 1 lists these parameters from 10 dances of Couvillon et al. (2014a) selected at random. Note that for the calculation of the Hafter, we have used 100 dances selected at random to calcu- late a mean µ = 1716.011 m and a mean σ = 147.246 m. We then use a computer program (see Supplementary Material) to calculate Hafter and consequently I. If we do that, we get Hafter = − 1024 X i = 1 1 1024 · eκ cos (i −µ) 2πI0(κ) log 1 1024 · eκ cos (i−µ) 2πI0(κ) (10) (10) where µ is the mean direction, κ is the concentration parameter and I0 is the modiﬁed Bessel function of order 0. We used the circular package in R to calculate these probability densities (see Supplementary Material). We ﬁnd that for µ = 0 and κ = 24.9 (Schürch et al., 2013), the entropy is Hafter = 4.92, (11) (11) and the information in the directional component is and the information in the directional component is (12) I = Hbefore −Hafter = 6.93 −4.92 = 2.01 natural units (12) Hafter = 6.41 (7) (7) or 2.90 bits. and hence 3. Distance Hbefore is then simply For the Hafter we have to discretize the continuous normal distri- bution and calculate the probability density at each step i Hafter = − 14,000 X i = 1 1 14,000 1 √ 2πσ 2 e−(i −µ)2 2σ2 log 1 14,000 1 √ 2πσ 2 e−(i −µ)2 2σ2 ( Hbefore = − 1024 X i = 1 1 1024 log 1 1024 = log 1024 = 6.93 (9) (9) (6) For the calculation of Hafter we employ the same method as in the distance situation, replacing the normal probability density function with the probability density function of the von Mises distribution: For the calculation of Hafter we employ the same method as in the distance situation, replacing the normal probability density function with the probability density function of the von Mises distribution: where µ is the mean and σ is the standard deviation of the nor- mal distribution. Both these parameters can be estimated from observed dances using our calibration curve (Schürch et al., 2013; Couvillon et al., 2014a,b). Brieﬂy, to get the probability distribu- tions of foraging distances in Couvillon et al. (2014b) and Couvil- lon et al. (2014a) we used a linear calibration model using Gibbs sampling (described in Schürch et al., 2013, R and jags scripts for the calibration are available as Supplementary Material). From the posterior samples of this model (N = 1000 per dance) we then calculated the mean and the standard deviation for a given dance. Table 1 lists these parameters from 10 dances of Couvillon et al. (2014a) selected at random. Note that for the calculation of the Hafter, we have used 100 dances selected at random to calcu- late a mean µ = 1716.011 m and a mean σ = 147.246 m. We then use a computer program (see Supplementary Material) to calculate Hafter and consequently I. If we do that, we get where µ is the mean and σ is the standard deviation of the nor- mal distribution. Both these parameters can be estimated from observed dances using our calibration curve (Schürch et al., 2013; Couvillon et al., 2014a,b). Brieﬂy, to get the probability distribu- tions of foraging distances in Couvillon et al. (2014b) and Couvil- lon et al. (2014a) we used a linear calibration model using Gibbs sampling (described in Schürch et al., 2013, R and jags scripts for the calibration are available as Supplementary Material). 6. Discussion Here we have shown, using previously published waggle dance calibration and waggle dance data, how to calculate the infor- mation content of the waggle dance. We also present an update on how much information is conveyed in waggle dances. In our ﬁrst approach, where we discretized the distance and angu- lar outcomes, we were able to calculate that the information about foraging distance conveyed by the dancing bees amounts to 4.53 bits, and that the angular information is 2.90 bits, for a summed information content of 7.43 bits. In a second approach, we have used simulated locations based on 10 waggle dances (N = 1000 per dance from Couvillon et al., 2014a) to show that the combined information content of the dance is about 7.3 bits, agreeing well with the single dance components measurement of information. We can again use a uniform prior expectation that each square has been visited, that is, our prior expectation of the probabil- ity that any square has been visited are assumed to be equal for all squares. We can calculate both Hbefore and Hafter, and then the information is the reduction of the uncertainty. The elec- tronic Supplementary Material provides an R script calculating the information on an ever ﬁner grid. Our numerical computa- tions demonstrate that for our prior assumption of uniform prob- ability within a 14 km radius, and as we choose ﬁner and ﬁner grid squares, the information content of the dance approaches a value around 7.3 bits (Figure 3). Note that for the calculation of information on the grid, we are now also limited by the number of simulated dances pre- dicted from the calibration curve, and the smallest grid size we have chosen here is approximately 110 m. If we choose smaller grid sizes for 1000 simulations per dance, the probability land- scape becomes discontinuous. More speciﬁcally, some of the grid squares will have no simulated dances on them, even though the probability that the general area had been visited was high. In More than half a century has passed since the last attempt to calculate the information in the dance (Haldane and Spurway, 1954). Haldane and Spurway’s seminal paper used data from von Frisch (1946). 4. Direction As for distance, we calculate the information of the directional component. Calculating Hbefore is once again straight-forward. March 2015 \| Volume 3 \| Article 22 Frontiers in Ecology and Evolution \| www.frontiersin.org 4 Schürch and Ratnieks Information in the waggle dance that situation our discrete calculations do not reﬂect the true uncertainty anymore. in the grid has been visited. If each dance is represented by many simulations, the probability that a grid square had been visited by that dance was calculated as the number of simulated dances falling on that square divided by the total number of simulated dances. 6. Discussion Since von Frisch and his pupils were more inter- ested in the principles of the dance language instead of quan- tifying the information, they sought to eliminate variation in their experiments as much as possible (Chittka and Dornhaus, 1999), which creates diﬃculties for the calculation of informa- tion, as such calculations depend on this variation (Shannon, 1948; Norwich, 1994). Furthermore, Haldane and Spurway did not calculate the information for the distance component (Hal- dane and Spurway, 1954), which we have now done. We thought it therefore prudent to calculate the information conveyed by dancing bees by using our recent calibration and waggle dance data (Schürch et al., 2013; Couvillon et al., 2014a). FIGURE 2 \| If we lay a grid over a ﬁnite landscape, we can calculate for each grid square the probability that a dance has pointed to it. In (A) we see depicted the prior probability assuming equal probability for each square within a 14 km radius. In (B,C) we depict the probabilities after a dance has been observed for 1750 and 875 m grid square length, respectively. This corresponds to the 3rd and 4th iteration in Figure 3. FIGURE 2 \| If we lay a grid over a ﬁnite landscape, we can calculate for each grid square the probability that a dance has pointed to it. In (A) we see depicted the prior probability assuming equal probability for each square within a 14 km radius. In (B,C) we depict the probabilities after a dance has been observed for 1750 and 875 m grid square length, respectively. This corresponds to the 3rd and 4th iteration in Figure 3. Our measurement for the angular information diﬀers slightly from Haldane and Spurways (2.9 vs. 2.0 Haldane and Spurway, 1954), which could potentially be explained by a few reasons. First, as we have pointed out before, Haldane and Spurway used FIGURE 3 \| (A) As the number of iterations increases and hence (B) the size of the squares in the grid decreases, the information gained by observing a dance approaches a value of around 7.3 bits. FIGURE 3 \| (A) As the number of iterations increases and hence (B) the size of the squares in the grid decreases, the information gained by observing a dance approaches a value of around 7.3 bits. 6. Discussion Frontiers in Ecology and Evolution \| www.frontiersin.org March 2015 \| Volume 3 \| Article 22 Frontiers in Ecology and Evolution \| www.frontiersin.org 5 Information in the waggle dance Schürch and Ratnieks 50◦, that bees communicate? Much will probably depend on the environment (Sherman and Visscher, 2002; Donaldson-Matasci and Dornhaus, 2012; Okada et al., 2012), or the beneﬁts of the spatial information may also depend on colony size (Donaldson- Matasci et al., 2013). For example, if a hive were situated in the middle of a large-scale farming landscape with mass ﬂowering crops, a dance with relatively little information might be informa- tive, whereas in a more fragmented landscape with small ﬂower patches, more information will be necessary to allow a dance fol- lower to ﬁnd an advertised resource. Future honey bee foraging models should incorporate variability in the dance’s informa- tion to investigate the relationship between spatial information content and adaptiveness of the dance. data that were not collected in a manner that would make it suitable for the calculation of information. Secondly, the data they used were from a diﬀerent bee sub-species. If there are dialects among bee species (Boch, 1956; Su et al., 2008), then the information contained in the dance might also diﬀer from species to species (but see Dyer and Seeley, 1991). p y y We cannot compare our calculated information for distance with other values in the literature. Additionally, as this calcu- lation depends on the prior distribution, comparing values will also only make sense if these prior assumptions are the same. For example, our decision to assume a uniform prior distribution is somewhat arbitrary, even if the interval over 0–14 km is based on the literature (Eckert, 1933), which probably gives a physiological upper limit for foraging distance. In our environment, in vicin- ity of the Laboratory of Apiculture and Social Insects in Sussex, bees seem not forage at distances greater than 7 km (Couvillon et al., 2014a,b). If we were to use only half the distance for our prior uniform distribution, the estimate of information changes accordingly (3.56 bits for a 7 km uniform prior). Of course, the same is true when using our second approach, where we calcu- lated the information from a grid. We therefore urge the reader to focus not only on the speciﬁc values presented in this or any other paper. References Donaldson-Matasci, M. C., and Dornhaus, A. (2012). How habitat aﬀects the beneﬁts of communication in collectively foraging honey bees. Behav. Ecol. Sociobiol. 66, 583–592. doi: 10.1007/s00265-011-1306-z R Core Team (2013). R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing. Donaldson-Matasci, M. C., DeGrandi-Hoﬀman, G., and Dornhaus, A. (2013). Big- ger is better: honeybee colonies as distributed information-gathering systems. Anim. Behav. 85, 585–592. doi: 10.1016/j.anbehav.2012.12.020 Agostinelli, C., and Lund, U. (2013). R Package Circular: Circular Statistics (ver- sion 0.4-7). Venice: Department of Environmental Sciences, Informatics and Statistics, Ca’ Foscari University; San Luis Obispo, CA: Department of Statistics, California Polytechnic State University. Dyer, F. C., and Seeley, T. D. (1991). Dance dialects and foraging range in three asian honey bee species. Behav. Ecol. Sociobiol. 28, 227–233. doi: 10.1007/BF00175094 Boch, R. (1956). Die Tänze der Bienen bei nahen und fernen Trachtquellen. Z. Vergl. Physiol. 38, 136–167. doi: 10.1007/BF00338624 Eckert, J. E. (1933). The ﬂight range of the honeybee. J. Agric. Res. 47, 257–285. Chittka, L., and Dornhaus, A. (1999). Comparisons in physiology and evolution, and why bees can do the things they do. Cienc. Int. 2, 1–17. Garbuzov, M., Schürch, R., and Ratnieks, F. L. W. (2014). Eating locally: dance decoding demonstrates that urban honey bees in Brighton, UK, forage mainly in the surrounding urban area. Urban Ecosyst. 1573–1642. doi: 10.1007/s11252- 014-0403-y Couvillon, M. J., Phillipps, H. L. F., Schürch, R., and Ratnieks, F. L. W. (2012). Working against gravity: horizontal honeybee waggle runs have greater angular scatter than vertical waggle runs. Biol. Lett. 8, 540–543. doi: 10.1098/rsbl.2012.0182 Haldane, J., and Spurway, H. (1954). A statistical analysis of communication in Apis mellifera and a comparison with communication in other animals. Insectes Soc. 1, 247–283. doi: 10.1007/BF02222949 Couvillon, M., Schürch, R., and Ratnieks, F. (2014a). Dancing bees com- municate a foraging preference for rural lands in high level agri- environment schemes. Curr. Biol. 24, 1212–1215. doi: 10.1016/j.cub.2014. 03.072 Norwich, K. H. (1994). Information, Sensation and Perception. San Diego, CA: Academic Press, Inc. Okada, R., Akamatsu, T., Iwata, K., Ikeno, H., Kimura, T., Ohashi, M., et al. (2012). Waggle dance eﬀect: dancing in autumn reduces the mass loss of a honeybee colony. J. Exp. Biol. 215(Pt 10), 1633–1641. doi: 10.1242/jeb.068650 Couvillon, M. J., Schüch, R., and Ratnieks, F. L. (2014b). Dancing bees provide an integrated seasonal picture of foraging challenges. PLoS ONE 9:e93495. doi: 10.1371/journal.pone.0093495 Preece, K., and Beekman, M. We thank Margaret Couvillon for sharing her waggle dance data. Despite the limitations outlined above, our calculations are a ﬁrst step, and important questions arise from the calculations. For example, how much information in a dance is useful to a colony? Is one bit of spatial information helpful, that is, ﬂy north or south? How useful are two bits that could communicate four directions unambiguously (north, east, south, west)? And how much better are the 2.9 bits, that is sectors of the circle of about RS has been funded by Rowse Honey. RS has been funded by Rowse Honey. 6. Discussion Clearly, more calibration data covering the whole diversity of honey bee races and species in a range of environ- ments will be necessary to get a clearer understanding on how much information is in the dance. Author Contributions FLWR contributed to the concept and the writing of the manuscript. RS contributed to the concept, the numerical calcu- lations, and the writing of the manuscript. Supplementary Material The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fevo.2015. 00022/abstract Acknowledgments We thank Margaret Couvillon for sharing her waggle dance data. References (2014). Honeybee waggle dance error: adaption or constraint? unravelling the complex dance language of honeybees. Anim. Behav. 94, 19–26. doi: 10.1016/j.anbehav.2014.05.016 Couvillon, M. (2012). The dance legacy of Karl von Frisch. Insect. Soc. 59, 297–306. doi: 10.1007/s00040-012-0224-z March 2015 \| Volume 3 \| Article 22 Frontiers in Ecology and Evolution \| www.frontiersin.org 6 Schürch and Ratnieks Information in the waggle dance Tautz, J., and Sandeman, D. C. (2003). Recruitment of honeybees to non-scented food sources. J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 189, 293–300. doi: 10.1007/s00359-003-0402-6 Schürch, R., and Couvillon, M. (2013). Too much noise on the dance ﬂoor: intra- and inter-dance angular error in honey bee waggle dances. Commun. Integr. Biol. 6, 1–3. doi: 10.4161/cib.22298 Schürch, R., Couvillon, M. J., Burns, D., Tasman, K., Waxman, D., and Ratnieks, F. L. (2013). Incorporating variability in honey bee waggle dance decoding improves the mapping of communicated resource locations. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 199, 1143–1152. doi: 10.1007/s00359- 013-0860-4 Towne, W. F., and Gould, J. L. (1988). The spatial precision of the honey bees’ dance communication. J. Insect Behav. 1, 129–155. doi: 10.1007/BF01052234 von Frisch, K. (1946). Die Tänze der Bienen. Österr. Zool. Z. 1, 1–148. doi: 10.1007/978-3-642-94916-6_2 von Frisch, K. (1967). The Dance Language and Orientation of Bees. Cambridge, MA: Harvard University Press. Seeley, T., and Visscher, P. (1988). Assessing the beneﬁts of cooperation in honey- bee foraging: search costs, forage quality, and competitive ability. Behav. Ecol. Soc. 22, 229–237. doi: 10.1007/BF00299837 Conﬂict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Shannon, C. E. (1948). A mathematical theory of communication. Bell Syst. Tech. J. 27, 379–423. doi: 10.1002/j.1538-7305.1948.tb01338.x Sherman, G., and Visscher, P. K. (2002). Honeybee colonies achieve ﬁtness through dancing. Nature 419, 920–922. doi: 10.1038/nature01127 Copyright © 2015 Schürch and Ratnieks. This is an open-access article dis- tributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publica- tion in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Su, S., Cai, F., Si, A., Zhang, S., Tautz, J., and Chen, S. Frontiers in Ecology and Evolution \| www.frontiersin.org March 2015 \| Volume 3 \| Article 22 References (2008). East learns from west: asiatic honeybees can understand dance language of european honeybees. PLoS ONE 3:e2365. doi: 10.1371/journal.pone.0002365 Tanner, D. A., and Visscher, P. K. (2010). Adaptation or constraint? reference- dependent scatter in honey bee dances. Behav. Ecol. Sociobiol. 64, 1081–1086. doi: 10.1007/s00265-010-0922-3 Tanner, D. A., and Visscher, P. K. (2010). Adaptation or constraint? reference- dependent scatter in honey bee dances. Behav. Ecol. Sociobiol. 64, 1081–1086. doi: 10.1007/s00265-010-0922-3 March 2015 \| Volume 3 \| Article 22 Frontiers in Ecology and Evolution \| www.frontiersin.org 7
https://openalex.org/W3035472985	https://zenodo.org/record/3595254/files/03_IJRG19_A12_2917.pdf	English	null	INSECT POLLINATORS DIVERSITY IN AVOCADO ORCHARD DURING FLOWERING PERIOD IN LUSHOTO DISTRICT TANZANIA	International journal of research - granthaalayah	2,020	cc-by	2,047	Abstract Insect pollinator diversity is key to ensuring adequate fruit yields within avocado orchards. Bee and non-bee insect species in avocado growing areas worldwide, has been considered as potential pollinators. Despite of this information in Tanzania there has been little research into which pollinator insect species diversity visit avocado flowers during flowering season. The study was conducted at Jaegetal avocado orchard from 450 trees of Hass variety planted at spacing of 10m x 10m. Sample size was 10 trees were used to generate pollinator’s population estimates. The orchard is located at S04˚47ʹ41.7ʹʹ and E038˚13ʹ11.8ʹʹ. Sweep net, forceps and aspirator were used to estimate pollinator’s diversity during avocado production season 2018/19. The results showed a total of 115 individuals were sampled and honey bees were more abundant flower visitors representing 60% of all insects recorded. The second species observed included the stingless bee representing (26%), followed by tachnid fly (6%) and hoverfly (6%). The least species in abundance was ants (1%) and wasps (1%). Mean population comparison there was significant differences P< 0.005.Understanding pollinators species diversity interaction between honey bee and other flower visitor’s abundances within and outside orchards could assist in improving pollination recommendations in avocado growing areas. Keywords: Avocado; Pollinator’s Abundance. Keywords: Avocado; Pollinator’s Abundance. Cite This Article: Christopher L Materu. (2019). “INSECT POLLINATORS DIVERSITY IN AVOCADO ORCHARD DURING FLOWERING PERIOD IN LUSHOTO DISTRICT TANZANIA.” International Journal of Research - Granthaalayah, 7(12), 20-24. https://doi.org/10.5281/zenodo.3595254. INSECT POLLINATORS DIVERSITY IN AVOCADO ORCHARD DURING FLOWERING PERIOD IN LUSHOTO DISTRICT TANZANIA Christopher L Materu 1 1 P O Box 6226, Dar Es Salaam Tanzania [Materu , Vol.7 (Iss.12): December 2019] [Materu , Vol.7 (Iss.12): December 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3595254 Science 1. Introduction Avocado (Persea americana), native to CentralAmerica and Mexico, is an evergreen subtropical fruiting tree grown for commercially in the tropics (Evans et al., 2010). Insect pollination has been shown to beessential for fruit production (Ish-Am & Lahav 2011). A wide range of insect pollinators has been reported worldwide including bees, wasps (Hymenoptera) and flies (Diptera) (Kevan & Baker, 1983; Perez-Balam et al., 2012). Pollinators including honeybee, wild pollinators requires diverse plants to provide sustainable biological insurance for pollination activity in avocado orchard (Ssymank et al., 2008). [20] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Materu , Vol.7 (Iss.12): December 2019] Honey bees as a native pollinator pollinate more than 80% of cultivated and non-cultivated plants. Enhancing bio diversity is an important to support stronger and more resilient ecosystems. The health of pollinator population, has, been impacted over recent decades by a variety of factors the loss, degradation, and fragmentation of habitat; diminished quantity and quality of food sources; reduced availability of sites for mating, nesting, migration; exposure to pesticides, increased adverse effects from pathogens, arthropod pests and parasites. Therefore, good knowledge on alternate food sources and nesting sites for pollinators is needed to modify agricultural land-use. Pesticide use has been reported to have negative impact to honey bee and other potential pollinators therefore, protecting of both wild and domesticated wild pollinators is an important in avocado production. Pesticide use in honeybee areas should be applied early morning or late evening when bee population activity is very low. Pesticide applications in avocado production may include different types of insecticides, fungicides, bactericides, herbicides, and others, each having different toxicity profile and impact on various insect species (Blacquiere et al., 2012).Good pollinator habitat sites suitable for bee foraging are open landscapes with good sun exposure and different types of plants including avocado. Large habitat types ie forest closer to avocado orchard are recommended than smaller and isolated orchard. Natural or artificial forest habitats with a variety of native flowering plants that have overlapping blooming times and that are adapted to local soils and climates are good source of nectar and pollen for pollinators (Black et al., 2007). Bee health is affected when pesticides area applied to manage threat pests (Biddinger et al., 2013). Various studies showed that pesticide use are not host specific to target organisms, pollute environment and also are harmful to human being (Hooven et al., 2013). 1. Introduction It is important to identify pollinator species diversity visiting avocado flowers during full bloom period. Identifying potential pollinators will assist to plan for sustainable enhancement of potential pollinators in avocado production in Tanzania and neighboring countries. 2. Materials and Methods The study area is located at S 04˚47ʹ41.7ʹʹ and E 038˚13ʹ11.8ʹʹ in Lushoto district. The insect pollinator’s estimation was carried on during the avocado season in September 2018. The size of the orchard is 4.5ha with a total of 450 trees of Hass variety planted at spacing of 10m by 10m. Two records were collected starting from 11:30AM to 17:00PM by two trained scientists during the study period. Random sampling, visual observations, sweep nets and forceps were used to estimate species diversity of pollinator visiting avocado flowers. Results During the study period honey bees were observed as an important pollinators and their efficiency was due to the large number of forager bees. Frequent foraging on open flowers for a short time indicates there is an efficient pollination. Different plants with different flowering period around avocado orchard enable bees to use little time for pollen and nectar collection. Furthermore, in the forest habitat destruction of the forest for different use lowers plant species diversity. Similarly, it means that changes for forest to other uses it reduce pollinator’s activity. Therefore, it is Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [21] [Materu , Vol.7 (Iss.12): December 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3595254 recommended to leave weeds hedges or trees for conserving potential pollinators in avocado growing areas. [Materu , Vol.7 (Iss.12): December 2019] recommended to leave weeds hedges or trees for conserving potential pollinators in avocado growing areas. Avocado requires insect pollination to maximize yields production. Furthermore, findings from this study showed that honey bee or sting bee (Apis mellifera) and non-sting bee (Meliponinae Apidae)as one of the potential pollinators in avocado orchard. Abundance of bee pollinators was high compared to non-bee pollinators indicating that bee has a major role in avocado crop pollination. The results showed that a total of 115 individuals were observed and honey bees were more abundant visitors representing 60% of all insects recorded. Common pollinators observed included the stingless bee (26%), followed by tachnid fly 6%, hoverfly (6%) and the least was wasps (1%) and ants (1%) Figure 1. Figure 1: Insects pollinators that were collected from Jaegetal avocado orchard during 2018/2019 season Figure 1: Insects pollinators that were collected from Jaegetal avocado orchard during 2018/2019 season 4. Conclusion Farmers are encouraged to plant different tree species or food crops to increase diversity of bee foraging as well as genetic variability in avocado growing areas. Furthermore, it was observed that orchard surrounded by perennial trees, grasses encourage bee visitation to their crops, or to leave part of their land fallow was an important to increase pollinator habitat (Vaughnet al., 2012). It is good to provide public education to consumers to choose products produced with good pollinator protection standards. This option will encourage conservation bodies in the country to reward honey bee produced from farm practices that preserve pollinator health. Different stakeholders including private sector, NGO can be involved, agricultural input suppliers, food retailers to strengthening capacity on pollinator surveys. Furthermore, to support recovery of endangered pollinator as well as plant species. To conduct regular surveys for pollinators before and after pesticide use is an important activity to measure the side effect caused by pesticides in avocado growing areas. 3. Results Discussions Findings from this work agreed to other studies carried out by Rader et al., (2009), he mentioned that the most effective pollinator species are those occurring in high abundance, actively moving from flower to flower and has a high visitation rate and transferring pollen grains from one tree to another. Efficient insect pollination can be estimated by counting or capture number of pollinator species visiting flowers during the bloom season (Pearce et al., 2012). Furthermore, in avocado growing areas our emphasis was given to honey bee population as a major pollinator (Abrol 2012). Full bloom was observed to attract different insect pollinators (Faegri & Pijl, 1979). Increase amount of food at a specified time observed to increase number of pollinators visiting avocado flowers, furthermore, increase of insect species diversity will increase pollination efficiency. [22] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Materu , Vol.7 (Iss.12): December 2019] [Materu , Vol.7 (Iss.12): December 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) Index Copernicus Value (ICV 2018): 86.20 DOI: 10.5281/zenodo.3595254 Acknowledgements The research team would like to acknowledge Tanzania Agricultural Research Institute (TARI) Mikocheni for their acceptance to participate in this particular research work, Dr Thomas from International Centre for Insect Physiology and Ecology (ICIPE) for their financial support. Lastly to the pest control staffs and farmers for their commitment during the study period. Without support from this identified team this work wouldn’t be possible to be carried out. Corresponding author. E-mail address: chrismateru@ yahoo.com p g E-mail address: chrismateru@ yahoo.com References [3] Blacquiere T, Smagghe G, van Gestel CAM, Mommaerts V: Neonicotinoids in bees: a review on concentration, side effects and risk assessment. Ecotoxicology 2012 http:// dx.doi.org/10.1007/s10646-012-0863-x. [3] Blacquiere T, Smagghe G, van Gestel CAM, Mommaerts V: Neonicotinoids in bees: a review on concentration, side effects and risk assessment. Ecotoxicology 2012 http:// dx.doi.org/10.1007/s10646-012-0863-x. [4] Biddinger DJ, Robertson J, Mullin C, Frazier J, Joshi NK, Vaughn M, Ashcraft S: Comparative toxicities and synergism of orchard pesticides to Apismellifera (L.) and Osmiacornifrons (Radoszkowski). PloS One 2013, 8:e72587 http://dx.doi.org/ 10.1371/journal.pone.0072587. [5] FAEGRI, K.; PIJL, L. VAN DER 1979. The Principies of PollinationEcology. 3111ed. Pergamon Press. Oxford. UK. 244 p. [6] Hooven LA, Sagili RR, Johansen E: How to Reduce Bee Poisoning from Pesticides. Oregon State University Extension Service. Oregon State University; 2013: PNW 591 [7] Kevan PG, Baker HG (1983) Insects as flower visitors and pollinators. Annual Review of Entomology28: 407-453. [8] Perez-Balam, J., J. J. G. Quezada-Euan, R. Alfaro-Bates, S. Medina, A.Soro, and R. J. Paxton. 2012. The contribution of honey bees, ﬂies andwasps to avocado (Persea americana) pollination in southern Mexico.J. Pollinat. Ecol. 8: 42–47. [9] Pearce, A. M., K. M. O’Neill, R. S. Miller, and S. Blodgett. 2012. Diversityof ﬂower visiting bees and their pollen loads on a wildﬂower seed farm inMontana. J. Kans. Entomol. Soc. 85: 97–108. [23] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Materu , Vol.7 (Iss.12): December 2019] [10] Proceedings VIIWorld Avocado Congress 2011 (Actas VII Congreso Mundial delAguacate 2011). Cairns, Australia.5 – 9 September 2011 p [11] Rader, R., W. Edwards, D. A. Wescott, S. A. Cunningham, and B. G.Howlett. 2011. Pollen transport differs among bees and ﬂies in a human modiﬁed landscape. Divers. Distrib.17: 519–529. [11] Rader, R., W. Edwards, D. A. Wescott, S. A. Cunningham, and B. G.Howlett. 2011. Pollen transport differs among bees and ﬂies in a human modiﬁed landscape. Divers. Distrib.17: 519–529. [12] Ssymank A, Kearns CA, Pape T, Thompson FC (2008) Pollinating flies (Diptera): a major ib i l di i d i l l d i Bi di i 9 86 89 [11] Rader, R., W. Edwards, D. A. Wescott, S. A. Cunningham, and B. G.Howlett. 2011. Pollen transport differs among bees and ﬂies in a human modiﬁed landscape. Divers. Distrib.17: 519–529. [12] Ssymank A, Kearns CA, Pape T, Thompson FC (2008) Pollinating flies (Diptera): a major contribution to plant diversity and agricultural production. Biodiversity 9: 86-89. Corresponding author. References transport differs among bees and ﬂies in a human modiﬁed landscape. Divers. Distrib.17: 519–529. [12] Ssymank A, Kearns CA, Pape T, Thompson FC (2008) Pollinating flies (Diptera): a major contribution to plant diversity and agricultural production. Biodiversity 9: 86-89. [12] Ssymank A, Kearns CA, Pape T, Thompson FC (2008) Pollinating flies (Diptera): a major contribution to plant diversity and agricultural production. Biodiversity 9: 86-89. [13] Vaughn M, Mader E, Guisse J, Goldetz-Dollar J, Borders B, Biddinger D, Gillis J: USDA-NRCS Conservation Cover (327) for Pollinators – Pennsylvania Installation Guide and Job Sheet. 2012: http://www.xerces.org/pollinator-conservation/agriculture/ pollinator-habitat-installation-guides/. [14] Vaughn M, Mader E, Guisse J, Goldetz-Dollar J, Borders B, Biddinger D, Gillis J: USDA-NRCS Hedgerow Plantings (422) for Pollinators – Pennsylvania Installation Guide and Job Sheet. 2012: http://www.xerces.org/pollinator-conservation/agriculture/ pollinator-habitat-installation-guides/. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [24]
https://openalex.org/W2786833399	https://www.frontiersin.org/articles/10.3389/fpls.2018.00012/pdf	English	null	Traversing the Links between Heavy Metal Stress and Plant Signaling	Frontiers in plant science	2,018	cc-by	19,897	Edited by: Jon Pittman, University of Manchester, United Kingdom Reviewed by: Rosario Vera-Estrella, Universidad Nacional Autónoma de México, Mexico Smita Kumar, National Botanical Research Institute (CSIR), India Edited by: Jon Pittman, University of Manchester, United Kingdom Reviewed by: Rosario Vera-Estrella, Universidad Nacional Autónoma de México, Mexico Smita Kumar, National Botanical Research Institute (CSIR), India Reviewed by: Rosario Vera-Estrella, Universidad Nacional Autónoma de México, Mexico Smita Kumar, National Botanical Research Institute (CSIR), India Correspondence: Alok K. Sinha alok@nipgr.ac.in Traversing the Links between Heavy Metal Stress and Plant Signaling Siddhi K. Jalmi, Prakash K. Bhagat, Deepanjali Verma, Stanzin Noryang, Sumaira Tayyeba, Kirti Singh, Deepika Sharma and Alok K. Sinha Plant Signaling, National Institute of Plant Genome Research, New Delhi, India Plants confront multifarious environmental stresses widely divided into abiotic and biotic stresses, of which heavy metal stress represents one of the most damaging abiotic stresses. Heavy metals cause toxicity by targeting crucial molecules and vital processes in the plant cell. One of the approaches by which heavy metals act in plants is by over production of reactive oxygen species (ROS) either directly or indirectly. Plants act against such overdose of metal in the environment by boosting the defense responses like metal chelation, sequestration into vacuole, regulation of metal intake by transporters, and intensiﬁcation of antioxidative mechanisms. This response shown by plants is the result of intricate signaling networks functioning in the cell in order to transmit the extracellular stimuli into an intracellular response. The crucial signaling components involved are calcium signaling, hormone signaling, and mitogen activated protein kinase (MAPK) signaling that are discussed in this review. Apart from signaling components other regulators like microRNAs and transcription factors also have a major contribution in regulating heavy metal stress. This review demonstrates the key role of MAPKs in synchronously controlling the other signaling components and regulators in metal stress. Further, attempts have been made to focus on metal transporters and chelators that are regulated by MAPK signaling. REVIEW published: 05 February 2018 doi: 10.3389/fpls.2018.00012 REVIEW Keywords: calcium signaling, chelators, heavy metals, hormones, MAPKs, metal transporters, metallothioneins, microRNAs INTRODUCTION Specialty section: This article was submitted to Plant Trafﬁc and Transport, a section of the journal Frontiers in Plant Science Heavy metals are essential to life only in trace amount while their excess amount causes cellular damage. The heavy metals present in environment aﬀecting the growth of many organisms are iron (Fe), arsenite (AsIII), arsenate (AsV), cadmium (Cd), chromium (Cr), lead (Pb), copper (Cu), mercury (Hg), aluminum (Al), etc. These metals have not only known to perturb animal kingdom but also plant kingdom. Their damaging impact on our agriculture has also been very well-documented (Tchounwou et al., 2012). At cellular level elevated quantity of heavy metals imposes damage by wide number of mechanisms. The most common mechanism is the production of reactive oxygen species (ROS) inducing oxidative stress, while others are inactivation of biomolecules by displacement of essential metal ions or by blocking essential functional groups (Stohs and Bagchi, 1995). Metals like As, Cd, Cr, Pb, Hg are able to work by displacing essential metal ions or blocking functional groups. Metals like Fe and Cu, which are redox active, generate ROS directly through redox reactions; in contrast, other metals like Pb, Cd, Ni, Al, Mn, and Zn generate ROS by indirect mechanisms. The indirect mechanism of ROS production includes stimulation of ROS producing enzymes like NADPH oxidases or displacing Received: 16 April 2017 Accepted: 03 January 2018 Published: 05 February 2018 Citation: Jalmi SK, Bhagat PK, Verma D, Noryang S, Tayyeba S, Singh K, Sharma D and Sinha AK (2018) Traversing the Links between Heavy Metal Stress and Plant Signaling. Front. Plant Sci. 9:12. doi: 10.3389/fpls.2018.00012 February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 1 Heavy Metal Stress and Plant Signaling Jalmi et al. essential cations from the binding sites of enzymes and inhibiting their activities. ROS at normal physiological level play essential role however its enhanced generation deteriorates functioning of cell (Cuypers et al., 2009). Plants show defense against these heavy metal ions by adsorbing them on to the chelating molecules [for e.g., phytochelatins (PCs), metallothionines, etc.] and by sequestration into the vacuoles (Figure 1). Many of the defense responses (not all) shown by the plants are due to the major contribution by signaling cascades, which perceive the signal from upstream receptors and transmit into the nucleus, thus regulating several defense related genes. The receptors that are known to perceive the signals and are well studied in plant stress and development include receptor like protein kinases (RLKs), ﬂagellin sensitive 2 (FLS2), EF-Tu receptor (EFR), ethylene resistance1/2 (ETR1/2), salt intolerance 1 (SIT1), ERECTA (ER), etc. (Rodriguez et al., 2010; Sinha et al., 2011; Jalmi and Sinha, 2015). The major signaling networks working in metals stresses in addition to the other environmental stresses are calcium signaling, hormone signaling and MAPK signaling. Calcium signaling employs multitude of calcium sensing proteins like Calmodulins (CaMs), CaM like proteins (CMLs), Calcineurin B-like proteins (CBLs), and Ca2+-dependent protein kinases (CDPKs) that bind to Ca2+ and trigger diﬀerent downstream signaling pathways (Luan et al., 2002; Sanders et al., 2002; Dodd et al., 2010; Steinhorst and Kudla, 2014). In case of hormone signaling there are diﬀerent plant hormones that play role in metal stress response (Cao et al., 2009; Vitti et al., 2013; Chen et al., 2014; Zhao et al., 2014). Of the several signaling modules, the most predominant and complex is the mitogen activated protein kinase (MAPK) signaling composed of three- tier phosphorylation module MAPKKKs (Mitogen Activated Protein Kinase Kinase Kinase), MAPKKs (Mitogen Activated Protein Kinase Kinase), and MAPKs (Mitogen Activated Protein Kinase) (Hamel et al., 2006). MAPKs are substantially known in providing tolerance against biotic and abiotic stress (Rodriguez et al., 2010; Rao et al., 2011; Sinha et al., 2011) (Figure 2). Plant Signaling in Response to Heavy Metals The inability of plants to escape from environmental stresses such as metal pollution has driven the evolution of multiple mechanisms to eﬃciently sense, respond, and therefore adapt to such stresses. Sensing of heavy metals by plants generates a response such as modulation of molecular and biochemical mechanisms of cell. Certainly, this response is evoked by important signal transduction network operated in plant cell formed by several signal transduction units. The ultimate response of plant is shown by synthesizing metal transporter proteins and metal binding proteins helping the plant to counteract excessive metal stress (Maksymiec, 2007; Peng and Gong, 2014; Singh S. et al., 2015). During environmental stress plants exhibit molecular response that helps them to adapt during various environmental calamities. Plant’s molecular response to metal stress is signiﬁed by the synthesis of signaling molecules and stress-related proteins like metal transporters and chelators. They tackle the heavy metal toxicity by chelating and sequestering them in the plant vacuoles, serving as temporary storage of essential as well as toxic metabolites (Sharma and Dietz, 2006; Verbruggen et al., 2009; Mendoza-Cózatl et al., 2011) (Figure 1). Based on this ability plants are now being widely used for removing heavy metals contamination from the environment by process of phytoremediation (Salt et al., 1998). In many crops, the early sign of metal toxicity is known to be similar to other environmental stresses like osmotic or dehydration stress, oxidative stress in addition to defects in nutrient balance, photosynthesis, and development (Chen et al., 2001; Yadav, 2010; Rucinska-Sobkowiak, 2016). This similarity of the response reﬂects interconnection between intricate signaling networks. The interplay and convergence of these signaling pathways ﬁnally results in regulation of various transcription factors activating several stress responsive genes. The genes that normally get regulated in context of metal stress include the genes for metal chelators and transporters (Singh S. et al., 2015) (Figure 2). Several signal transduction units operate in response to heavy metal toxicity, with diﬀerent signaling pathways acting in response to diﬀerent species and concentrations of metals. Some of these signaling pathways are discussed in detail below. Transport of heavy metals required for their relocation is performed by transporters localized in the parenchyma cells of xylem and companion cells of phloem. Majority of loading and unloading of the metal ions in xylem and phloem is done by the transporters. Citation: In case of hormone signaling there are diﬀerent plant hormones that play role in metal stress response (Cao et al., 2009; Vitti et al., 2013; Chen et al., 2014; Zhao et al., 2014). Of the several signaling modules, the most predominant and complex is the mitogen activated protein kinase (MAPK) signaling composed of three- tier phosphorylation module MAPKKKs (Mitogen Activated Protein Kinase Kinase Kinase), MAPKKs (Mitogen Activated Protein Kinase Kinase), and MAPKs (Mitogen Activated Protein Kinase) (Hamel et al., 2006). MAPKs are substantially known in providing tolerance against biotic and abiotic stress (Rodriguez et al., 2010; Rao et al., 2011; Sinha et al., 2011) (Figure 2). This review will majorly emphasize on impact and mechanism of action of heavy metals, diﬀerent signaling modules and other regulators triggered by heavy metal stress, the impact of plant signaling on downstream defense responses and the fragmentary work performed on regulation of metal transporters by MAPKs that still remains unexplored in plants. Frontiers in Plant Science \| www.frontiersin.org Citation: ATP-binding cassette (ABC) transporters, present at plasma membrane and on tonoplast membrane of cell (Park et al., 2012; Singh S. et al., 2015) (Figure 1). In addition, Cys-rich metal binding peptides like PCs or metallothionines, nicotinamide, and glutathione are also important players of metal transport (Figure 1). Studies suggesting the role of metal transporters and Cys-rich metal binding peptides in arsenic metal uptake, transport, and detoxiﬁcation have been very well-described by Kumar et al. (2015). Apart from the transporters and chelators, vacuole sequestration capacity (VSC) is very much important in metal allocation. Interaction between membrane localized transporters and ion chelators adjust the VSC in response to changing environment (Peng and Gong, 2014). The regulation of the VSC will decide the toxicity of heavy metals to the plants. It is important to study the regulatory mechanisms of VSC and its ultimate impact on metal transport and sequestration. Additionally, study of metals signal perception and transmission by the plants in regulating the metal transport is also important. essential cations from the binding sites of enzymes and inhibiting their activities. ROS at normal physiological level play essential role however its enhanced generation deteriorates functioning of cell (Cuypers et al., 2009). Plants show defense against these heavy metal ions by adsorbing them on to the chelating molecules [for e.g., phytochelatins (PCs), metallothionines, etc.] and by sequestration into the vacuoles (Figure 1). Many of the defense responses (not all) shown by the plants are due to the major contribution by signaling cascades, which perceive the signal from upstream receptors and transmit into the nucleus, thus regulating several defense related genes. The receptors that are known to perceive the signals and are well studied in plant stress and development include receptor like protein kinases (RLKs), ﬂagellin sensitive 2 (FLS2), EF-Tu receptor (EFR), ethylene resistance1/2 (ETR1/2), salt intolerance 1 (SIT1), ERECTA (ER), etc. (Rodriguez et al., 2010; Sinha et al., 2011; Jalmi and Sinha, 2015). The major signaling networks working in metals stresses in addition to the other environmental stresses are calcium signaling, hormone signaling and MAPK signaling. Calcium signaling employs multitude of calcium sensing proteins like Calmodulins (CaMs), CaM like proteins (CMLs), Calcineurin B-like proteins (CBLs), and Ca2+-dependent protein kinases (CDPKs) that bind to Ca2+ and trigger diﬀerent downstream signaling pathways (Luan et al., 2002; Sanders et al., 2002; Dodd et al., 2010; Steinhorst and Kudla, 2014). Plant Signaling in Response to Heavy Metals Prominent groups of transporters maintaining physiological concentration of heavy metals are: zinc–iron permease (ZIP), heavy metal transport ATPase (CPx- and P1B-ATPase), natural resistant associated macrophage protein (NRAMP), cation diﬀusion facilitator (CDF), and February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 2 Jalmi et al. Heavy Metal Stress and Plant Signaling FIGURE 1 \| Metal detection, plant signaling, and sequestration. Different transporters are involved in metal ion uptake. Elevated level of heavy metals triggers different signaling modules which transmit the signals inside cell, thus triggering defense response. The toxicity of these metals inside the cell is sequestered by metal chelators like phytochellatins and metallothionines. The chelated metals are then ultimately transported to the vacuoles with the help of metal transporters present on the vacuole membrane. PC, phytochelatins; MT, metallothionines; GSH, Glutathiones. FIGURE 1 \| Metal detection, plant signaling, and sequestration. Different transporters are involved in metal ion uptake. Elevated level of heavy metals triggers different signaling modules which transmit the signals inside cell, thus triggering defense response. The toxicity of these metals inside the cell is sequestered by metal chelators like phytochellatins and metallothionines. The chelated metals are then ultimately transported to the vacuoles with the help of metal transporters present on the vacuole membrane. PC, phytochelatins; MT, metallothionines; GSH, Glutathiones. FIGURE 1 \| Metal detection, plant signaling, and sequestration. Different transporters are involved in metal ion uptake. Elevated level of heavy metals triggers different signaling modules which transmit the signals inside cell, thus triggering defense response. The toxicity of these metals inside the cell is sequestered by metal chelators like phytochellatins and metallothionines. The chelated metals are then ultimately transported to the vacuoles with the help of metal transporters present on the vacuole membrane. PC, phytochelatins; MT, metallothionines; GSH, Glutathiones. FIGURE 2 \| Crosstalk of signaling pathways and its ultimate response in heavy metal stress. This ﬁgure displays the involvement of several signaling components working during metal stress. Sensing of signiﬁcant level of heavy metals by plants initiates signaling network causing activation of various metal responsive transcription factors. These transcription factors (TFs) regulate the expression of metal responsive and other stress related genes ultimately helping the plant to counteract stressed situation. These stress related genes are mainly metal transporters, phytochellatins, metallothionine, antioxidant genes, and miRNA genes (MIR genes). MAPK Signaling in Heavy Metal Stress These studies provide a speculation and link of MAPK cascades that might work in diﬀerent metal stress depending upon the activation by the ROS molecules produced. Furthermore, MEKK1-MKK4/MKK5- MPK3/MPK6 module working downstream of receptor FLS2 and receptor like kinase (RLKs), eventually giving resistance against pathogen is very well known (Asai et al., 2002). These RLKs are also reported to be regulated by Cd2+ speculating the involvement of similar MAPK cascade working downstream of RLK in metal stress. Recent study reported that MEKK1-MKK5-MPK6 mediates salt induced expression of iron superoxide dismutase gene further inducing ROS production. Iron superoxide dismutases (Fe-SOD) are the metal binding SOD and require Fe3+ metal ion as cofactor (Myouga et al., 2008; Xing et al., 2015). These reports suggest involvement of MAPKs in mediating metal stress however a detail study of a complete MAPK cascade working in heavy metal stress is required. There are plenty of reports showing the activation of MAPKs in response to heavy metals like Cd, Cu, and As (Jonak et al., 2004; Yeh et al., 2007; Ding et al., 2011; Rao et al., 2011; Smeets et al., 2013), however studies in response to other metals such as Pb, Zn, Fe are very scant. Likewise, in depth investigation to decipher a complete MAPK signaling cascade in response to speciﬁc metal stress still remains elusive. In Arabidopsis, the best-characterized MAPKs are MPK3 and MPK6, which are activated by diverse stimuli are also known to induced by CdCl2 and CuSO4 (Asai et al., 2002; Pitzschke et al., 2009; Liu et al., 2010; Takahashi et al., 2011; Sethi et al., 2014). Similarly in rice, OsMSRMK2 (OsMPK3 homolog), OsMSRMK3 (OsMPK7 homolog), and OsWJUMK1 (OsMPK20-4 homolog) transcripts increased in response to Cu2+ and Cd2+ treatment in leaves and roots (Yeh et al., 2007; Rao et al., 2011). In Alfalfa, activation of four distinct MAPKs: SIMK, MMK2, MMK3, and SAMK was demonstrated in response to CuCl2 or CdCl2. SAMK and SIMK are the orthologs of rice OsMPK3 and OsMPK6, respectively. Higher concentrations of CuCl2 induced the activity of SIMK, MMK2, and MMK3, and to a lesser extent of SAMK while CdCl2 showed a similar but delayed MAPK activation (Jonak et al., 2004). Copper- mediated induction of SIMKK speciﬁcally activated SIMK and SAMK and not MMK2 and MMK3 manifesting speciﬁcity in the signaling cascades in response to diﬀerent metals (Jonak et al., 2004; Opdenakker et al., 2012) (Table 1). MAPK Signaling in Heavy Metal Stress by three MAPKs with approximate molecular weights of 34, 40, and 42 kDa in rice roots (Lin et al., 2005). Pb stress leads to the upregulation of four MAPKs such as MAPKKK7, MAPK6, MAPK18, and MAPK20 in radish (Wang et al., 2013). Arsenite severely aﬀect the growth of rice seedlings. OsMKK4 and OsMPK3 transcripts were found to be induced in arsenite treated rice leaves and roots (Table 1). In-silico homology modeling and docking analysis supported OsMKK4–OsMPK3 interaction (Rao et al., 2011), suggesting the role of this MAPK module in arsenic stress. Accumulating evidences suggest that metal ions such as arsenic and chromium are able to induce reactive oxygen and nitrogen species, thereby altering nitric oxide (NO) induced cell signaling. NO has been shown to modulate the activity of MAPK, NO donors, and recombinant NOS were shown to cause the activation of SIPK (Rao et al., 2011). g g y MAPKs are some of the most important and highly conserved signaling molecules that function in response to many diverse stresses and during many developmental pathways (Sinha et al., 2011). MAPK cascade consists of three tier components MAPKKKs, MAPKKs, and MAPKs mediating phosphorylation reactions from upstream receptor to downstream target (Hamel et al., 2006). MAPK signaling mediates the transmission of stress related signals thus regulating large number of cellular processes (Hamel et al., 2006; Rodriguez et al., 2010). Among abiotic stresses, heavy metal stress has conferred profound eﬀect on MAPK signaling pathways. MAPKs are known to be activated by perception of speciﬁc metal ligand and also by ROS molecules produced in the metal stress (Jonak et al., 2004; Smeets et al., 2013; Jalmi and Sinha, 2015). Heavy metal induced ROS production is already known in plants and the role of these ROS molecules in activating MAPK signaling is very well-accepted. In Arabidopsis, two important completely characterized MAPK cascade MEKK1-MKK4/5- MPK3/6 (Asai et al., 2002) and MEKK1-MKK2-MPK4/6 are known to work downstream of ROS, participating in both abiotic and biotic stress signaling (Pitzschke et al., 2009; Jalmi and Sinha, 2015). Apart from this, MAPK cascades also exert positive feedback regulation on ROS production. A cascade OXI1-MPK6 activated by ROS also positively regulates ROS production (Asai et al., 2008). MEKK1-MKK4-MPK3/6 is known to act upstream of NADPH oxidase stimulating ROS production in pathogen attack and H2O2 produced is in turn known to activate MPK3 and MPK6 (Kovtun et al., 2000). Plant Signaling in Response to Heavy Metals The ROS produced in response to metal stress either by respiratory burst oxidase homolog (RBOH) activity or by alteration in electron transport is also known to activate signal transduction. This ﬁgure also exhibits the crosstalk between different signaling modules and the feedback regulation of MAPK cascade by miRNA. P = phosphorylated. FIGURE 2 \| Crosstalk of signaling pathways and its ultimate response in heavy metal stress. This ﬁgure displays the involvement of several signaling components working during metal stress. Sensing of signiﬁcant level of heavy metals by plants initiates signaling network causing activation of various metal responsive transcription factors. These transcription factors (TFs) regulate the expression of metal responsive and other stress related genes ultimately helping the plant to counteract stressed situation. These stress related genes are mainly metal transporters, phytochellatins, metallothionine, antioxidant genes, and miRNA genes (MIR genes). The ROS produced in response to metal stress either by respiratory burst oxidase homolog (RBOH) activity or by alteration in electron transport is also known to activate signal transduction. This ﬁgure also exhibits the crosstalk between different signaling modules and the feedback regulation of MAPK cascade by miRNA. P = phosphorylated. February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 3 Heavy Metal Stress and Plant Signaling Jalmi et al. MAPK Signaling in Heavy Metal Stress Beside the activation of MAPKs by Cu2+ and Cd metals, there are several other heavy metals that cause the activation of MAPKs, but are not studied in detailed aspect. In yeast, Al3+ tolerance was provided by over-expression of a MAP kinase gene in Al3+-sensitive mutant, indicating the association of MAPK with Al3+-resistance (Schott and Gardner, 1997). Similarly, in wheat root apex Al3+ treatment led to the activation of 48- kDa MAPK, playing signiﬁcant role in transmitting Al related signals and Al-resistant in wheat (Mossor-Pietraszewska, 2001). In rice, a 42-kDa MAPK found to activate myelin basic protein (MBP) in response to iron. Pre-treatment of rice roots with an antioxidant, glutathione (GSH), decreased iron-induced root cell death, and MAPK activation, demonstrating the involvement of ROS induced MAPK activation in iron-triggered signaling (Tsai and Huang, 2006). Although, Zn is a non-redox metal, however MAPK activation by Zn results from the activation of oxidative stress in rice. Zn stimulates a rapid activation of MBP Calcium Signaling in Heavy Metal Stress Calcium Signaling in Heavy Metal Stress The calcium ion (Ca2+) as corroborated by diﬀerent studies acts as a universal secondary messenger in the normal functioning of plants as well as in response to various environmental stresses (Sanders et al., 2002). The cytosolic free Ca2+ concentration changes in response to various stress stimuli triggering complex interactions and signal transduction pathways (Rudd and Franklin-Tong, 2001). This transient increase in the cytosolic concentration is perceived by highly sensitive calcium sensing proteins that mediate this chemical signal into a biological February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 4 Heavy Metal Stress and Plant Signaling Jalmi et al. TABLE 1 \| Signaling components involved in metal stress. Heavy metal Plant Signaling components MAPK Calcium Hormone Cd Arabidopsis MEKK1, MPK3, MPK6 (Jonak et al., 2004; Liu et al., 2010) Unknown Auxin: 2PAT1, 2CYP79B2, 2CYP79B3, 2YUCCA, 2GH3, 2TIR1, 1,2,3PINs, 2ABCB, 2ARFs, 3AXR3/IAA17 (1Hu et al., 2013; 2Wang et al., 2015; 3Yuan and Huang, 2016) Cytokinin: IPT, CKX (Vitti et al., 2013) Ethylene: 4,5ACS, 5ACC, 4ETR2, 4ERF1,5, 5GSH1, 5GSH2 (4Weber et al., 2006; 5Schellingen et al., 2015) Rice MAPK2, MPK3, MPK6, MSRMK3, WJUMK (Agrawal et al., 2003; Yeh et al., 2007; Rao et al., 2011) Unknown Auxin: MAPK3/6/7, YUCCA, PINs, ARF, and IAA (Zhao et al., 2014) M. sativa SAMK, SIMK, MMK2, MMK3 (Jonak et al., 2004) Unknown Unknown Zea mays MPK3 (Wang et al., 2010) Unknown Unknown Radish Unknown Ca2+/CaM (Rivetta et al., 1997) Unknown B. natalensis, R. crispus Unknown Unknown Cytokinin: PI-55, INCYDE (Gemrotová et al., 2013) Glycin max Unknown Unknown Ethylene: ACS, MAPK2, MAPKK2 Chmielowska-Bak et al., 2014 Cu Arabidopsis MPK3, MPK6 (Liu et al., 2010; Schellingen et al., 2015) Unknown Auxin: 2PAT1, 2CYP79B2, 2CYP79B3, 2YUCCA, 2GH3, 2TIR1, 2PINs, 2ABCB, 2ARFs, 1,2DR5 (1 Peto et al., 2011; 2Wang et al., 2015) Ethylene: 4COPT5, 3ACS, 3ERF (3Weber et al., 2006; 4Carrió-Seguí et al., 2015) Rice MAPK2, MPK3, MPK6, MSRMK3, WJUMK (Agrawal et al., 2003; Yeh et al., 2007; Rao et al., 2011) Unknown Unknown M. sativa SIMKK, SAMK, SIMK, MMK2, MMK3 (Jonak et al., 2004) Unknown Unknown As Arabidopsis Unknown Unknown Auxin: AUX1, PIN1, PIN2 (Krishnamurthy and Rathinasabapathi, 2013) Ethylene: ERFs (Fu et al., 2014) Rice MKK4, MPK3, MPK4 (Rao et al., 2011) CaM, CaM kinase, CaM like protein (Chakrabarty et al., 2009; Huang et al., 2012) Unknown B. Calcium Signaling in Heavy Metal Stress juncea 46Kda MAPK (Gupta et al., 2009) Unknown Unknown Al Arabidopsis Unknown Unknown Auxin: 1,2PIN2, 2AUX1 (1Shen et al., 2008; 2Sun et al., 2010) Wheat Unknown Myosin, Calpain, Phospholipase C, Phospholipase A2 Unknown TABLE 1 \| Signaling components involved in metal stress. 5GSH1, 5GSH2 (4Weber et al., 2006; 5Schellingen et al., 2015) Rice MAPK2, MPK3, MPK6, MSRMK3, WJUMK (Agrawal et al., 2003; Yeh et al., 2007; Rao et al., 2011) Unknown Auxin: MAPK3/6/7, YUCCA, PINs, ARF, and IAA (Zhao et al., 2014) M. sativa SAMK, SIMK, MMK2, MMK3 (Jonak et al., 2004) Unknown Unknown Zea mays MPK3 (Wang et al., 2010) Unknown Unknown Radish Unknown Ca2+/CaM (Rivetta et al., 1997) Unknown B. natalensis, R. crispus Unknown Unknown Cytokinin: PI-55, INCYDE (Gemrotová et al., 2013) Glycin max Unknown Unknown Ethylene: ACS, MAPK2, MAPKK2 Chmielowska-Bak et al., 2014 Cu Arabidopsis MPK3, MPK6 (Liu et al., 2010; Schellingen et al., 2015) Unknown Auxin: 2PAT1, 2CYP79B2, 2CYP79B3, 2YUCCA, 2GH3, 2TIR1, 2PINs, 2ABCB, 2ARFs, 1,2DR5 (1 Peto et al., 2011; 2Wang et al., 2015) Ethylene: 4COPT5, 3ACS, 3ERF (3Weber et al., 2006; 4Carrió-Seguí et al., 2015) Rice MAPK2, MPK3, MPK6, MSRMK3, WJUMK (Agrawal et al., 2003; Yeh et al., 2007; Rao et al., 2011) Unknown Unknown M. sativa SIMKK, SAMK, SIMK, MMK2, MMK3 (Jonak et al., 2004) Unknown Unknown As Arabidopsis Unknown Unknown Auxin: AUX1, PIN1, PIN2 (Krishnamurthy and Rathinasabapathi, 2013) Ethylene: ERFs (Fu et al., 2014) Rice MKK4, MPK3, MPK4 (Rao et al., 2011) CaM, CaM kinase, CaM like protein (Chakrabarty et al., 2009; Huang et al., 2012) Unknown B. juncea 46Kda MAPK (Gupta et al., 2009) Unknown Unknown Al Arabidopsis Unknown Unknown Auxin: 1,2PIN2, 2AUX1 (1Shen et al., 2008; 2Sun et al., 2010) Wheat Unknown Myosin, Calpain, Phospholipase C, Phospholipase A2 (Jones and Kochian, 1997) Unknown T. aestivum 48Kda MAPK, 42Kda Protein kinase (Osawa and Matsumoto, 2001) Unknown Ethylene: 3ALMT1, 4ACS, 4ACO (3Tian et al., 2014; 4Yu et al., 2016) M. sativa Unknown Unknown Auxin: AUX1, PIN2 (Wang S. et al., 2016) (Continued) Auxin: 2PAT1, 2CYP79B2, 2CYP79B3, 2YUCCA, 2GH3, 2TIR1, 2PINs, 2ABCB, 2ARFs, 1,2DR5 (1 Peto et al., 2011; 2Wang et al., 2015) Ethylene: 4COPT5, 3ACS, 3ERF (3Weber et al., 2006; 4Carrió-Seguí et al., 2015) Unknown Frontiers in Plant Science \| www.frontiersin.org February 2018 \| Volume 9 \| Article 12 Heavy Metal Stress and Plant Signaling Jalmi et al. Calcium Signaling in Heavy Metal Stress TABLE 1 \| Continued Heavy metal Plant Signaling components MAPK Calcium Hormone Hg Arabidopsis Unknown Unknown Auxin: PAT1, CYP79B2, CYP79B3, YUCCA, GH3, TIR1, PINs, ABCB, ARFs (Wang et al., 2015) Rice MSRMK2, MSRMK3, WJUMK (Agrawal et al., 2003) Unknown Ethylene: OsACS2, OsACO1, OsACO2, OsACO5 and OsACO6, 5 MAPKKK, 1 MAPKK and 2 MAPK (Chen et al., 2014) M. sativa Unknown Unknown Ethylene: ACCS, ACCO, AP2, ERF1 (Montero-Palmero et al., 2014) Pb Arabidopsis Unknown CNGC1 (Sunkar et al., 2000) Auxin: PAT1, CYP79B2, CYP79B3, YUCCA, GH3, TIR1, PINs, ABCB, ARFs (Wang et al., 2015) Ethylene: EIN2 (Cao et al., 2009) Rice 34Kda, 40Kda & 42Kda MAPK (Huang and Huang, 2008) CDPK-like Kinase (Huang and Huang, 2008) Unknown tobacco Unknown CBP4 (Arazi et al., 1999; Sunkar et al., 2000) Unknown R. sativus MAPKKK7, MAPK6, MAPK18, MAPK20 (Wang et al., 2013) Unknown Unknown Zn Arabidopsis Unknown Unknown Auxin: PAT1, CYP79B2, CYP79B3, YUCCA, GH3, TIR1, PINs, ABCB, ARFs (Wang et al., 2015) Rice 34Kda, 40Kda & 42Kda MAPK (Lin et al., 2005) Unknown Auxin: MAPK3/6/7, YUCCA, PINs, ARF, and IAA (Zhao et al., 2014) Wheat Unknown Myosin, Calpain, Phospholipase A2 (Jones and Kochian, 1997) Unknown Cr Zea mays MPK5 (Ding et al., 2009) Unknown Unknown Foxtail millet Unknown TPC1, MRC5, CaM (Fang H. et al., 2014) Unknown Mn Arabidopsis Unknown ECA1 (Wu et al., 2002) Unknown Ni Arabidopsis Unknown Unknown Auxin: PAT1, CYP79B2, CYP79B3, YUCCA, GH3, TIR1, PINs, ABCB, ARFs (Wang et al., 2015) Tobacco Unknown CBP4 (Arazi et al., 1999) Unknown Ba Faba bean Unknown Ca2+ channels (Hamilton et al., 2001) Unknown B Barley Unknown Calmodulin, Ca2+- binding proteins (Tombuloglu et al., 2015) Unknown response. Plants harbor myriads of calcium sensing proteins such as Calmodulins (CaMs), CaM like proteins (CMLs), Calcineurin B-like proteins (CBLs), and Ca2+-dependent protein kinases (CDPKs) that bind to Ca2+ and trigger diﬀerent downstream signaling pathways (Luan et al., 2002; Sanders et al., 2002; Steinhorst and Jörg, 2003; Dodd et al., 2010). Multiple studies in diﬀerent plant species, such as chickpea, Glycine max, Vitis vinifera, and tomato have been carried out response. Plants harbor myriads of calcium sensing proteins such as Calmodulins (CaMs), CaM like proteins (CMLs), Calcineurin B-like proteins (CBLs), and Ca2+-dependent protein kinases (CDPKs) that bind to Ca2+ and trigger diﬀerent downstream response. Calcium Signaling in Heavy Metal Stress Plants harbor myriads of calcium sensing proteins such as Calmodulins (CaMs), CaM like proteins (CMLs), Calcineurin B-like proteins (CBLs), and Ca2+-dependent protein kinases (CDPKs) that bind to Ca2+ and trigger diﬀerent downstream signaling pathways (Luan et al., 2002; Sanders et al., 2002; Steinhorst and Jörg, 2003; Dodd et al., 2010). Multiple studies in diﬀerent plant species, such as chickpea, Glycine max, Vitis vinifera, and tomato have been carried out February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 6 Heavy Metal Stress and Plant Signaling Jalmi et al. to discern the contribution of Ca2+-binding like proteins and Ca2+ sensing proteins in augmented tolerance to various abiotic stresses (Tripathi et al., 2009; Li Z. Y. et al., 2012; de la Torre et al., 2013). There have been reports demonstrating that the application of Ca2+ exogenously can modulate the physiological and biochemical responses in order to alleviate the heavy metal stress. The activity of antioxidant enzymes such as ascorbate peroxidase, glutathione reductase, and superoxide dismutase has been shown to be enhanced upon the application of exogenous Ca2+ (Ahmad et al., 2015). Though there have been several reports that substantiate the role of Ca2+ and Ca2+-dependent signaling pathways in imparting tolerance to heavy metal stresses in plants, our understanding of the mechanisms by which these responses are regulated is still meager and invites further elaboration. Arazi et al. that transgenic tobacco plants expressing the plasma membrane associated NtCBP4 (Nicotiana tobacum calmodulin- binding protein) exhibit higher levels of tolerance to Ni toxicity. Contrastingly, the same plants were found to be hypersensitive to Pb2+, depicting an exclusion of Ni2+ and augmented accumulation of Pb2+ as compared to wild type plants (Arazi et al., 1999) (Table 1). In foxtail millet (Setaria italica), hydrogen sulﬁde was found to interact with Ca2+ signaling in imparting improved tolerance to Chromium (Cr VIIV)-mediated heavy metal stress. It has also been discerned that Ca2+ provides tolerance against Cr stress by enhancing the activity of antioxidant enzymes (Fang H. et al., 2014). Additionally, an involvement of CDPKs has also been suggested through transcriptional proﬁling of the rice roots exposed to long or short durations of Cr stress. Increasing Cr(VI) concentration was found to be correlated with an increase in CDPK-like protein activity, reﬂecting the role of Ca2+ signaling in the stress response (Huang et al., 2014) (Table 1). Calcium Signaling in Heavy Metal Stress Cd is one of the heavy metals physiochemical properties very similar to that of calcium (Choong et al., 2014). This naturally results in an exchangeability of the two ions in Ca2+ binding proteins and studies have provided evidence that Cd displaced Ca2+ from its binding sites in calmodulin, sarcolemma and troponin C in vitro (Langer and Nudd, 1983; Chao et al., 1984; Ellis et al., 1984). The high similarity in the ionic radii of Ca2+ and Cd indicates a possibility of Cd uptake through receptor- or voltage-gated Ca2+ channels and this uptake could possibly be inhibited (at least to some extent) by blocking the Ca2+ channels (Choong et al., 2014). Plants exposed to cadmium exhibit a higher level of intracellular Ca2+, inducing adaptive mechanisms in order to mitigate the toxic eﬀects of the heavy metal (Yang and Poovaiah, 2003). One of the mechanisms used for the increase in Ca2+ level is the production of IP3 which triggers the release of sequestered calcium from the intracellular calcium reserves as indicated in few reports (Smith et al., 1989). A study of Brassica juncea in consistence with the above report proves that application of Ca2+ attenuates the toxicity and makes the plant withstand the deleterious eﬀects of Cd consequently improving the growth and seed quality of the plants (Ahmad et al., 2015). Moreover, studies on diﬀerent plant species have demonstrated that exogenous application of calcium and silica, calcium and spermidine and Ca2+ and/or K2+ can promote the alleviation of cadmium toxicity and reduction in metal accumulation (Siddiqui et al., 2012; Srivastava et al., 2015; Gong et al., 2016). p g Diﬀerential expression of Calmodulins in response to arsenic stress indicates the possible role of Ca2+ signaling components in the stress response (Chakrabarty et al., 2009). Besides, a study has demonstrated that cytosolic free Ca2+ played a key role in the regulation of root activity, metal contents and biomass in close relation to lanthanum (La) dose and acid rain strength. The adverse eﬀects on the roots caused by acid rain could be alleviated by low concentrations of LaIII and synergistic eﬀects on the roots were observed upon combined exposures at higher concentrations of La(III) and acid rain (Zhang et al., 2016). The release of intracellular Ca2+, the subsequent activation of Ca2+ channels and the generation of H2O2 was observed in response to elevated levels of Cu2+ in the marine alga, Ulva compressa. Frontiers in Plant Science \| www.frontiersin.org Hormone Signaling in Heavy Metal Stress This inhibition of primary root elongation by Cu is also due to the modulation of auxin redistribution by PIN1 (Yuan and Huang, 2016). Further, Al was also studied to inhibit root growth by inhibiting the transport of PIN2 vesicles from plasma membranes to endosomes, further disturbing IAA synthesis in apical buds and imbalance of IAA transportation and distribution in roots (Shen et al., 2008; Wang S. et al., 2016). It was further revealed that aux1-7 and pin2 mutants exhibited better tolerance to Al3+ than wild-type plants implying the plausible involvement of AUX1 and PIN2 proteins in Al3+ induced inhibition of root elongation. Apart from this, Cd disrupts the maintenance of auxin homeostasis in Arabidopsis seedlings by increasing IAA oxidase activity and altering the expression of several auxin biosynthetic and catabolic genes (Hu et al., 2013). Cd- mediated up-regulation of biosynthesic gene NITRILASE (NIT) resulted in increased IAA concentration in Arabidopsis roots promoting lateral root growth, thus protecting roots from Cd (Vitti et al., 2013). Moreover, recent report revealed the inhibition of root meristem growth through Cd-induced NO accumulation, which in turn represses auxin transport and stabilizes AUX/IAA proteins to repress auxin signaling (Yuan and Huang, 2016). A positive role for auxin transport through AUX1 on plant tolerance to As stress via ROS-mediated signaling was also disclosed in a study (Krishnamurthy and Rathinasabapathi, 2013) (Table 1). Cytokinins (CKs) are N6-prenylated adenine derivatives involved in the regulation of plant growth and development and in biotic and abiotic stresses (Perilli et al., 2010). There are reports of CKs in plants activated upon heavy metal stress that are able to alleviate heavy metal induced toxicity. The inhibition of photosynthetic pigment and chloroplast membranes by Cd was restored by CKs, increasing photosynthetic capacity and primary metabolite levels (Piotrowska-Niczyporuk et al., 2012). Exogenous kinetin application can also modulate antioxidant enzyme activity, proline, free amino acids, and soluble sugars that counteracted Cd caused inhibitory eﬀects on growth and photosynthesis (Al-Hakimi, 2007). Ethylene (ET) is a gaseous plant hormone regulating various important growth aspects. It is biosynthesized by ACC synthase (ACCS) that convert AdoMet to ACC, while ACC oxidase (ACCO) catalyzes the conversion of ACC to ethylene. ACCS and ACCO are encoded by multigene families and regulated by many biotic and abiotic factors (Kende, 1993). Several reports support the role for ethylene in the regulation of plant metal stress responses. Hormone Signaling in Heavy Metal Stress report unveiled the interplay of auxin/cytokinin and MAPKs, in which OsMKK4/5-OsMPK3/6 was elucidated as key player in auxin/cytokinin interaction regulating the expression pattern of OsPIN1b/9 (Singh P. et al., 2015). However, the involvement of MAPK signaling regulating auxin response in metal stress is still uncertain. Appealing study performed in rice displayed relationship between auxin signaling and MAPK signaling in Cd stress. It was analyzed that expression of most of the key genes of auxin signaling including YUCCA, PIN, ARF, IAA, and cell cycle related genes was negatively regulated by MAPK in Cd stress (Zhao et al., 2014). This certainly implicates the major role of MAPK signaling in regulating auxin signaling in heavy metal stress. Hormone Signaling in Heavy Metal Stress The root architecture is of great importance in plant grown in metal-polluted areas, as the remodeling of root architecture in response to metals can be used as a strategy to escape from heavy metal stress. Interestingly, auxin, ethylene, and cytokinin modulate patterning (Vanstraelen and Benková, 2012) and lateral root formation (De Smet et al., 2015). Thus, there are several studies reporting the involvement of these phytohormones in remodeling of the root system architecture in response to heavy metal stress. Auxin is an essential plant growth hormone playing role in developmental as well as environmental stress responses. It directly aﬀects plant responses to metal stresses by modulating auxin homeostasis including auxin stability, transport, and redistribution (Potters et al., 2007). Basipetal auxin transport through the outer root cell layers is mediated by AUX1 and PIN2 (Marchant et al., 1999; Rashotte et al., 2000). The regulation of auxin signaling in heavy metal stress is evident by various studies conducted over the years. Recently, it was reported that in response to metal stress plants regulate the location and accumulation of auxin by diﬀerential and dynamic expression of auxin-related genes like Phosphoribosyl Anthranilate Transferase 1 (PAT1), CYP79B2 and CYP79B3, YUCCA (YUC), Gretchen Hagen (GH3) genes, (TIR1), PIN family, and ABCB family (Wang et al., 2015) (Table 1). Cu2+ toxicity in Arabidopsis leads to changes in auxin and cytokinin accumulations and mitotic activity within the primary and secondary root tips (Lequeux et al., 2010). It is also reported that in excess of Cu2+ lack of auxin leads to an increase in NO levels thereby diminishing root elongation (Peto et al., 2011). Calcium Signaling in Heavy Metal Stress It was evidenced that the gene expression of antioxidant system is regulated via cross-talk among the various cellular signals and levels of Ca2+, NO, and H2O2 (González et al., 2012). It is established that in response to environmental changes CDPK work together with MAPK for transmission of signals to adapt against changing environment (Takahashi et al., 2011; Wurzinger et al., 2011; Opdenakker et al., 2012) (Figure 2). A CDPK, CPK18 was found to be an upstream kinase of MPK5 in rice, wherein MPK5 was phosphorylated on Threonine 14 and 32 by CPK18 (Xie et al., 2014). Also, MKK3 together with Ca/CaM is known to activate MPK8, which negatively regulates the expression of RBOHD (NADPH oxidase) in response to mechanical stress (Takahashi et al., 2011). A study suggest that Ca2+ is involved in Pb2+-mediated cell death and triggering of MAPK activity via CDPK pathway by enhancing the activity of CDPK like kinase (Huang and Huang, 2008). Besides this, the role of calmodulins has been reported to modulate MAPK signaling pathway (Tebar et al., 2002), which deﬁnes a possibility of their interplay in response to metal stress. All these ﬁndings outline a vital function for the Ca2+ regulatory loop, which is critical for maintaining the redox homeostasis of the cell and ion balance in response to heavy metal stress. In animals this crosstalk has been elaborately studied in metal stress than in plants. Hence, it will be highly advantageous to study the importance of this signaling crosstalk and further the regulation of Ca2+ signaling in heavy metal stress in plants. An interesting study on Arabidopsis seedlings has shown that Ca2+ mitigates the toxic eﬀects of Cd through maintaining auxin homeostasis indicating a crosstalk between signaling pathways in order to combat heavy metal stress (Zhao et al., 2015). Moreover, studies on yeast cells have proposed the role of Ca2+-ATPases (Pmr1p and Pmc1p) of vacuolar and Golgi membrane in coping with Cd toxicity. This is achieved through cooperation with a Glutathione-conjugated transporter Ycf1p whose activity is controlled by phosphorylation once again insinuating an interface between diﬀerent signaling pathways in response to environmental stresses (Mielniczki-Pereira et al., 2011). The Ca2+/Calmodulin system is also involved in response to toxicity mediated by heavy metals other than Cd such as Pb2+ and nickel (Ni2+) (Ahmad et al., 2015). Frontiers in Plant Science \| www.frontiersin.org Calcium Signaling in Heavy Metal Stress It was demonstrated by February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 7 Heavy Metal Stress and Plant Signaling Jalmi et al. Hormone Signaling in Heavy Metal Stress This suggests that decrease in the amount of miR192 leads to the accumulation of ABC transcripts which eventually leads to Cd sequestration by ABC transporter during Cd stress (Ding et al., 2011; Tang et al., 2014; He et al., 2016) (Figure 3). g Like the interplay of MAPK with auxin signaling, there are also evidences of involvement of MAPKs in ethylene biosynthesis and signaling, however its importance in metal stress is still unknown (Opdenakker et al., 2012). Two important MAPKs, MPK3 and MPK6 are known to be responsible for phosphorylation of ACS2 and ACS6, thus increasing ethylene production (Liu and Zhang, 2004; Li G. et al., 2012). This ACS2/6 is very well reported to be induced by metal stress. Further, putresciene, an essential signaling molecule involved in modulating plant resistance to Al stress by inhibiting ACS and ET production (Yu et al., 2016), was found to be regulated by AtMPK3/6 (Kim et al., 2013). Moreover, a MAPK cascade MKK9-MPK3/6 acting downstream of ethylene receptor CTR1 was found to control a key transcription factor EIN3 involved in ethylene biosynthesis (Xu et al., 2008; Yoo et al., 2008). Roots of rice plants exposed to Cr showed an increased expression of the EIN3 and EIN4 genes (Trinh et al., 2014) suggesting the putative role of this MAPK cascade heavy metal induced ET biosynthesis. Recently, Chmielowska-Bak et al. (2014) revealed that cadmium causes induction of ethylene responsive genes and MAPKs in soybean seedlings also suggesting an elevation in MAPKK2 gene expression. Further, Schellingen et al. (2015) suggested a link between MPK3/6 mediated ROS production and ethylene signaling during Cd stress in Arabidopsis. g Contemplating these studies carefully we found that cadmium stress downregulates major miRNAs like miR159 and miR166 in most of the plant species (Figure 3). The targets of several Cd responsive miRNAs, including miR398 and miR408 have been shown to target the heavy metal detoxiﬁcation genes. The Cu–Zn superoxide dismutase (CSD), is an essential enzyme for the detoxiﬁcation of superoxide radicals and reduced accumulation of respective miRNAs lead to the accumulation of these scavengers during stress, hence protecting the plants against heavy metal induced oxidative damage. Recent studies have identiﬁed many As responsive miRNAs by deep sequencing in rice and mustard (Liu and Zhang, 2012; Srivastava et al., 2013; Pandey et al., 2015; Sharma et al., 2015) (Figure 3). Hormone Signaling in Heavy Metal Stress The eﬀects of metal stress on ethylene production in plants are both metal and concentration dependent (Thao et al., 2015; Keunen et al., 2016) (Table 1). Major ﬁve ET synthesis genes from rice OsACS2, OsACO1, OsACO2, OsACO5, and OsACO6 (Chen et al., 2014) along with transcription factors AP2 and ERF1 from Medicago sativa (Montero-Palmero et al., 2014) were found to be upregulated in Hg treatment. However in rice, genes involved in cytokinin signaling (OsRR1, 3, 4, 6, and 11) were down regulated, suggesting both ET and CK may regulate the Hg-induced inhibition of rice root growth (Chen et al., 2014). Additionally, Cu and Al were also found to increase ACS transcript level in Arabidopsis, Medicago truncatula and Lotus japonicus (Weber et al., 2006; Sun et al., 2010). There was inhibition in root growth under Al stress which was correlated to enhanced ethylene production upon Al treatment (Sun et al., 2010). Recently, it was revealed that in wheat ET negatively regulates Al-induced eﬄux of malate ions using ET8, which is an important mechanism for Al tolerance (Tian et al., 2014; Yu et al., 2016) (Table 1). Besides the eﬀect of heavy metals on ethylene synthesis, they even exert eﬀect on ethylene signaling. Cu treatment increases expression of number of ethylene responsive factors like ERF1, ERF2, and ERF5 (Weber et al., 2006). Apart from these, Cd was exhibited to establish its role in regulating ethylene synthetic genes (ACS2 and ACS6) along with MAPK cascades, MAPK signaling is established in inﬂuencing auxin signaling and its transport (Mockaitis and Howell, 2000). A captivating February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 8 Heavy Metal Stress and Plant Signaling Jalmi et al. NO generation, and polyamine metabolism (Chmielowska-Bak et al., 2014; Schellingen et al., 2015) (Table 1). Plant exposed to excess concentration of Cd, employed diﬀerential regulation of miRNAs. For example in rice, Cd exposure modulates expression of various novel and evolutionarily conserved miRNAs. Upon exposure to Cd, miR441 expression was signiﬁcantly upregulated while 12 miRNAs were found to be down-regulated. Among the down-regulated miRNAs, miR192 predicted to target ABC transporter, which is shown to be involved in heavy metal transport. Overexpression of miR192 signiﬁcantly reduced rice seed germination and seedling growth under Cd stress compared to wild-type plants. Regulation of microRNAs during Heavy Metal Stress Besides the contribution of signaling pathways in transmitting heavy metal related stimuli and regulating the plant response, other regulators like small RNAs are majorly found to have profound eﬀect on metal stress response. Small RNAs such as microRNAs are a 20–24 nucleotide non-coding RNAs that regulate the gene expression at post-transcriptional level by targeting mRNA degradation or by translation repression (Raghuram et al., 2014). It has been shown that diﬀerent miRNA families are diﬀerentially regulated temporally as well as spatially, diﬀering in concentration from species to species (He et al., 2016). All these data indicate that diﬀerential regulation of any miRNA depends upon the function of miRNA target, physiology, and metabolism of the plant. g Aluminum (Al) is being considered as a major limiting factor for plant development interfering with cellular redox equilibrium. Similar to other metals, Al stress also downregulates most of the miRNAs in rice such as miR156, miR395, miR398, miR159 except miR399, miR166, miR168 which showed upregulation (Lima et al., 2011). Contrastingly, in maize the similar miRNAs showed upregulation except miR171 and miR396 which showed downregulation (Kong et al., 2014) (Figure 3). MicroRNAs also play a key role in metal complexation wherein two important genes ATP sulfurylase (APS) and SULTR2:1 were identiﬁed as targets of miR395 which is reported to induce by Al stress, Cd stress, and sulfur deﬁciency. Both of these genes lead to the production of GSH and PCs which are chief molecules in metal chelation (Matthewman et al., 2012) (Table 2). Recently genome wide, transcriptome analysis, and high throughput sequencing have been used to identify the microRNAs, which are responsive to heavy metal stress in many plant species. It has been shown that various conserved miRNAs are diﬀerentially regulated during the normal and stress conditions (Figure 3). Diﬀerential expression of miRNAs in heavy metal stress indicates the possible involvement of miRNAs in heavy metal stress detoxiﬁcation and tolerance (Ding et al., 2011; Liu and Zhang, 2012; Zhou et al., 2012; Bukhari et al., 2015; Noman and Aqeel, 2017). Here, we have focused on studies showing diﬀerential expression of conserved miRNA in metal stress and regulation of signaling pathway by miRNAs or vice versa in response to heavy metals. Other metals such as mercury, lead, and chromium have also been shown to aﬀect miRNA expression. Mercury treatment diﬀerentially regulated miRNAs in M. Hormone Signaling in Heavy Metal Stress They have reported that the expression of miR172 was signiﬁcantly down-regulated whereas miR393, miR397, and miR408 were upregulated. Studies revealed that miR408 has a direct role in targeting Cu containing proteins or superoxide dismutase (Ma et al., 2015). Also, it has been reported that during heavy metal stress, ROS leads to the induction of lipid peroxidation and downregulation of miR397, which has been shown to target laccase. This may lead to positive regulation of lignin biosynthesis through the accumulation of laccase enzymes (Jones-Rhoades and Bartel, 2004) (Figure 3). Frontiers in Plant Science \| www.frontiersin.org Regulation of microRNAs during Heavy Metal Stress truncatula where miR156, miR172, miR164, miR169, miR398 were downregulated whereas miR167 and miR172 were upregulated (Zhou et al., 2012). Cotton February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 9 Jalmi et al. Heavy Metal Stress and Plant Signaling FIGURE 3 \| Differential expression of heavy metal responsive microRNAs in plants. The ﬁgure represents the data taken from genome wide study of differentially expressing miRNAs in different plant species. Green color and red color indicates up regulated and down regulated miRNAs respectively. FIGURE 3 \| Differential expression of heavy metal responsive microRNAs in plants. The ﬁgure represents the data taken from genome wide study of differentially expressing miRNAs in different plant species. Green color and red color indicates up regulated and down regulated miRNAs respectively. seedlings treated with Pb showed downregulation of miR156, miR398, miR399 and upregulation of miR162, miR167, miR169, miR395, miR396, and miR397 (He et al., 2014). Chromium showed upregulation of miR156, miR167, miR169, miR171, and downregulation of miR166 (Bukhari et al., 2015) (Figure 3). Among the heavy metal responsive miRNAs, miR156, miR159, miR166, and miR398 were shown to be diﬀerentially regulated. The currently available data about miRNA targets suggests that most of the miRNAs such as miR169, miR390, miR394, mir395, miR397, miR399, and miR528 are directly involved in the heavy metal stress tolerance by regulating the transcripts of ROS scavenging enzymes, laccases, or metal transporters. Apart from their direct involvement in heavy metal stress, some of the miRNAs play important role in plant growth and development. For example, miR156 regulate the important transitions in shoot development while miR159 is known to February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 10 Heavy Metal Stress and Plant Signaling Jalmi et al. TABLE 2 \| Heavy metal responsive conserved miRNAs, their targets, and functions. miRNAs Targets Target functions References miR156 SQUAMOSA-PROMOTER BINDING PROTEIN (SBP)-like proteins (SPL) Floral development Schwab et al., 2005 miR159 MYB transcription factors Floral development Achard et al., 2004 miR160 ARF transcription factors Auxin signaling, Floral organ development Wang et al., 2005 miR162 DCL1 Micro RNA biogenesis Xie et al., 2003 miR164 NAC, CUC genes Drought resistance, Leaf margin serration Nikovics et al., 2006; Fang Y. Regulation of microRNAs during Heavy Metal Stress et al., 2014 miR165/166 HD-ZIP transcription factors, KANADI Root development Singh et al., 2017 miR167 ARF transcription factors Auxin signaling Wang et al., 2015 miR168 AGO1 MicroRNA pathway Vaucheret et al., 2004 miR169 Nuclear factor Y Drought resistance Li et al., 2008 miR171 GRAS domain transcription factors/SCARECROW-like (SCL) Floral development Ma et al., 2014 miR172 APETELA2-like transcription factors Transcriptional regulation, Developmental phase transition Aukerman and Sakai, 2003 miR319 TCP family transcription factor JA biosynthesis, Senescence Schommer et al., 2008 miR390 Stress-responsive leucine-rich repeat receptor-like kinase(SRK), ARF Cd stress tolerance Fahlgren et al., 2006; Ding et al., 2016 miR393 TIR1, AFB family Auxin signaling Chen et al., 2011 miR394 LEAF CURLING RESPONSIVENESS (LCR) Abiotic stress tolerance Song et al., 2016 miR395 ATP sulfurylases (APS), ARABIDOPSIS SULFATE TRANSPORTER 68 (AST68) Sulfate assimilation Matthewman et al., 2012 miR396 GROWTH-REGULATING FACTOR(GRF) TFs, bHLH74 Cell proliferation regulation Debernardi et al., 2012 miR397 Laccase Lignin biosynthesis Jones-Rhoades and Bartel, 2004 miR398 Cu–Zn superoxide dismutase (CSD) ROS response Jones-Rhoades and Bartel, 2004 miR399 ubiquitin-conjugating enzyme E2 24 (UBC24)/PHOS2 Phosphate starvation Chiou et al., 2006 miR408 Cu containing proteins, Cu/Zn superoxide dismutase, Cu chaperon Abiotic stress tolerance Ma et al., 2015 miR441 Unknown – – miR444 MADS-box TFs Root development Wang H. et al., 2016 miR528 MATE transporter family, Cu binding protein(CBF) Enhances Tolerance to Salinity Stress and Nitrogen Starvation, Arsenite Tolerance Liu et al., 2015; Yuan et al., 2015 miR529 SPL family Phase transition Morea et al., 2016 miR818 Serine/threonine kinase Flowering time regulation Liu and Zhang, 2012; Ding et al., 2013 miR827 ubiquitin E3 ligase Suppress immune responses Hewezi et al., 2016 miR2111 PHO2 and GmPT5 Responses to phosphate starvation Xu et al., 2013 miR2118 MEL1 gene, TIR-NBS-LRR Determinate fate acquisition of panicle meristems, drought stress responses Wu et al., 2015; Ta et al., 2016 TABLE 2 \| Heavy metal responsive conserved miRNAs, their targets, and functions. Xu et al., 2013 inhibit growth and promote programmed cell death by regulating R2R3 MYB transcription factors (Alonso-Peral et al., 2010; Xu et al., 2016) (Table 2). miR166 regulate diverse aspects like formation of apical and lateral meristem, leaf polarity vascular development, and ﬂoral development while miR398 is a major plant stress regulator (Jung and Park, 2007). Diﬀerential regulation of these important miRNAs by heavy metals might severely aﬀect plant development by altering various mechanisms (Table 2). by OXI upon Cd and Cu treatment (Smeets et al., 2013). Frontiers in Plant Science \| www.frontiersin.org Modulation of Transcription Factors during Heavy Metal Stress WRKY transcription factors speciﬁcally bind to W-box in the promoters of many genes that are responsive to many biotic and abiotic environmental stress factors. Opdenakker et al. (2012) have reported signiﬁcantly higher expression of members of WRKY family upon Cu and Cd metal exposures. They found that the transcription factors WRKY22, WRKY25, and WRKY29 were overexpressed in response to short-term exposure of roots to Cu. In contrast, only the expression of WRKY25 and WRKY29 aﬀected upon exposure to Cd over a period of 24 hr (Opdenakker et al., 2012). In Cd treated T. caerulescens, expression of WRKY53 was found to be highly induced (Wei et al., 2008). Previous reports also showed that the ﬂagellin induced MAPK cascade MEKK1-MKK4/MKK5-MPK3/MPK6 activates WRKY22 and its close homolog WRKY29 (Asai et al., 2002). Also there are reports showing SA dependent activation of WRKY25 and WRKY33 by MKS1 that directly interacts with MPK4 and negatively regulates defense responses in plants (Andreasson et al., 2005). In accordance with this, a tobacco WRKY1 was found to be phosphorylated by the defense-activating MAP-kinase SIPK (Menke et al., 2005). Also, it was reported that MPK3 and MPK6 phosphorylates WRKY33 and induces phytoalexin biosynthesis in Arabidopsis (Mao et al., 2011). Previous studies revealed that MEKK1 directly interact with WRKY53 on the protein level and also bind to its promoter (Miao et al., 2007). Most recently, expression of WRKY25, a downstream target for MPK4 was found to be up-regulated following Cd exposure (Smeets et al., 2013). Overall, these reports suggest that WRKY transcription factors can work coordinately with MAPK cascade during heavy metal stress tolerance. Heavy metal toxicity is the serious problem of the modern world. For combating heavy metal stress, plants have evolved numerous detoxiﬁcation and mobilization mechanisms as described earlier. Beside these, activation of complex signaling network is another important factor in heavy metal stress tolerance. Genome wide expression analysis have also reported modulation in the expression of transcription factor families upon exposure to heavy metals (Yanhui et al., 2006; Ogawa et al., 2009; Shim et al., 2009; Farinati et al., 2010; Wang et al., 2010; Smeets et al., 2013). Several studies have reported that upon heavy metal exposure, MAPK signaling cascade activates the downstream transcription factor targets (Figure 2). Modulation of Transcription Factors during Heavy Metal Stress The transcription factors such as MYB (MYeloBlastosis), WRKY (containing a conserved WRKYGQK domain and a zinc ﬁnger- like motif), ZAT (C2H2-type zinc ﬁnger transcription factor), bZIP (basic region leucine ZIPper), AP2 (Activator Protein 2), ERF (ethylene-responsive factor), and DREB (dehydration responsive element-binding protein), have been identiﬁed as potential downstream targets of MAPKs (Roelofs et al., 2008; Li et al., 2016). Transcription factors are important regulators of gene expression aﬀecting many developmental processes and defense responses in plants (Yanhui et al., 2006). Studies have showed that upon exposure to Cd, expression of most of the transcription factors belonging to MYB, AP2, DREB, WRKY, and NAC up- regulates at diﬀerent time intervals in rice (Ogawa et al., 2009). The MYB family is one of the largest families of transcription factors having diverse functions in eukaryotes (Dubos et al., 2010). Previous report have shown that MYBs such as MYB4, MYB28, MYB43, MYB48, MYB72, and MYB124 were highly induced in Cd and Zn metal stresses in Arabidopsis (van de Mortel et al., 2008). They have also found that the MYB72 loss of function mutant exhibits increased metal sensitivity in Arabidopsis than the related Zn/Cd-hyper accumulator Thlaspi caerulescens. In another study, it was reported that Cd inactivates MYB2 by induction of NO production which causes nitrosylation of cysteine residues in the MYB2 transcription factor in Arabidopsis (Serpa et al., 2007). Recent reports have also established role of OsMYB45 in Cd toxicity (Hu et al., 2017). They found that mutation in OsMYB45 resulted in Cd hypersensitive phenotype with signiﬁcant increase in H2O2 content in the leaves of mutant and decrease in CAT activity as compared to the wild-type. In recent times, Wang F. Z. et al. (2017) have established the role of rice MYB transcription factor OsARM1 (ARSENITE-RESPONSIVE MYB1) that regulates As-associated transporters genes. He found that OsARM1 binds to the conserved MYB binding sites in the promoters of OsLsi1, OsLsi2, and OsLsi6, which encode key As transporters and aﬀects their expression. Several studies Plant bZIP transcription factors are another class that provides defense against various environmental stresses including heavy metal stress. Reports have suggested induced expression of bZIP transcription factors upon Cd exposure (Ramos et al., 2007). Previous studies showed that the bZIP transcription factor from B. juncea, BjCdR15, is a regulator of Cd uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants (Farinati et al., 2010). Regulation of microRNAs during Heavy Metal Stress OXI is a component of MAPK cascade working upstream to MPK6, regulating ROS production (Asai et al., 2008) (Figure 2). Apart from this, several transcription factors which are known to be downstream target of MAPKs have also been found to be target of miRNA. Transcription factors of Squamosa promoter binding like protein (SPL) family, known to be involved in ﬂower development, were studied to be the targets of miR156/157 and are regulated by Cd, Hg, and Al (Zhao et al., 2014) (Table 2). Additionally, SPLs binds to Cu responsive elements in the promoter of miR398 gene (Cu-responsive gene), A connection between miRNA and MAPK signaling was deciphered by a study which showed regulation of miR398b/c February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 11 Heavy Metal Stress and Plant Signaling Jalmi et al. suggest MYB TFs to be the downstream targets of MAPKs. Most recently, Li et al. (2016) found that MPK4 induced by light, regulates photoprotective anthocyanin biosynthesis by regulating MYB75/PAP1 transcription factor (Li et al., 2016). An altered gene expression and activity of MYBs as well as MAPKs in Cd stress gives us a clue of MYB being potential substrates of MAPKs during heavy metal stress. regulating the expression of miR398 in Cu stress (Yamasaki et al., 2009). These studies uncover the concept of how plants exhibit interaction among diﬀerent components in responding against the environmental stress. Modulation of Transcription Factors during Heavy Metal Stress In Soybean, Cd treatment signiﬁcantly up-regulates bZIP62 expression while the expression of bZIP44 and bZIP78 is down-regulated (Chmielowska-Bak et al., 2014). Likewise, bZIP1 from Tamarix hispida showed increased expression in Cd stress in tobacco (Wang et al., 2010). Recent study on a novel bZIP gene, BnbZIP3 from ramie (Boehmeria nivea) plant has showed that it positively regulates heavy metal stress tolerance by improving root growth upon overexpression (Huang et al., 2016). Though the direct link for bZIP transcription factors with MAPKs was not discovered in context to heavy metal stress but a report suggests that Arabidopsis bZIP transcription factor VIP1 (VirE1-Interacting Protein 1) localization and VirE2/T-DNA February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 12 Heavy Metal Stress and Plant Signaling Jalmi et al. complex nuclear import may require phosphorylation by MPK3 (Djamei et al., 2007). on these MAPKs in number of species. Up-regulation of MPK3 and MPK6 in these heavy metals gives us a clue about their function in metal homeostasis by either regulating downstream metal transporters or chelators that function in response to Cd and Cu. However, studies on ultimate eﬀect of their activation on regulation of metal transporters, other TFs and proteins are still elusive in plants. Even though, there are signiﬁcant reports on regulation of metal transporters by MAPKs in animals which gives an idea about this crosstalk occurring even in plants. j Apart from the above mentioned transcription factors, Cd also modulate expression of AP2/ERF family members, namely, ERF1 and ERF5 in Arabidopsis (Herbette et al., 2006). Similarly, Cd induces ERFs in A. thaliana and A. halleri (Weber et al., 2006). It has been reported that dehydration-responsive element-binding protein (DREB) transcription factors which are members of ERF family of transcription factors gets up-regulated upon heavy metal treatment. Cd leads to elevated expression of DREB1A and DREB1B in rice (Ogawa et al., 2009) while inhibited expression of transcription factors belonging to DRRB family was found in Solanum torvum plants (Yamaguchi et al., 2010). Recent studies have demonstrated that MPK3 and MPK6 regulates ethylene signaling by regulating ERF104 and Ethylene-Insensitive 3 (EIN3) which enhances the expression of ERF104 (Yoo et al., 2008; Bethke et al., 2009). In a recent study, a zinc ﬁnger transcription factor (C2H2-type), ZAT12 expression modulated upon short-term exposure to Cu while no such inﬂuence was observed upon long-term Cd exposure (Opdenakker et al., 2012). Modulation of Transcription Factors during Heavy Metal Stress Recently, Arabidopsis ZAT6 was found to be positive regulator of Cd tolerance through the glutathione-dependent pathway (Chen et al., 2016). They identiﬁed that ZAT6 positively regulates expression of PCs synthesis pathway genes such as GSH1, GSH2, PCS1, and PCS2. Studies through protein-protein interaction also showed ZAT10 as direct substrate for MPK3 and MPK6 (Nguyen et al., 2012), suggesting involvement of MAPKs in regulation of heavy metal stress via ERFs and ZAT transcription factors. g g p A number of metal transporters involved in metal ion homeostasis have been identiﬁed from diﬀerent plants. The major groups of metal transporters studied are ZIP, heavy metal transport ATPase (CPx- and P1B-ATPase), NRAMP, CDF, and ABC transporters (Park et al., 2012; Singh S. et al., 2015). Several studies report their role in metal translocation and uptake based on expression pattern in diﬀerent heavy metals. ZIP members were the ﬁrst to be reported in plants, having ability to transport divalent cations like Zn2+, Fe2+, Mn2+, and Cd (Eide et al., 1996). IRT1 gene from Arabidopsis belonging to ZIP family is major transporter of iron leading to high aﬃnity Fe uptake. In iron limiting environment IRT1 is present only in roots and is studied to be induced within 24 h of iron deﬁcient conditions. Plants overexpressing IRT1 accumulate high levels of Cd and Zn along with Fe (Connolly et al., 2002). Other ZIP members ZIP1 and ZIP2 were studied to be Zn and Mn transporters in roots contributing to remobilization of Mn/Zn from vacuole to cytoplasm in root stellar cells and further movement from root stele to xylem parenchyma. According to their role, the expression of both the genes is mainly localized to the root stele (Milner et al., 2013). An interesting study on iron deﬁciency induced ethylene production in Arabidopsis reports the role of MPK3 and MPK6 in iron transport. The expression of iron transport and chelator genes (IRT1, FRO2, and FIT) was down regulated in mpk3 and mpk6 mutants under Fe deﬁciency (Ye et al., 2015), which suggests a possibility of these iron responsive genes working downstream of MAPK cascade (Table 3). This also suggests a crosstalk of MAPK signaling and hormone pathways in metal translocation in plants. Whilst in plants, there is single report of MAPK involvement in regulating metal transporter; in animals this concept is well-explored. Modulation of Transcription Factors during Heavy Metal Stress It is not only the MAPKs known for regulating metal transporters but metal transporters are also equally involved in activating MAPKs. A report on chicken cell line suggest a role of ZIP transporter ZIP9/SLC39A9 in elevating intracellular zinc level and thereby regulating the activation of Erk MAPK signaling (Taniguchi et al., 2013) (Table 3). Altogether, heavy metal stress activates various signaling components including MAPK cascades. Though, the reports on involvement of MAPKs upstream to transcription factors are rather scarce under metal stress but the above data demonstrated that they indeed interacts with transcription factors and mediates heavy metal stress tolerance response in plants. Frontiers in Plant Science \| www.frontiersin.org MAPK Signaling in Metal Sequestration and Transport Encounter of heavy metal by plant roots generates many responses. This starts with binding of metal to the root cell wall and exudates, followed by metal inﬂux across the plasma membrane. The high degree of metal inﬂux is taken care by eﬄux of metal ions into the apoplast and chelation in the cytoplasm by PCs, metallothionines, organic, and amino acids. These metal ligand complexes are transported to the tonoplast and sequestered into the vacuoles (Sharma and Dietz, 2006; Verbruggen et al., 2009; Mendoza-Cózatl et al., 2011) (Figure 1). There are several molecules involved in this whole process of metal uptake, transportation, chelation, and sequestration. These are metal transporters and chelators accomplishing their task and protecting plants in metal toxicity (Singh S. et al., 2015). Other metal transporter family NRAMP functions in diverse organisms ranging from bacteria to humans. In plants there are two subfamilies of NRAMP genes and several of them upregulate in Fe, Mn, and Cd deﬁciency. NRAMP proteins are studied to be localized on intracellular membranes of plastid and vacuolar membrane (Thomine and Schroeder, 2004). Expression analysis of NRAMP in plants suggests that unlike ZIP family (expressed mainly in roots) these metal transporters are expressed both in root and shoot, thus participating in metal homeostasis in all plant tissues. However, functional characterization of plant NRAMP transporters remains limited. Couple of studies MAPKs are one of the important signaling modules transmitting various stress related signals and are also known to get activated by heavy metal stress as discussed previously. The best characterized MAPKs MPK3 and MPK6 are the ones that are known to get expressed and activated by wide range of metals. Cadmium and copper have shown profound eﬀect February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 13 Heavy Metal Stress and Plant Signaling Jalmi et al. TABLE 3 \| Metal transporters regulating MAPK signaling and vice versa. Family Metal transporter Metal ions MAPKs Model organism Reference METAL TRANSPORTERS REGULATING MAPKs 1 ZIP family ZIP9/SLC39A9 Zn ERK MAPKs Chicken Taniguchi et al., 2013 2 NRAMP family NRAMP1 (SLC11A1) – P38MAPK Mammals Moisan et al., 2006 3 CTR family CTR1 Cu MEK1-ERK1 Mammals Tsai et al., 2012; Turski et al., 2012 4 CDF family ZnT1 Zn, Co, Cd Raf1-MEK-ERK C. MAPK Signaling in Metal Sequestration and Transport elegans Jirakulaporn and Muslin, 2004 5 Metallothioneins Zn P38 MAPK Mammals Rice et al., 2016 MAPKs REGULATING METAL TRANSPORTERS 1 ZIP family IRT1 Fe MPK3, MPK6 Arabidopsis Ye et al., 2015 2 NRAMP family NRAMP1 – P38MAPK, p42/44 MAPK Mammals Zhang et al., 2000 3 ABC family MRP1 – ERK/MAPK pathway Mammals El Azreq et al., 2012 4 ABCA1, ABCG1 Ras-MAPK pathway Mammals Mulay et al., 2013 5 Zn transporter family Zrc1 Zn Pbs2-Hog1-RCK1/RCK2 Yeast Bilsland et al., 2004 6 Phytochelatins – Unknown MAPK S. mansoni Rigouin et al., 2013 Model organism Reference METAL TRANSPORTERS REGULATING MAPKs MAPKs REGULATING METAL TRANSPORTERS In mammals, a study on high aﬃnity copper transporter CTR1 was reported to activate MAPK cascade, wherein the mutation of CTR1 and Cu chelators reduces the activation of Erk1 (MAPK) by MEK1 (MAPKK) (Table 3). This is due to the fact that Erk1 phosphorylation by MEK1 requires Cu binding which diminishes in ctr1 mutant (Tsai et al., 2012; Turski et al., 2012). Likewise, another report suggest that CDF proteins which are famous for the transport of Zn2+, Co2+, Cd in plants, modulates the activity of Raf1-MEK-ERK pathway in C. elegans. Its homolog in mammals, ZnT1 binds directly to the Raf1 in its regulatory domain thus activating it (Jirakulaporn and Muslin, 2004) (Table 3). Besides this, there are wide array of metal transporters that mediate Ca transport and its sequestration into the vacuole. Cation/proton exchangers (CAX) and isoforms have broad speciﬁcity and are widely implicated in Ca transport and other heavy metals like Mn2+ and Cd. CAX have been identiﬁed in salt tolerance, cadmium transport and tolerance. In mammals NHE1, one of the CAX was found to regulate MAPKs wherein it inhibited ERK1/2 and stimulated JNK1/2 activity (Pedersen et al., 2007). in animal suggest the regulation of NRAMP1 transporter by MAPKs. This ion transporter was known to work downstream of p38 and p42/44 MAPK pathway activated upon proinﬂammatory mediators and bacterial infection in mammalian cells (Zhang et al., 2000). Also, another study implied the role of NRAMP1 in modulation of MAPK pathway (Moisan et al., 2006) (Table 3). ( ) ABC transporters comprises of largest family, classiﬁed into eight subfamilies playing roles in diverse cellular processes like nutrient uptake, osmotic homeostasis, hormone transport, pathogen resistance, fatty acid import, and metal tolerance (Park et al., 2012). Frontiers in Plant Science \| www.frontiersin.org MAPK Signaling in Metal Sequestration and Transport Arabidopsis ABC transporter, AtPDR8 is identiﬁed as cadmium extrusion pump conferring resistance to heavy metal Cd and Pb (Kim et al., 2007). Owing to their metal transportation capacity ABC C-type transporters AtABCC1 and AtABCC2 have been identiﬁed as major phytochelatin-heavy metal(oid) complex transporters. In recent study in wheat, expression of 13 ABC transporter genes was analyzed in diﬀerent metals, which suggested that these genes were diﬀerentially regulated by Cd, indicating their participation in Cd uptake, transport, and sequestration (Wang X. et al., 2017). Yet another transporter of ABC family named as MRP1 (multidrug associated protein) is known to be regulated in ERK/MAPK pathway dependent manner in leukemic T cells (El Azreq et al., 2012). ABC transporters are known for their role in vacuole sequestration in plants. Besides this Ras/MAPK pathway is also reported to regulate ABC metal transporters (ABCA1 and ABCG1) in human hepatic cells (Mulay et al., 2013) (Table 3). A study in yeast proves the fact of activation of metal transporter by MAPKs more ﬁrmly. A MAPK cascade consisting of Pbs2-Hog1-Rck1/Rck2 in yeast is studied to activate a transcription factor (Yap2) and Zn transporter (Zrc1) thereby providing oxidative stress resistance (Bilsland et al., 2004) (Table 3). Apart from the transporters activating MAPKs or vice versa, there are also studies suggesting the activation of MAPKs by metal chelators. The important metal chelators are PCs and metallothioneins and these are small cystein rich peptides with metal binding capacity (Singh S. et al., 2015). Two reports suggest MAPK activation by metallothioneins mainly by the release of the metal ions chelated (Chung et al., 2008; Rice et al., 2016). Additionally, PCs and phytochelatin synthase are hypothesized to be acting downstream of MAPK pathway in plants and human parasite respectively (Rigouin et al., 2013) (Table 3). REFERENCES Aukerman, M. J., and Sakai, H. (2003). Regulation of ﬂowering time and ﬂoral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 15, 2730–2741. doi: 10.1105/tpc.016238 Achard, P., Herr, A., Baulcombe, D. C., and Harberd, N. P. (2004). Modulation of ﬂoral development by a gibberellin-regulated microRNA. Development 131, 3357–3365. doi: 10.1242/dev.01206 Bethke, G., Unthan, T., Uhrig, J. F., Pöschl, Y., Gust, A. A., Scheel, D., et al. (2009). Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Proc. Natl. Acad. Sci. U.S.A. 106, 8067–8072. doi: 10.1073/pnas.0810206106 Agrawal, G. K., Iwahashi, H., and Rakwal, R. (2003). Rice MAPKs. Biochem. Biophys. Res. Commun. 302, 171–180. doi: 10.1016/S0006-291X(03)00174-8 Bilsland, E., Molin, C., Swaminathan, S., Ramne, A., and Sunnerhagen, P. (2004). Rck1 and Rck2 MAPKAP kinases and the HOG pathway are required for oxidative stress resistance. Mol. Microbiol. 53, 1743–1756. doi: 10.1111/j.1365-2958.2004.04238.x Ahmad, A., Hadi, F., and Ali, N. (2015). Eﬀective phytoextraction of cadmium (Cd) with increasing concentration of total phenolics and free proline in Cannabis sativa (L) plant under various treatments of fertilizers, plant growth regulators and sodium salt. Int. J. Phytoremed. 17, 56–65. doi: 10.1080/15226514.2013.828018 Bukhari, S. A., Shang, S., Zhang, M., Zheng, W., Zhang, G., Wang, T. Z., et al. (2015). Genome-wide identiﬁcation of chromium stress-responsive micro RNAs and their target genes in tobacco (Nicotiana tabacum) roots. Environ. Toxicol. Chem. 34, 2573–2582. doi: 10.1002/etc.3097 Al-Hakimi, A. M. A. (2007). Modiﬁcation of cadmium toxicity in pea seedlings by kinetin. Plant Soil Environ. 53, 129–135. doi: 10.4236/ajps.2016.712153 Alonso-Peral, M. M., Li, J., Li, Y., Allen, R. S., Schnippenkoetter, W., Ohms, S., et al. Alonso-Peral, M. M., Li, J., Li, Y., Allen, R. S., Schnippenkoetter, W., Ohms, S., et al. (2010). The microRNA159-regulated GAMYB-like genes inhibit growth and promote programmed cell death in Arabidopsis. Plant Physiol. 154, 757–771. doi: 10.1104/pp.110.160630 Cao, S., Chen, Z., Liu, G., Jiang, L., Yuan, H., Ren, G., et al. (2009). The Arabidopsis Ethylene-Insensitive 2 gene is required for lead resistance. Plant Physiol. Biochem. 47, 308–312. doi: 10.1016/j.plaphy.2008.12.013 Andreasson, E., Jenkins, T., Brodersen, P., Thorgrimsen, S., Petersen, N. H., Zhu, S., et al. (2005). The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J. 24, 2579–2589. doi: 10.1038/sj.emboj.7600737 Carrió-Seguí, A., Garcia-Molina, A., Sanz, A., and Peñarrubia, L. (2015). Defective copper transport in the copt5 mutant aﬀects cadmium tolerance. Plant Cell Physiol. CONCLUSION From this review it is implied that metal exerts tremendous eﬀect on plant by modulating its functioning at various levels. Metal stress activates several signaling pathways, known to have important role in imparting resistance against environmental stresses. Of these, an important signaling pathway contributing Another metal transporter involves CTR transporter having an important role in maintaining Cu homeostasis in various species. CTR transporters are either plasma membrane proteins transporting Cu from extracellular spaces to cytosol or lysosome membrane proteins transporting Cu from lysosome to cytosol. February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 14 Heavy Metal Stress and Plant Signaling Jalmi et al. majorly in managing stress response is MAPK signaling pathway. Activation of signaling pathways magniﬁes the activation and functioning of various downstream components like transcription factors and other cytosolic protein thereby altering the expression of genes. In this review, the impact of heavy metals on activation of MAPK, calcium and hormone signaling along with other regulators like transcription factors and miRNAs is certainly reported. Several studies performed throughout diﬀerent scientiﬁc groups suggest important role of MAPK signaling in heavy metal stress. However, a detailed evaluation of complete MAPK cascade working in combating heavy metal stress is required. Further, this review compiles the results revealing interplay of MAPK signaling with calcium, auxin, and ethylene signaling in response to heavy metal stress. All these ﬁndings outline the regulatory function of MAPKs acting either upstream or downstream to other signaling molecules. In animals this regulatory network has been elaborately studied in metal stress than in plants. Hence, it will be highly advantageous to study the importance of this signaling crosstalk in heavy metal stress in plants. Additionally, this review also summarizes interplay between MAPK signaling and other regulators like miRNAs and transcription factors in conveying a response against metal stress. However, scarce reports on regulatory network of MAPKs with transcription factors suggest a need for more in depth experiments in response to heavy metal stress in plants. Though, there are ample numbers of reports on activation of diﬀerent signaling components, studies on deciphering of a complete regulatory network in heavy metal stress in plants are still lacking. Furthermore, the studies on activation of MAPKs by metals and metal transporters and in turn their regulation by MAPKs in animals and yeast, suggests occurrence of this phenomenon even in plants. CONCLUSION However, the fragmentary work performed keeps this area mysterious in plants. Exploring the regulators of these metal transporters will contribute signiﬁcantly in unraveling the mechanisms of metal stress tolerance in plants. ACKNOWLEDGMENTS Authors thank Department of Biotechnology, Government of India, Department of Science and Technology, Government of India and core grant of National Institute of Plant Genome Research, India. FUNDING This work is funded by Department of Biotechnology, Government of India, Department of Science and Technology, Government of India and core grant of National Institute of Plant Genome Research, India. AUTHOR CONTRIBUTIONS SJ, PB, DV, SN, ST, and DS: wrote the manuscript; SJ: conceptualize the overall structure, prepared the illustrations, and edited the manuscript; KS and PB: prepared ﬁgures; AS: conceptualized, edited and approved the ﬁnal manuscript. REFERENCES H., et al. (2011). Regulation of auxin response by miR393-targeted transport inhibitor response protein1 is involved in normal development in Arabidopsis. Plant Mol. Biol. 77, 619–629. doi: 10.1007/s11103-011-9838-1 Fahlgren, N., Montgomery, T. A., Howell, M. D., Allen, E., Dvorak, S. K., Alexander, A. L., et al. (2006). Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA aﬀects developmental timing and patterning in Arabidopsis. Curr. Biol. 16, 939–944. doi: 10.1016/j.cub.2006.03.065 619–629. doi: 10.1007/s11103-011-9838-1 Chiou, T. J., Aung, K., Lin, S. I., Wu, C. C., Chiang, S. F., and Su, C. L. (2006). Regulation of phosphate homeostasis by microRNA in Arabidopsis. Plant Cell 18, 412–421. doi: 10.1105/tpc.105.038943 Fang, H., Jing, T., Liu, Z., Zhang, L., Jin, Z., and Pei, Y. (2014). Hydrogen sulﬁde interacts with calcium signaling to enhance the chromium tolerance in Setaria italica. Cell Calcium 56, 472–481. doi: 10.1016/j.ceca.2014. 10.004 Chmielowska-Bak, J., Gzyl, J., Rucinska-Sobkowiak, R., Arasimowicz-Jelonek, M., and Deckert, J. (2014). The new insights into cadmium sensing. Front. Plant Sci. 5:245. doi: 10.3389/fpls.2014.00245 Fang, Y., Xie, K., and Xiong, L. (2014). Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice. J. Exp. Bot. 65, 2119–2135. doi: 10.1093/jxb/eru072 Choong, G., Liu, Y., and Templeton, D. M. (2014). Interplay of calcium and cadmium in mediating cadmium toxicity. Chem. Biol. Interact. 211, 54–65. doi: 10.1016/j.cbi.2014.01.007 Farinati, S., DalCorso, G., Varotto, S., and Furini, A. (2010). The Brassica juncea BjCdR15, an ortholog of Arabidopsis TGA3, is a regulator of cadmium uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants. New Phytol. 185, 964–978. doi: 10.1111/j.1469-8137.2009.03132.x Chung, R. S., Hidalgo, J., and West, A. K. (2008). New insight into the molecular pathways of metallothionein-mediated neuroprotection and regeneration. J. Neurochem. 104, 14–20. doi: 10.1111/j.1471-4159.2007. 05026.x Connolly, E. L., Fett, J. P., and Guerinot, M. L. (2002). Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. Plant Cell 14, 1347–1357. doi: 10.1105/tpc. 001263 Fu, S. F., Chen, P. Y., Nguyen, Q. T. T., Huang, L. Y., Zeng, G. R., Huang, T. L., et al. (2014). Transcriptome proﬁling of genes and pathways associated with arsenic toxicity and tolerance in Arabidopsis. BMC Plant Biol. 14:94. doi: 10.1186/1471-2229-14-94 Cuypers, A., Smeets, K., and Vangronsveld, J. (2009). “Heavy metal stress in plants,” in Plant Stress Biology: From Genomics to Systems Biology, ed H. Hirt (Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA), 161–178. Gemrotová, M., Kulkarni, M. REFERENCES 56, 442–454. doi: 10.1093/pcp/pcu180 Arazi, T., Sunkar, R., Kaplan, B., and Fromm, H. (1999). A tobacco plasma membrane calmodulin-binding transporter confers Ni2+ tolerance and Pb2+ hypersensitivity in transgenic plants. Plant J. 20, 171–182. doi: 10.1046/j.1365-313x.1999.00588.x Chakrabarty, D., Trivedi, P. K., Misra, P., Tiwari, M., Shri, M., Shukla, D., et al. (2009). Comparative transcriptome analysis of arsenate and arsenite stresses in rice seedlings. Chemosphere 74, 688–702. doi: 10.1016/j.chemosphere.2008.09.082 Asai, S., Ohta, K., and Yoshioka, H. (2008). MAPK signaling regulates nitric oxide and NADPH oxidase dependent oxidative bursts in Nicotiana benthamiana. Plant Cell 20, 1390–1406. doi: 10.1105/tpc.107.055855 Chao, S. H., Suzuki, Y., Zysk, J. R., and Cheung, W. Y. (1984). Activation of calmodulin by various metal cations as a function of ionic radius. Mol. Pharmacol. 26, 75–82. Asai, T., Tena, G., Plotnikova, J., Willmann, M. R., Chiu, W. L., Gomez-Gomez, L., et al. (2002). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415, 977–983. doi: 10.1038/415977a Chen, C. T., Chen, L., Lin, C. C., and Kao, C. H. (2001). Regulation of proline accumulation in detached rice leaves exposed to excess copper. Plant Sci. 160, 283–290. doi: 10.1016/S0168-9452(00)00393-9 Frontiers in Plant Science \| www.frontiersin.org February 2018 \| Volume 9 \| Article 12 15 Heavy Metal Stress and Plant Signaling Jalmi et al. Chen, J., Yang, L., Yan, X., Liu, Y., Wang, R., Fan, T., et al. (2016). Zinc-Finger transcription factor ZAT6 positively regulates cadmium tolerance through the glutathione-dependent pathway in Arabidopsis. Plant Physiol. 171, 707–719. doi: 10.1104/pp.15.01882 Eide, D., Broderius, M., Fett, J., and Guerinot, M. L. (1996). A novel iron-regulated metal transporter from plants identiﬁed by functional expression in yeast. Proc. Natl. Acad. Sci. U.S.A. 93, 5624–5628. doi: 10.1073/pnas.93.11.5624 El Azreq, M. A., Naci, D., and Aoudjit, F. (2012). Collagen/β1 integrin signaling up-regulates the ABCC1/MRP-1 transporter in an ERK/MAPK- dependent manner. Mol. Biol. Cell 23, 3473–3484. doi: 10.1091/mbc.E12- 02-0132 Chen, Y. A., Chi, W. C., Trinh, N. N., Huang, L. Y., Chen, Y. C., Cheng, K. T., et al. (2014). Transcriptome proﬁling and physiological studies reveal a major role for aromatic amino acids in mercury stress tolerance in rice seedlings. PLoS ONE 9:e95163. doi: 10.1371/journal.pone. 0095163 Ellis, P. D., Strang, P., and Potter, J. D. (1984). Cadmium-substituted skeletal troponin C. Cadmium-113 NMR spectroscopy and metal binding investigations. J. Biol. Chem. 259, 10348–10356. Chen, Z. H., Bao, M. L., Sun, Y. Z., Yang, Y. J., Xu, X. H., Wang, J. REFERENCES G., Stirk, W. A., Strnad, M., Van Staden, J., and Spíchal, L. (2013). Seedlings of medicinal plants treated with either a cytokinin antagonist (PI-55) or an inhibitor of cytokinin degradation (INCYDE) are protected against the negative eﬀects of cadmium. Plant Growth Regul. 71, 137–145. doi: 10.1007/s10725-013-9813-8 Debernardi, J. M., Rodriguez, R. E., Mecchia, M. A., and Palatnik, J. F. (2012). Functional specialization of the plant miR396 regulatory network through distinct microRNA–target interactions. PLoS Genet. 8:e1002419. doi: 10.1371/journal.pgen.1002419 Gong, X., Liu, Y., Huang, D., Zeng, G., Liu, S., Tang, H., et al. (2016). Eﬀects of exogenous calcium and spermidine on cadmium stress moderation and metal accumulation in Boehmeria nivea (L.) Gaudich. Environ. Sci. Pollut. Res. 23, 8699–8708. doi: 10.1007/s11356-016-6122-6 de la Torre, F., Gutiérrez-Beltrán, E., Pareja-Jaime, Y., Chakravarthy, S., Martin, G. B., and del Pozo, O. (2013). The tomato calcium sensor Cbl10 and its interacting protein kinase Cipk6 deﬁne a signaling pathway in plant immunity. Plant Cell 25, 2748–2764. doi: 10.1105/tpc.113.113530 González, A., Cabrera Mde, L., Henríquez, M. J., Contreras, R. A., Morales, B., and Moenne, A. (2012). Cross talk among calcium, hydrogen peroxide, and nitric oxide and activation of gene expression involving calmodulins and calcium- dependent protein kinases in Ulva compressa exposed to copper excess. Plant Physiol. 158, 1451–1462. doi: 10.1104/pp.111.191759 De Smet, S., Cuypers, A., Vangronsveld, J., and Remans, T. (2015). Gene networks involved in hormonal control of root development in Arabidopsis thaliana: a framework for studying its disturbance by metal stress. Int. J. Mol. Sci. 16, 19195–19224. doi: 10.3390/ijms160 819195 Gupta, M., Sharma, P., Sarin, N. B., and Sinha, A. K. (2009). Diﬀerential response of arsenic stress in two varieties of Brassica juncea L. Chemosphere 74, 1201–1208. doi: 10.1016/j.chemosphere.2008.11.023 Ding, H., Zhang, A., Wang, J., Lu, R., Zhang, H., Zhang, J., et al. (2009). Identity of an ABA-activated 46 kDa mitogen-activated protein kinase from Zea mays leaves: partial puriﬁcation, identiﬁcation and characterization. Planta 230, 239–251. doi: 10.1007/s00425-009-0938-y Hamel, L. P., Nicole, M. C., Sritubtim, S., Morency, M. J., Ellis, M., Ehlting, J., et al. (2006). Ancient signals: comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci. 11, 192–198. doi: 10.1016/j.tplants.2006. 02.007 Ding, Y., Chen, Z., and Zhu, C. (2011). Microarray-based analysis of cadmium- responsive microRNAs in rice (Oryza sativa). J. Exp. Bot. 62, 3563–3573. doi: 10.1093/jxb/err046 Hamilton, D. W., Hills, A., and Blatt, M. R. (2001). REFERENCES (2015). miR408 is involved in abiotic stress responses in Arabidopsis. Plant J. 84, 169–187. doi: 10.1111/tpj.12999 Kim, D. Y., Bovet, L., Maeshima, M., Martinoia, E., and Lee, Y. (2007). The ABC transporter AtPDR8 is a cadmium extrusion pump conferring heavy metal resistance. Plant J. 50, 207–218. doi: 10.1111/j.1365-313X.2007.03044.x Ma, Z., Hu, X., Cai, W., Huang, W., Zhou, X., Luo, Q., et al. (2014). Arabidopsis miR171-targeted scarecrow-like proteins bind to GT cis-elements and mediate gibberellin-regulated chlorophyll biosynthesis under light conditions. PLoS Genet. 10:e1004519. doi: 10.1371/journal.pgen.1004519 Kim, S. H., Kim, S. H., Yoo, S. J., Min, K. H., Nam, S. H., Cho, B. H., et al. (2013). Putrescine regulating by stress-responsive MAPK cascade contributes to bacterial pathogen defense in Arabidopsis. Biochem. Biophys. Res. Commun. 437, 502–508. doi: 10.1016/j.bbrc.2013.06.080 Maksymiec, W. (2007). Signaling responses in plants to heavy metal stress. Acta Physiol. Plant. 29, 177–187. doi: 10.1007/s11738-007-0036-3 Kong, X., Zhang, M., Xu, X., Li, X., Li, C., and Ding, Z. (2014). System analysis of microRNAs in the development and aluminium stress responses of the maize root system. Plant Biotechnol. J. 12, 1108–1121. doi: 10.1111/pbi.12218 Mao, G., Meng, X., Liu, Y., Zheng, Z., Chen, Z., and Zhang, S. (2011). Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell 23, 1639–1653. doi: 10.1105/tpc.111.084996 Kovtun, Y., Chiu, W. L., Tena, G., and Sheen, J. (2000). Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. U.S.A. 97, 2940–2945. doi: 10.1073/pnas.97.6.2940 Marchant, A., Kargul, J., May, S. T., Muller, P., Delbarre, A., Perrot-Rechenmann, C., et al. (1999). AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues. EMBO J. 18, 2066–2073. doi: 10.1093/emboj/18.8.2066 Krishnamurthy, A., and Rathinasabapathi, B. (2013). Auxin and its transport play a role in plant tolerance to arsenite-induced oxidative stress in Arabidopsis thaliana. Plant Cell Environ. 36, 1838–1849. doi: 10.1111/pce.12093 Matthewman, C. A., Kawashima, C. G., Húska, D., Csorba, T., Dalmay, T., and Kopriva, S. (2012). miR395 is a general component of the sulfate assimilation regulatory network in Arabidopsis. FEBS Lett. 586, 3242–3248. doi: 10.1016/j.febslet.2012.06.044 Kumar, S., Dubey, R. S., Tripathi, R. D., Chakrabarty, D., and Trivedi, P. K. (2015). Omics and biotechnology of arsenic stress and detoxiﬁcation in plants: current updates and prospective. Environ. Int. 74, 221–230. doi: 10.1016/j.envint.2014.10.019 Mendoza-Cózatl, D. G., Jobe, T. O., Hauser, F., and Schroeder, J. I. (2011). REFERENCES Extracellular Ba2+ and voltage interact to gate Ca2+ channels at the plasma membrane of stomatal guard cells. FEBS Lett. 491, 99–103. doi: 10.1016/S0014-5793(01)02176-7 Ding, Y., Qu, A., Gong, S., Huang, S., Lv, B., and Zhu, C. (2013). Molecular identiﬁcation and analysis of Cd-responsive microRNAs in rice. J. Agricult. Food Chem. 61, 11668–11675. doi: 10.1021/jf401359q He, Q., Zhu, S., and Zhang, B. (2014). MicroRNA–target gene responses to lead- induced stress in cotton (Gossypium hirsutum L.). Funct. Integr. Genomics 14, 507–515. doi: 10.1007/s10142-014-0378-z Ding, Y., Ye, Y., Jiang, Z., Wang, Y., and Zhu, C. (2016). MicroRNA390 Is involved in cadmium tolerance and accumulation in rice. Front. Plant Sci. 7:235. doi: 10.3389/fpls.2016.00235 He, X., Zheng, W., Cao, F., and Wu, F. (2016). Identiﬁcation and comparative analysis of the microRNA transcriptome in roots of two contrasting tobacco genotypes in response to cadmium stress. Sci. Rep. 6:32805 doi: 10.1038/srep32805 Djamei, A., Pitzschke, A., Nakagami, H., Rajh, I., and Hirt, H. (2007). Trojan horse strategy in Agrobacterium transformation: abusing MAPK defense signaling. Science 318, 453–456. doi: 10.1126/science.1148110 Herbette, S., Taconnat, L., Hugouvieux, V., Piette, L., Magniette, M. L., Cuine, S., et al. (2006). Genome-wide transcriptome proﬁling of the early cadmium response of Arabidopsis roots and shoots. Biochimie 88, 1751–1765. doi: 10.1016/j.biochi.2006.04.018 Dodd, A. N., Kudla, J., and Sanders, D. (2010). The language of calcium signaling. Annu. Rev. Plant Biol. 61, 593–620. doi: 10.1146/annurev-arplant-070109-104628 Hewezi, T., Piya, S., Qi, M., Balasubramaniam, M., Rice, J. H., and Baum, T. J. (2016). Arabidopsis miR827 mediates post-transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., and Lepiniec, L. (2010). MYB transcription factors in Arabidopsis. Trends Plant Sci. 15, 573–581. doi: 10.1016/j.tplants.2010.06.005 February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 16 Heavy Metal Stress and Plant Signaling Jalmi et al. Lequeux, H., Hermans, C., Lutts, S., and Verbruggen, N. (2010). Response to copper excess in Arabidopsis thaliana: impact on the root system architecture, hormone distribution, lignin accumulation and mineral proﬁle. Plant Physiol. Biochem. 48, 673–682. doi: 10.1016/j.plaphy.2010. 05.005 nematode Heterodera schachtii to enhance susceptibility. Plant J. 88, 179–192. doi: 10.1111/tpj.13238 nematode Heterodera schachtii to enhance susceptibility. Plant J. 88, 179–192. doi: 10.1111/tpj.13238 Hu, S., Yu, Y., Chen, Q., Mu, G., Shen, Z., and Zheng, L. (2017). OsMYB45 plays an important role in rice resistance to cadmium stress. REFERENCES Plant Sci. 264, 1–8. doi: 10.1016/j.plantsci.2017.08.002 Li, G., Meng, X., Wang, R., Mao, G., Han, L., Liu, Y., et al. (2012). Dual- level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis. PLoS Genet. 8:e1002767. doi: 10.1371/journal.pgen.1002767 Hu, Y. F., Zhou, G., Na, X. F., Yang, L., Nan, W. B., Liu, X., et al. (2013). Cadmium interferes with maintenance of auxin homeostasis in Arabidopsis seedlings. J. Plant Physiol. 170, 965–975. doi: 10.1016/j.jplph.2013.02.008 Huang, C., Zhou, J., Jie, Y., Xing, H., Zhong, Y., She, W., et al. (2016). A ramie (Boehmeria nivea) bZIP transcription factor BnbZIP3 positively regulates drought, salinity and heavy metal tolerance. Mol. Breed. 36:120. doi: 10.1007/s11032-016-0470-2 Li, S., Gao, J., Yin, K., Wang, R., Wang, C., Petersen, M., et al. (2016). MYB75 Phosphorylation by MPK4 is required for light-induced anthocyanin accumulation in Arabidopsis. Plant Cell 16:130. doi: 10.1105/tpc.16.00130 Huang, J., Zhang, Y., Peng, J. S., Zhong, C., Yi, H. Y., Ow, D. W., et al. (2012). Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis. Plant Physiol. 158, 1779–1788. doi: 10.1104/pp.111.192872 Li, W. X., Oono, Y., Zhu, J., He, X. J., Wu, J. M., Iida, K., et al. (2008). The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. Plant Cell 20, 2238–2251. doi: 10.1105/tpc.108.059444 Huang, T. L., and Huang, H. J. (2008). ROS and CDPK-like kinase-mediated activation of MAP kinase in rice roots exposed to lead. Chemosphere 71, 1377–1385. doi: 10.1016/j.chemosphere.2007.11.031 Li, Z. Y., Xu, Z. S., He, G. Y., Yang, G. X., Chen, M., Li, L. C., et al. (2012). Overexpression of soybean GmCBL1 enhances abiotic stress tolerance and promotes hypocotyl elongation in Arabidopsis. Biochem. Biophys. Res. Commun. 427, 731–736. doi: 10.1016/j.bbrc.2012.09.128 Huang, T. L., Huang, L. Y., Fu, S. F., Trinh, N. N., and Huang, H. J. (2014). Genomic proﬁling of rice roots with short-and long-term chromium stress. Plant Mol. Biol. 86, 157–170. doi: 10.1007/s11103-014-0219-4 Lima, J., Arenhart, R. A., Margis-Pinheiro, M., and Margis, R. (2011). Aluminum triggers broad changes in microRNA expression in rice roots. Genet. Mol. Res. 10, 2817–2832. doi: 10.4238/2011.November.10.4 Jalmi, S. K., and Sinha, A. K. (2015). ROS mediated MAPK signaling in abiotic and biotic stress-striking similarities and diﬀerences. Front. Plant Sci. 6:769. doi: 10.3389/fpls.2015.00769 Lin, C. W., Chang, H. B., and Huang, H. J. (2005). REFERENCES Zinc induces mitogen-activated protein kinase activation mediated by reactive oxygen species in rice roots. Plant Physiol. Biochem. 43, 963–968. doi: 10.1016/j.plaphy.2005.10.001 Jirakulaporn, T., and Muslin, A. J. (2004). Cation diﬀusion facilitator proteins modulate Raf-1 activity. J. Biol. Chem. 279, 27807–27815. doi: 10.1074/jbc.M401210200 Liu, Q., Hu, H., Zhu, L., Li, R., Feng, Y., Zhang, L., et al. (2015). Involvement of miR528 in the regulation of arsenite tolerance in rice (Oryza sativa L). J. Agric. Food Chem. 63, 8849–8861. doi: 10.1021/acs.jafc.5b04191 Jonak, C., Nakagami, H., and Hirt, H. (2004). Heavy metal stress. activation of distinct mitogen-activated protein kinase pathways by copper and cadmium. Plant Physiol. 136, 3276–3283. doi: 10.1104/pp.104.045724 j Liu, Q., and Zhang, H. (2012). Molecular identiﬁcation and analysis of arsenite stress-responsive miRNAs in rice. J. Agric. Food Chem. 60, 6524–6536. doi: 10.1021/jf300724t Jones, D. L., and Kochian, L. V. (1997). Aluminum interaction with plasma membrane lipids and enzyme metal binding sites and its potential role in Al cytotoxicity. FEBS Lett. 400, 51–57. doi: 10.1016/S0014-5793(96)01319-1 Liu, X. M., Kim, K. E., Kim, K. C., Nguyen, X. C., Han, H. J., Jung, M. S., et al. (2010). Cadmium activates Arabidopsis MPK3 and MPK6 via accumulation of reactive oxygen species. Phytochemistry 71, 614–618. doi: 10.1016/j.phytochem.2010.01.005 Jones-Rhoades, M. W., and Bartel, D. P. (2004). Computational identiﬁcation of plant microRNAs and their targets, including a stress-induced miRNA. Mol. Cell 14, 787–799. doi: 10.1016/j.molcel.2004.05.027 Liu, Y., and Zhang, S. (2004). Phosphorylation of 1-aminocyclopropane-1- carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell 16, 3386–3399. doi: 10.1105/tpc.104.026609 Jung, J. H., and Park, C. M. (2007). MIR166/165 genes exhibit dynamic expression patterns in regulating shoot apical meristem and ﬂoral development in Arabidopsis. Planta 225, 1327–1338. doi: 10.1007/s00425-006-0439-1 Luan, S., Kudla, J., Rodriguez-Concepcion, M., Yalovsky, S., and Gruissem, W. (2002). Calmodulins and calcineurin B–like proteins calcium sensors for speciﬁc signal response coupling in plants. Plant Cell 14, S389–S400. doi: 10.1105/tpc.001115 Kende, H. (1993). Ethylene biosynthesis. Annu. Rev. Plant Biol. 44, 283–307. doi: 10.1146/annurev.pp.44.060193.001435 Kende, H. (1993). Ethylene biosynthesis. Annu. Rev. Plant Biol. 44, 283–307. doi: 10.1146/annurev.pp.44.060193.001435 Keunen, E., Schellingen, K., Vangronsveld, J., and Cuypers, A. (2016). Ethylene and metal stress: small molecule, big impact. Front. Plant Sci. 7:23. doi: 10.3389/fpls.2016.00023 Keunen, E., Schellingen, K., Vangronsveld, J., and Cuypers, A. (2016). Ethylene and metal stress: small molecule, big impact. Front. Plant Sci. 7:23. doi: 10.3389/fpls.2016.00023 Ma, C., Burd, S., and Lers, A. REFERENCES Long-distance transport, vacuolar sequestration, tolerance, and transcriptional responses induced by cadmium and arsenic. Curr. Opin. Plant Biol. 14, 554–562. doi: 10.1016/j.pbi.2011.07.004 Langer, G. A., and Nudd, L. M. (1983). Eﬀects of cations, phospholipases, and neuraminidase on calcium binding to “gas-dissected” membranes from cultured cardiac cells. Circ. Res. 53, 482–490. doi: 10.1161/01.RES.53.4.482 February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 17 Heavy Metal Stress and Plant Signaling Jalmi et al. Pedersen, S. F., Darborg, B. V., Rasmussen, M., Nylandsted, J., and Hoﬀmann, E. K. (2007). The Na+/H+ exchanger, NHE1, diﬀerentially regulates mitogen- activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells. Cell. Physiol. Biochem. 20, 735–750. doi: 10.1159/000 110434 Menke, F. L. H., Kang, H. G., Chen, Z., Park, J. M., Kumar, D., and Klessig, D. F. (2005). Tobacco transcription factor WRKY1 is phosphorylated by the MAP kinase SIPK and mediates HR-like cell death in tobacco. Mol. Plant Microbe Interact. 18, 1027–1034. doi: 10.1094/MPMI-18-1027 Miao, Y., Laun, T. M., Smykowski, A., and Zentgraf, U. (2007). Arabidopsis MEKK1 can take a short cut: it can directly interact with senescence-related WRKY53 transcription factor on the protein level and can bind to its promoter. Plant Mol. Biol. 65, 63–76. doi: 10.1007/s11103-007-9198-z Peng, J. S., and Gong, J. M. (2014). Vacuolar sequestration capacity and long-distance metal transport in plants. Front. Plant Sci. 5:19. doi: 10.3389/fpls.2014.00019 Plant Mol. Biol. 65, 63–76. doi: 10.1007/s11103-007-9198-z Perilli, S., Moubayidin, L., and Sabatini, S. (2010). The molecular basis of cytokinin function. Curr. Opin. Plant Biol. 13, 21–26. doi: 10.1016/j.pbi.2009.09.018 Mielniczki-Pereira, A. A., Hahn, A. B. B., Bonatto, D., Riger, C. J., Eleutherio, E. C. A., and Henriques, J. A. P. (2011). New insights into the Ca2+-ATPases that contribute to cadmium tolerance in yeast. Toxicol. Lett. 207, 104–111. doi: 10.1016/j.toxlet.2011.08.023 Peto, A., Lehotai, N., Lozano-Juste, J., León, J., Tari, I., Erdei, L., et al. (2011). Involvement of nitric oxide and auxin in signal transduction of copper-induced morphological responses in Arabidopsis seedlings. Ann. Bot. 108, 449–457. doi: 10.1093/aob/mcr176 Milner, M. J., Seamon, J., Craft, E., and Kochian, L. V. (2013). Transport properties of members of the ZIP family in plants and their role in Zn and Mn homeostasis. J. Exp. Bot. 64, 369–381. doi: 10.1093/jxb/ers315 Piotrowska-Niczyporuk, A., Bajguz, A., Zambrzycka, E., and Godlewska- Zyłkiewicz, B. (2012). Phytohormones as regulators of heavy metal biosorption and toxicity in green alga Chlorella vulgaris (Chlorophyceae). Plant Physiol. REFERENCES Biochem. 52, 52–65. doi: 10.1016/j.plaphy.2011.11.009 p j Mockaitis, K., and Howell, S. H. (2000). Auxin induces mitogenic activated protein kinase (MAPK) activation in roots of Arabidopsis seedlings. Plant J. 24, 785–796. doi: 10.1046/j.1365-313x.2000.00921.x Pitzschke, A., Djamei, A., Bitton, F., and Hirt, H. (2009). A major role of the MEKK1–MKK1/2–MPK4 pathway in ROS signalling. Mol. Plant 2, 120–137. doi: 10.1093/mp/ssn079 Moisan, J., Thuraisingam, T., Henault, J., De Sanctis, J., and Radzioch, D. (2006). Role of SLC11A1 (formerly NRAMP1) in regulation of signal transduction induced by Toll-like receptor 7 ligands. FEMS Immunol. Med. Microbiol. 47, 138–147. doi: 10.1111/j.1574-695X.2006.00077.x Potters, G., Pasternak, T. P., Guisez, Y., Palme, K. J., and Jansen, M. A. (2007). Stress-induced morphogenic responses: growing out of trouble? Trends Plant Sci. 12, 98–105. doi: 10.1016/j.tplants.2007.01.004 Montero-Palmero, M. B., Martín-Barranco, A., Escobar, C., and Hernández, L. E. (2014). Early transcriptional responses to mercury: a role for ethylene in mercury-induced stress. New Phytol. 201, 116–130. doi: 10.1111/nph.12486 Raghuram, B., Sheikh, A. H., and Sinha, A. K. (2014). Regulation of MAP kinase signaling cascade by microRNAs in Oryza sativa. Plant signal. Behav. 9:e972130. doi: 10.4161/psb.29804 Morea, E. G. O., da Silva, E. M., e Silva, G. F. F., Valente, G. T., Rojas, C. H. B., Vincentz, M., et al. (2016). Functional and evolutionary analyses of the miR156 and miR529 families in land plants. BMC Plant Biol. 16:40. doi: 10.1186/s12870-016-0716-5 Ramos, J., Clemente, M. R., Naya, L., Loscos, J., Perez-Rontome, C., Sato, S., et al. (2007). Phytochelatin synthases of the model legume Lotus japonicus. a small multigene family with diﬀerent responses to cadmium and alternatively spiced variants. Plant Physiol. 143, 110–118. doi: 10.1104/pp.106.090894 Mossor-Pietraszewska, T. (2001). Eﬀect of aluminium on plant growth and metabolism. Acta Biochim. Pol. 48, 673–686. variants. Plant Physiol. 143, 110–118. doi: 10.1104/pp.106.090894 Rao, K. P., Vani, G., Kumar, K., Wankhede, D. P., Misra, M., Gupta, M., et al. (2011). Arsenic stress activates MAP kinase in rice roots and leaves. Arch. Biochem. Biophys. 506, 73–82. doi: 10.1016/j.abb.2010.11.006 Mulay, V., Wood, P., Manetsch, M., Darabi, M., Cairns, R., Hoque, M., et al. (2013). Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells. PLoS ONE 8:e62667. doi: 10.1371/journal.pone. 0062667 Rashotte, A. M., Brady, S. R., Reed, R. C., Ante, S. J., and Muday, G. K. (2000). Basipetal auxin transport is required for gravitropism in roots of Arabidopsis. Plant Physiol. REFERENCES 122, 481–490. doi: 10.1104/pp.122.2.481 Myouga, F., Hosoda, C., Umezawa, T., Iizumi, H., Kuromori, T., Motohashi, R., et al. (2008). A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis. Plant Cell 20, 3148–3162. doi: 10.1105/tpc.108.061341 Rice, J. M., Zweifach, A., and Lynes, M. A. (2016). Metallothionein regulates intracellular zinc signaling during CD4+ T cell activation. BMC Immunol. 17:13. doi: 10.1186/s12865-016-0151-2 Rigouin, C., Nylin, E., Cogswell, A. A., Schaumlöﬀel, D., Dobritzsch, D., and Williams, D. L. (2013). Towards an understanding of the function of the phytochelatin synthase of Schistosoma mansoni. PLoS Negl. Trop. Dis. 7:e2037. doi: 10.1371/journal.pntd.0002037 Nguyen, X. C., Kim, S. H., Lee, K., Kim, K. E., Liu, X. M., Han, H. J., et al. (2012). Identiﬁcation of a C2H2-type zinc ﬁnger transcription factor (ZAT10) from Arabidopsis as a substrate of MAP kinase. Plant Cell Rep. 31, 737–745. doi: 10.1007/s00299-011-1192-x Rivetta, A., Negrini, N., and Cocucci, M. (1997). Involvement of Ca2+- calmodulin in Cd2+ toxicity during the early phases of radish (Raphanus sativus L.) seed germination. Plant Cell Environ. 20, 600–608. doi: 10.1111/j.1365-3040.1997.00072.x Nikovics, K., Blein, T., Peaucelle, A., Ishida, T., Morin, H., Aida, M., et al. (2006). The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis. Plant Cell 18, 2929–2945. doi: 10.1105/tpc.106.045617 Rodriguez, M. C., Petersen, M., and Mundy, J. (2010). Mitogen-activated protein kinase signaling in plants. Annu. Rev. Plant Biol. 61, 621–649. doi: 10.1146/annurev-arplant-042809-112252 Noman, A., and Aqeel, M. (2017). miRNA-based heavy metal homeostasis and plant growth. Environ. Sci. Pollut. Res. Int. 24, 10068–10082. doi: 10.1007/s11356-017-8593-5 Ogawa, I., Nakanishi, H., Mori, S., and Nishizawa, N. K. (2009). Time course analysis of gene regulation under cadmium stress in rice. Plant Soil 325:97. doi: 10.1007/s11104-009-0116-9 Roelofs, D., Aarts, M. G. M., Schat, H., and Van Straalen, N. M. (2008). Functional ecological genomics to demonstrate general and speciﬁc responses to abiotic stress. Funct. Ecol. 22, 8–18. doi: 10.1111/j.1365-2435.2007.01312.x Opdenakker, K., Remans, T., Vangronsveld, J., and Cuypers, A. (2012). Mitogen- activated protein (MAP) kinases in plant metal stress: regulation and responses in comparison to other biotic and abiotic stresses. Int. J. Mol. Sci. 13, 7828–7853. doi: 10.3390/ijms13067828 Rucinska-Sobkowiak, R. (2016). Water relations in plants subjected to heavy metal stresses. Acta Physiol. Plant. 38:257. doi: 10.1007/s11738-016-2277-5 Rudd, J. J., and Franklin-Tong, V. E. (2001). Unravelling response-speciﬁcity in Ca2+ signalling pathways in plant cells. New Phytol. 151, 7–33. REFERENCES Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice. Plant Cell 21, 4031–4043. doi: 10.1105/tpc.109.066902 Tchounwou, P. B., Yedjou, C. G., Patlolla, A. K., and Sutton, D. J. (2012). Heavy metal toxicity and the environment. Mol. Clin. Environ.Toxicol. 101, 133–164. doi: 10.1007/978-3-7643-8340-4_6 Siddiqui, M. H., Al-Whaibi, M. H., Sakran, A. M., Basalah, M. O., and Ali, H. M. (2012). Eﬀect of calcium and potassium on antioxidant system of Vicia faba L. under cadmium stress. Int. J. Mol. Sci. 13, 6604–6619. doi: 10.3390/ijms13066604 Tebar, F., Lladó, A., and Enrich, C. (2002). Role of calmodulin in the modulation of the MAPK signalling pathway and the transactivation of epidermal growth factor receptor mediated by PKC. FEBS Lett. 517, 206–210. doi: 10.1016/S0014-5793(02)02624-8 Singh, A., Roy, S., Singh, S., Das, S. S., Gautam, V., Yadav, S., et al. (2017). Phytohormonal crosstalk modulates the expression of miR166/165s, target class III HD-ZIPs, and KANADI genes during root growth in Arabidopsis thaliana. Sci. Rep. 7:3408. doi: 10.1038/s41598-017-03632-w Thao, N. P., Khan, M. I., Thu, N. B., Hoang, X. L., Asgher, M., Khan, N. A., et al. (2015). Role of ethylene and its cross talk with other signaling molecules in plant responses to heavy metal stress. Plant Physiol. 169, 73–84. doi: 10.1104/pp.15.00663 Singh, P., Mohanta, T. K., and Sinha, A. K. (2015). Unraveling the intricate nexus of molecular mechanisms governing rice root development: OsMPK3/6 and auxin–cytokinin interplay. PLoS ONE 10:e0123620. doi: 10.1371/journal.pone.0123620 Thomine, S., and Schroeder, J. I. (2004). “Plant metal transporters with homology to proteins of the NRAMP family,” in The NRAMP family, eds M. Cellier and P. Gros (Austin: Andes/Kluwer Series), 113–121. Singh, S., Parihar, P., Singh, R., Singh, V. P., and Prasad, S. M. (2015). Heavy metal tolerance in plants: role of transcriptomics, proteomics, metabolomics, and ionomics. Front. Plant Sci. 6:1143. doi: 10.3389/fpls.2015.01143 Tian, Q., Zhang, X., Ramesh, S., Gilliham, M., Tyerman, S. D., and Zhang, W. H. (2014). Ethylene negatively regulates aluminium-induced malate eﬄux from wheat roots and tobacco cells transformed with TaALMT1. J. Exp. Bot. 65, 2415–2426. doi: 10.1093/jxb/eru123 Sinha, A. K., Jaggi, M., Raghuram, B., and Tuteja, N. (2011). Mitogen-activated protein kinase signaling in plants under abiotic stress. Plant Signal. Behav. 6, 196–203. doi: 10.4161/psb.6.2.14701 Tombuloglu, G., Tombuloglu, H., Sakcali, M. S., and Unver, T. (2015). High- throughput transcriptome analysis of barley (Hordeum vulgare) exposed to excessive boron. Gene 557, 71–81. REFERENCES Aluminium-induced inhibition of root elongation in Arabidopsis is mediated by ethylene and auxin. J. Exp. Bot. 61, 347–356. doi: 10.1093/jxb/erp306 Serpa, V., Vernal, J., Lamattina, L., Grotewold, E., Cassia, R., and Terenzi, H. (2007). Inhibition of AtMYB2 DNA-binding by nitric oxide involves cysteine S-nitrosylation. Biochem. Biophys. Res. Commun. 361, 1048–1053. doi: 10.1016/j.bbrc.2007.07.133 Sunkar, R., Kaplan, B., Bouche, N., Arazi, T., Dolev, D., Talke, I. N., et al. (2000). Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance. Plant J. 24, 533–542. doi: 10.1046/j.1365-313x.2000. 00901.x Sethi, V., Raghuram, B., Sinha, A. K., and Chattopadhyay, S. (2014). A mitogen- activated protein kinase cascade module, MKK3-MPK6 and MYC2, is involved in blue light-mediated seedling development in Arabidopsis. Plant Cell 26, 3343–3357. doi: 10.1105/tpc.114.128702 Ta, K. N., Sabot, F., Adam, H., Vigouroux, Y., De Mita, S., Ghesquière, A., et al. (2016). miR2118-triggered phased siRNAs are diﬀerentially expressed during the panicle development of wild and domesticated African rice species. Rice 9:10. doi: 10.1186/s12284-016-0082-9 Sharma, D., Tiwari, M., Lakhwani, D., Tripathi, R. D., and Trivedi, P. K. (2015). Diﬀerential expression of microRNAs by arsenate and arsenite stress in natural accessions of rice. Metallomics 7, 174–187. doi: 10.1039/C4MT 00264D Takahashi, F., Mizoguchi, T., Yoshida, R., Ichimura, K., and Shinozaki, K. (2011). Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis. Mol. Cell 41, 649–660. doi: 10.1016/j.molcel.2011. 02.029 Sharma, S. S., and Dietz, K. J. (2006). The signiﬁcance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress. J. Exp. Bot. 57, 711–726. doi: 10.1093/jxb/erj073 Tang, M., Mao, D., Xu, L., Li, D., Song, S., and Chen, C. (2014). Integrated analysis of miRNA and mRNA expression proﬁles in response to Cd exposure in rice seedlings. BMC Genomics 15:835. doi: 10.1186/1471-2164-15-835 Shen, H., Hou, N., Schlicht, M., Wan, Y., Mancuso, S., and Baluska, F. (2008). Aluminium toxicity targets PIN2 in Arabidopsis root apices: eﬀects on PIN2 endocytosis, vesicular recycling, and polar auxin transport. Chin. Sci. Bull. 53, 2480–2487. doi: 10.1007/s11434-008-0332-3 Taniguchi, M., Fukunaka, A., Hagihara, M., Watanabe, K., Kamino, S., Kambe, T., et al. (2013). Essential role of the zinc transporter ZIP9/SLC39A9 in regulating the activations of Akt and Erk in B-cell receptor signaling pathway in DT40 cells. PLoS ONE 8:e58022. doi: 10.1371/journal.pone.0058022 Shim, D., Hwang, J. U., Lee, J., Lee, S., Choi, Y., An, G., et al. (2009). REFERENCES doi: 10.1046/j.1469-8137.2001.00173.x Osawa, H., and Matsumoto, H. (2001). Possible involvement of protein phosphorylation in aluminum-responsive malate eﬄux from wheat root apex. Plant Physiol. 126, 411–420. doi: 10.1104/pp.126.1.411 Salt, D. E., Smith, R. D., and Raskin, I. (1998). Phytoremediation. Annu. Rev. Plant Biol. 49, 643–668. doi: 10.1146/annurev.arplant.49.1.643 Pandey, C., Raghuram, B., Sinha, A. K., and Gupta, M. (2015). miRNA plays a role in the antagonistic eﬀect of selenium on arsenic stress in rice seedlings. Metallomics 7, 857–866. doi: 10.1039/C5MT00013K Sanders, D., Pelloux, J., Brownlee, C., and Harper, J. F. (2002). Calcium at the crossroads of signaling. Plant Cell 14, S401–S417. doi: 10.1105/tpc.002899 Schellingen, K., Van Der Straeten, D., Remans, T., Loix, C., Vangronsveld, J., and Cuypers, A. (2015). Ethylene biosynthesis is involved in the early oxidative challenge induced by moderate Cd exposure in Arabidopsis thaliana. Environ. Exp. Bot. 117, 1–11. doi: 10.1016/j.envexpbot.2015. 04.005 Park, J., Song, W. Y., Ko, D., Eom, Y., Hansen, T. H., Schiller, M., et al. (2012). The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury. Plant J. 69, 278–288. doi: 10.1111/j.1365-313X.2011.04789.x February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 18 Heavy Metal Stress and Plant Signaling Jalmi et al. Steinhorst, L., and Jörg, K. (2003). Calcium-a central regulator of pollen germination and tube growth. Biochimica et Biophysica Acta 1833, 1573–1581. doi: 10.1016/j.bbamcr.2012.10.009 Schommer, C., Palatnik, J. F., Aggarwal, P., Chételat, A., Cubas, P., Farmer, E. E., et al. (2008). Control of jasmonate biosynthesis and senescence by miR319 targets. PLoS Biol. 6:e230. doi: 10.1371/journal.pbio.0060230 g j Schott, E. J., and Gardner, R. C. (1997). Aluminum-sensitive mutants of Saccharomyces cerevisiae. Mol. Gen. Genet. 254, 63–72. doi: 10.1007/s004380050391 Steinhorst, L., and Kudla, J. (2014). Signaling in cells and organisms–calcium holds the line. Curr. Opin. Plant Biol. 22, 14–21. doi: 10.1016/j.pbi.2014.08.003 p j p Stohs, S. J., and Bagchi, D. (1995). Oxidative mechanisms in the toxicity of metal Stohs, S. J., and Bagchi, D. (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321–336. doi: 10.1016/0891-5849(94)00159-H , J , g , ( ) y ions. Free Radic. Biol. Med. 18, 321–336. doi: 10.1016/0891-5849(94)00159-H Schwab, R., Palatnik, J. F., Riester, M., Schommer, C., Schmid, M., and Weigel, D. (2005). Speciﬁc eﬀects of microRNAs on the plant transcriptome. Dev. Cell 8, 517–527. doi: 10.1016/j.devcel.2005.01.018 Sun, P., Tian, Q. Y., Chen, J., and Zhang, W. H. (2010). REFERENCES B., Qi, H., et al. (2017). OsARM1, an R2R3 MYB transcription factor, is involved in regulation of the response to arsenic stress in rice. Front. Plant Sci. 30:1868. doi: 10.3389/fpls.2017.01868 Yadav, S. K. (2010). Heavy metals toxicity in plants: an overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants. South Afr. J. Bot. 76, 167–179. doi: 10.1016/j.sajb.2009.10.007 Wang, H., Jiao, X., Kong, X., Humaira, S., Wu, Y., Chen, X., et al. (2016). A signaling cascade from miR444 to RDR1 in rice antiviral RNA silencing pathway. Plant Physiol. 170, 2365–2377. doi: 10.1104/pp.15.01283 Yamaguchi, H., Fukuoka, H., Arao, T., Ohyama, A., Nunome, T., Miyatake, K., et al. (2010). Gene expression analysis in cadmium-stressed roots of a low cadmium- accumulating solanaceous plant, Solanum torvum. J. Exp. Bot. 61, 423–437. doi: 10.1093/jxb/erp313 Wang, J. W., Wang, L. J., Mao, Y. B., Cai, W. J., Xue, H. W., and Chen, X. Y. (2005). Control of root cap formation by microRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17, 2204–2216. doi: 10.1105/tpc.105.033076 Yamasaki, H., Hayashi, M., Fukazawa, M., Kobayashi, Y., and Shikanai, T. (2009). SQUAMOSA promoter binding protein–like7 is a central regulator for copper homeostasis in Arabidopsis. Plant Cell 21, 347–361. doi: 10.1105/tpc.108.060137 Wang, R., Wang, J., Zhao, L., Yang, S., and Song, Y. (2015). Impact of heavy metal stresses on the growth and auxin homeostasis of Arabidopsis seedlings. Biometals 28, 123–132. doi: 10.1007/s10534-014-9808-6 Yang, T., and Poovaiah, B. W. (2003). Calcium/calmodulin- mediated signal network in plants. Trends Plant Sci. 8, 505–512. doi: 10.1016/j.tplants.2003.09.004 Wang, S., Ren, X., Huang, B., Wang, G., Zhou, P., and An, Y. (2016). Aluminium- induced reduction of plant growth in alfalfa (Medicago sativa) is mediated by interrupting auxin transport and accumulation in roots. Sci. Rep. 6:30079. doi: 10.1038/srep30079 Yanhui, C., Xiaoyuan, Y., Kun, H., Meihua, L., Jigang, L., Zhaofeng, G., et al. (2006). The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol. Biol. 60, 107–124. doi: 10.1007/s11103-005-2910-y Wang, X., Wang, C., Sheng, H., Wang, Y., Zeng, J., Kang, H., et al. (2017). Transcriptome-wide identiﬁcation and expression analyses of ABC transporters in dwarf polish wheat under metal stresses. Biol. Plant. 61, 293–304. doi: 10.1007/s10535-016-0697-0 Ye, L., Li, L., Wang, L., Wang, S., Li, S., Du, J., et al. (2015). MPK3/MPK6 are involved in iron deﬁciency-induced ethylene production in Arabidopsis. Front. Plant Sci. 6:953. REFERENCES Eﬀects of cadmium exposure on growth and metabolic proﬁle of bermudagrass [Cynodon dactylon (L.) Pers.]. PLoS ONE 9:e115279. doi: 10.1371/journal.pone.0115279 van de Mortel, J. E., Schat, H., Moerland, P. D., Ver Loren van Themaat, E., Van Der Ent, S., Blankestijn, H., et al. (2008). Expression diﬀerences for genes involved in lignin, glutathione and sulphate metabolism in response to cadmium in Arabidopsis thaliana and the related Zn/Cd- hyperaccumulator Thlaspi caerulescens. Plant Cell Environ. 31, 301–324. doi: 10.1111/j.1365-3040.2007.01764.x Xie, Z., Kasschau, K. D., and Carrington, J. C. (2003). Negative feedback regulation of dicer-like1 in Arabidopsis by microRNA-guided mRNA degradation. Curr. Biol. 13, 784–789. doi: 10.1016/S0960-9822(03)00281-1 Vanstraelen, M., and Benková, E. (2012). Hormonal interactions in the regulation of plant development. Annu. Rev. Cell Dev. Biol. 28, 463–487. doi: 10.1146/annurev-cellbio-101011-155741 Xing, Y., Chen, W., Jia, W., and Zhang, J. (2015). Mitogen-activated protein kinase kinase 5 (MKK5)-mediated signalling cascade regulates expression of iron superoxide dismutase gene in Arabidopsis under salinity stress. J. Exp. Bot. 66, 5971–5981. doi: 10.1093/jxb/erv305. Vaucheret, H., Vazquez, F., Crété, P., and Bartel, D. P. (2004). The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 18, 1187–1197. doi: 10.1101/gad.1201404 Xu, F., Liu, Q., Chen, L., Kuang, J., Walk, T., Wang, J., et al. (2013). Genome- wide identiﬁcation of soybean microRNAs and their targets reveals their organ-speciﬁcity and responses to phosphate starvation. BMC Genomics 14:66. doi: 10.1186/1471-2164-14-66 Verbruggen, N., Hermans, C., and Schat, H. (2009). Mechanisms to cope with arsenic or cadmium excess in plants. Curr. Opin. Plant Biol. 12, 364–372. doi: 10.1016/j.pbi.2009.05.001 Xu, J., Li, Y., Wang, Y., Liu, H., Lei, L., Yang, H., et al. (2008). Activation of MAPK kinase 9 induces ethylene and camalexin biosynthesis and enhances sensitivity to salt stress in Arabidopsis. J. Biol. Chem. 283, 26996–27006. doi: 10.1074/jbc.M801392200 Vitti, A., Nuzzaci, M., Scopa, A., Tataranni, G., Remans, T., Vangronsveld, J., et al. (2013). Auxin and cytokinin metabolism and root morphological modiﬁcations in Arabidopsis thaliana seedlings infected with Cucumber mosaic virus (CMV) or exposed to cadmium. Int. J. Mol. Sci. 14, 6889–6902. doi: 10.3390/ijms14046889 j Xu, M., Hu, T., Zhao, J., Park, M. Y., Earley, K. W., Wu, G., et al. (2016). Developmental functions of miR156-regulated squamosa promoter binding protein-like (SPL) genes in Arabidopsis thaliana. PLoS Genet. 12:e1006263. doi: 10.1371/journal.pgen.1006263 Wang, F. Z., Chen, M. X., Yu, L. J., Xie, L. J., Yuan, L. REFERENCES doi: 10.3389/fpls.2015.00953 Wang, Y., Gao, C., Liang, Y., Wang, C., Yang, C., and Liu, G. (2010). A novel bZIP gene from Tamarix hispida mediates physiological responses to salt stress in tobacco plants. J. Plant Physiol. 167, 222–230. doi: 10.1016/j.jplph.2009. 09.008 Yeh, C. M., Chien, P. S., and Huang, H. J. (2007). Distinct signalling pathways for induction of MAP kinase activities by cadmium and copper in rice roots. J. Exp. Bot. 58, 659–671. doi: 10.1093/jxb/erl240 Yoo, S. D., Cho, Y. H., Tena, G., Xiong, Y., and Sheen, J. (2008). Dual control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signalling. Nature 451, 789–795. doi: 10.1038/nature06543 Wang, Y., Xu, L., Chen, Y., Shen, H., Gong, Y., Limera, C., et al. (2013). Transcriptome proﬁling of radish (Raphanus sativus L.) root and identiﬁcation of genes involved in response to lead (Pb) stress with next generation sequencing. PLoS ONE 8:e66539. doi: 10.1371/journal.pone.0066539 Yu, Y., Jin, C., Sun, C., Wang, J., Ye, Y., Zhou, W., et al. (2016). Inhibition of ethylene production by putrescine alleviates aluminium-induced root inhibition in wheat plants. Sci. Rep. 6:8888. doi: 10.1038/srep18888 Weber, M., Trampczynska, A., and Clemens, S. (2006). Comparative transcriptome analysis of toxic metal responses in Arabidopsis thaliana and the Cd2+- hypertolerant facultative metallophyte Arabidopsis halleri. Plant Cell Environ. 29, 950–963. doi: 10.1111/j.1365-3040.2005.01479.x Yuan, H. M., and Huang, X. (2016). Inhibition of root meristem growth by cadmium involves nitric oxide-mediated repression of auxin accumulation and signalling in Arabidopsis. Plant Cell Environ. 39, 120–135. doi: 10.1111/pce.12597 Wei, W., Zhang, Y., Han, L., Guan, Z., and Chai, T. (2008). A novel WRKY transcriptional factor from Thlaspi caerulescens negatively regulates the osmotic stress tolerance of transgenic tobacco. Plant Cell Rep. 27, 795–803. doi: 10.1007/s00299-007-0499-0 Yuan, S., Li, Z., Li, D., Yuan, N., Hu, Q., and Luo, H. (2015). Constitutive expression of rice MicroRNA528 alters plant development and enhances tolerance to salinity stress and nitrogen starvation in creeping bentgrass. Plant Physiol. 169, 576–593. doi: 10.1104/pp.15.00899 Wu, B. F., Li, W. F., Xu, H. Y., Qi, L. W., and Han, S. Y. (2015). Role of cin- miR2118 in drought stress responses in Caragana intermedia and tobacco. Gene 574, 34–40. doi: 10.1016/j.gene.2015.07.072 Zhang, G., Wu, H., Ross, C. R., and Ernest, J. (2000). Cloning of porcine. Infect. Immun. 68, 1086–1093. doi: 10.1128/IAI.68.3.1086-1093.2000 Wu, Z., Liang, F., Hong, B., Young, J. C., Sussman, M. R., Harper, J. F., et al. (2002). REFERENCES doi: 10.1016/j.gene.2014.12.012 Smeets, K., Opdenakker, K., Remans, T., Forzani, C., Hirt, H., Vangronsveld, J., et al. (2013). The role of the kinase OXI1 in cadmium- and copper-induced molecular responses in Arabidopsis thaliana. Plant Cell Environ. 36, 1228–1238. doi: 10.1111/pce.12056 Trinh, N. N., Huang, T. L., Chi, W. C., Fu, S. F., Chen, C. C., and Huang, H. J. (2014). Chromium stress response eﬀect on signal transduction and expression of signaling genes in rice. Physiol. Plant. 150, 205–224. doi: 10.1111/ppl. 12088 Smith, J. B., Dwyer, S. D., and Smith, L. (1989). Cadmium evokes inositol polyphosphate formation and calcium mobilization. evidence for a cell surface receptor that cadmium stimulates and zinc antagonizes. J. Biol. Chem. 264, 7115–7118. Tripathi, V., Parasuraman, B., Laxmi, A., and Chattopadhyay, D. (2009). CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants. Plant J. 58, 778–790. doi: 10.1111/j.1365-313X.2009.03812.x Song, J. B., Gao, S., Wang, Y., Li, B. W., Zhang, Y. L., and Yang, Z. M. (2016). miR394 and its target gene LCR are involved in cold stress response in Arabidopsis. Plant Gene 5, 56–64. doi: 10.1016/j.plgene.2015. 12.001 Tsai, C. Y., Finley, J. C., Ali, S. S., Patel, H. H., and Howell, S. B. (2012). Copper inﬂux transporter 1 is required for FGF, PDGF and EGF-induced MAPK signaling. Biochem. Pharmacol. 84, 1007–1013. doi: 10.1016/j.bcp.2012. 07.014 Srivastava, R. K., Pandey, P., Rajpoot, R., Rani, A., Gautam, A., and Dubey, R. S. (2015). Exogenous application of calcium and silica alleviates cadmium toxicity by suppressing oxidative damage in rice seedlings. Protoplasma 252, 959–975. doi: 10.1007/s00709-014-0731-z Tsai, T. M., and Huang, H. J. (2006). Eﬀects of iron excess on cell viability and mitogen-activated protein kinase activation in rice roots. Physiol. Plant. 127, 583–592. doi: 10.1111/j.1399-3054.2006.00696.x Srivastava, S., Srivastava, A. K., Suprasanna, P., and D’Souza, S. F. (2013). Identiﬁcation and proﬁling of arsenic stress-induced microRNAs in Brassica juncea. J. Exp. Bot. 64, 303–315. doi: 10.1093/jxb/ers333 Turski, M. L., Brady, D. C., Kim, H. J., Kim, B. E., Nose, Y., Counter, C. M., et al. (2012). A novel role for copper in Ras/mitogen-activated protein kinase signaling. Mol. Cell. Biol. 32, 1284–1295. doi: 10.1128/MCB.05722-11 February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 19 Heavy Metal Stress and Plant Signaling Jalmi et al. Xie, Y., Hu, L., Du, Z., Sun, X., Amombo, E., Fan, J., et al. (2014). Zhao, F. Y., Wang, K., Zhang, S. Y., Ren, J., Liu, T., and Wang, X. (2014). Crosstalk between ABA, auxin, MAPK signaling, and the cell cycle in cadmium-stressed rice seedlings. Acta physiol. Plant. 36, 1879–1892. doi: 10.1007/s11738-014-1564-2 Frontiers in Plant Science \| www.frontiersin.org Zhou, Z. S., Zeng, H. Q., Liu, Z. P., and Yang, Z. M. (2012). Genome-wide identiﬁcation of Medicago truncatula microRNAs and their targets reveals their diﬀerential regulation by heavy metal. Plant Cell Environ. 35, 86–99. doi: 10.1111/j.1365-3040.2011. 02418.x (DSE), Exophiala pisciphila: evidence from RNA-seq data. Microbiol. Res. 170 27–35. doi: 10.1016/j.micres.2014.09.005 (DSE), Exophiala pisciphila: evidence from RNA-seq data. Microbiol. Res. 170 27–35. doi: 10.1016/j.micres.2014.09.005 REFERENCES An endoplasmic reticulum-bound Ca2+/Mn2+ pump, ECA1, supports plant growth and confers tolerance to Mn2+ stress. Plant Physiol. 130, 128–137. doi: 10.1104/pp.004440 Zhang, X., Wang, L., Zhou, A., Zhou, Q., and Huang, X. (2016). Alterations in cytosol free calcium in horseradish roots simultaneously exposed to lanthanum (III) and acid rain. Ecotoxicol. Environ. Saf. 126, 62–70. doi: 10.1016/j.ecoenv.2015.12.014 Wurzinger, B., Mair, A., Pﬁster, B., and Teige, M. (2011). Cross-talk of calcium- dependent protein kinase and MAP kinase signaling. Plant Signal. Behav. 6, 8–12. doi: 10.4161/psb.6.1.14012 Zhao, D., Li, T., Shen, M., Wang, J., and Zhao, Z. (2015). Diverse strategies conferring extreme cadmium (Cd) tolerance in the dark septate endophyte February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 20 Jalmi et al. Heavy Metal Stress and Plant Signaling Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Zhao, F. Y., Wang, K., Zhang, S. Y., Ren, J., Liu, T., and Wang, X. (2014). Crosstalk between ABA, auxin, MAPK signaling, and the cell cycle in cadmium-stressed rice seedlings. Acta physiol. Plant. 36, 1879–1892. doi: 10.1007/s11738-014-1564-2 Copyright © 2018 Jalmi, Bhagat, Verma, Noryang, Tayyeba, Singh, Sharma and Sinha. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zhou, Z. S., Zeng, H. Q., Liu, Z. P., and Yang, Z. M. (2012). Genome-wide identiﬁcation of Medicago truncatula microRNAs and their targets reveals their diﬀerential regulation by heavy metal. Plant Cell Environ. 35, 86–99. doi: 10.1111/j.1365-3040.2011. 02418.x February 2018 \| Volume 9 \| Article 12 Frontiers in Plant Science \| www.frontiersin.org 21
https://openalex.org/W2969528754	https://madoc.bib.uni-mannheim.de/51990/1/Epperson_et_al-2019-Journal_of_Economic_Surveys.pdf	English	null	MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS: A REVIEW	Journal of economic surveys	2,019	cc-by	16,548	Fraunhofer Institute for Microstructure of Materials and Systems IMWS Abstract. This paper provides a synthesis of the experimental literature on matching subsidies in the context of charitable giving. We classify results according to four different outcome variables frequently considered in the literature and address (i) short-term effects of linear matching, (ii) the role of the matching rate, (iii) context-dependence of behavioural responses, (iv) the relevance of the price of giving, (v) long-term effects and (vi) nonlinear matching schemes. Based on this comprehensive review, we highlight several avenues for future research, such as putting stronger emphasis on competition in fundraising, long-term effects or heterogeneity in responses. Keywords. Charitable giving; Experiments; Fundraising; Matching subsidy; Voluntary public good provision doi: 10.1111/joes.12337 Corresponding author contact email: raphael.epperson@gess.uni-mannheim.de; Tel: +49-621/181-1772. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1 24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. f y p y y This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Christiane Reif Christiane Reif Fraunhofer Institute for Microstructure of Materials and Systems IMWS Christiane Reif Fraunhofer Institute for Microstructure of Materials and Systems IMW Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS: A REVIEW Raphael Epperson* University of Mannheim Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 f y ( ) , , pp C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution Licen f y ( ) , , pp C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution Lic distribution and reproduction in any medium, provided the original work is properly cited. 1. Introduction Fundraisers commonly apply matching subsidies to stimulate giving. The underlying idea is that a third party supplements the individual’s contribution and thereby reduces the effective price of giving. Since large amounts of money are involved in such fundraising campaigns and nonproﬁt organizations are crucial to provide goods and services the private economy often fails to provide, it is of great importance to have a solid understanding of the effect of matching subsidies on fundraising success. Therefore, we present a synthesis of the experimental literature on matching subsidies in the context of charitable giving. In a ﬁrst step, we disentangle the different ways in which effectiveness is assessed and classify the empirical evidence accordingly. On this basis, we summarize the short-term effects of matching on the different outcome variables – namely charity receipts, checkbook giving, extensive and intensive margin – taking into account that a large donation by a third party might alternatively be used to announce a lead donor gift (Section 3.1). To allow the reader to easily access the available evidence as well as the key characteristics of the respective studies, we additionally provide detailed tables on the short-term effects found in the experimental literature (Table A1 for laboratory experiments and Table A2 for ﬁeld experiments). Afterwards, we discuss the relevance of the matching rate (Section 3.2) as well as contextual factors (Section 3.3), and infer whether an effect of matching solely results from a change in the price of giving (Section 3.4). To complement the review, we also pay particular attention to long-term 2 EPPERSON AND REIF effects (Section 3.5) and recent alternative designs of matching schemes (Section 3.6). We provide a short summary on each of these aspects at the end of the respective sections. Our review is related to that of Vesterlund (2016), who tackles the more general question of why people give to charities and provides an overview on different fundraising mechanisms. Although her broad review considers short- and long-term effects of matching subsidies, these are addressed rather brieﬂy. Devoting a complete literature review to the effectiveness of matching in the charitable giving context allows us to extend the important work of Vesterlund (2016) in several dimensions. First, we offer a more comprehensive picture on the impact of matching, for example, by classifying and structuring results according to the different outcome variables considered, taking into account contextual factors and incorporating new evidence. 2. Fundamentals of Matching Subsidies We follow the deﬁnition of matching subsidies shaped by the empirical literature on charitable giving: a matching subsidy supplements the contributed money (e.g. at a linear rate) and both the individual’s contribution as well as the matching payment are given to the charity. It is precisely this approach which is the focus of the literature review. We exclude rebates, which use subsidy payments to refund a fraction of the donor’s contribution, while the charity still receives the entire amount contributed. Although rebates are commonly deﬁned as being realized without any delay and are, therefore, strategically equivalent to matches, the charitable giving literature shows that subjects do not react the same way to rebates as they do to matches. For instance, a well-established result is that rebates raise considerably less money (see e.g. Eckel and Gossman, 2003; Davis et al., 2005; Eckel and Gossman, 2006a, 2006b; Davis, 2006; Eckel and Gossman, 2008; Lukas et al., 2010; Bekkers, 2015; Eckel and Gossman, 2017). Therefore, including both subsidy types would require reviewing the empirical evidence for each separately, exceeding the scope of the review. The majority of the empirical literature considers linear matching schemes. In this case, a third party – often an anonymous donor – offers to match the individuals’ contributions to the charity at a matching rate of m > 0.1 Based on the matching rate, each individual i decides on her contribution di.2 Thus, the charity receives di ∗(1 + m) and the implied price of giving, which is the amount of money a donor needs to give so that the charity receives one dollar, can be expressed as pi = 1 1+m . 1+m There are four outcome variables, each of which can be used to assess the effectiveness of a matching subsidy: charity receipts, checkbook giving, extensive and intensive margin. We use the following deﬁnitions and formally summarize them in Table 1. In line with Davis et al. (2005), we deﬁne charity receipts as the amount of money the charity receives including the matching subsidy. As control and treatment groups often differ in the exact group size, evidence is commonly based on the charity receipts per individual. Although charity receipts constitute a highly relevant outcome variable, fundraisers or policy makers might be speciﬁcally interested in the effect on individuals’ contributions, excluding the matching payment. Following Eckel and Grossman (2008), we refer to this as checkbook giving. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. 1. Introduction Second, we elaborate whether the decrease in the price of giving is the single driver of the observed matching impact and tackle recent advances with respect to the design of matching subsidies. Finally, the detailed review allows us to identify research gaps and formulate avenues for future work on matching subsidies, while also serving as a source of information for practitioners. In the following section, we offer a more detailed description of matching subsidies in the charitable giving context and introduce the four outcome variables commonly analysed in the literature. Based on these outcome variables, we discuss the experimental evidence on the effectiveness of matching in detail in Section 3. Finally, we conclude and highlight additional opportunities for future research. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 3 Table 1. Outcome Variables. Charity receipts Checkbook giving Extensive margin Intensive margin Amount the charity receives Amount given excluding the match Likelihood of giving a positive amount Checkbook giving of contributors E[di(1 + m)\|m, xi] E[di\|m, xi] Pr(di > 0\|m, xi) E[di\|m, xi, di⟩0] Note: di is the individual’s contribution, m is the matching rate and xi contains other factors which might be considered and do not need to be individual speciﬁc. Table 1. Outcome Variables. Note: di is the individual’s contribution, m is the matching rate and xi contains other factors which might be considered and do not need to be individual speciﬁc. checkbook giving, referred to as crowding out, whereas one below –1 represents crowding in. A price elasticity of –1 correspondingly indicates that checkbook giving is not affected and the percentage increase in charity receipts mirrors the matching rate, for example, a matching rate of 0.5 increases charity receipts by 50%. While charity receipts depend on checkbook giving, checkbook giving itself aggregates two behavioural responses: (i) the likelihood of giving, which we refer to as extensive margin and (ii) the checkbook giving conditional on contributing a positive amount denoted as intensive margin or conditional checkbook giving. Therefore, a lack of sensitivity on the level of checkbook giving might be explained by the absence of any effect on both margins, or for example, an increase in the likelihood of contributing paired with a counterbalanced decrease in conditional checkbook giving. The extensive margin is often of particular interest, as former donors are more likely to give in the future than those who have never donated to the charity (Landry et al., 2010; Adena and Huck, 2019), even if the initial economic incentive, that is, a lottery, has been removed (Landry et al., 2010). This aspect also emphasizes why focusing on short-term effects alone is not sufﬁcient when assessing the effectiveness of matching subsidies. The effect of linear matching subsidies on these four outcome variables depends on individuals’ preferences, which are usually not known. As there are several motives for giving to charities (Bekkers and Wiepking, 2011; Vesterlund, 2016), various predictions can arise (Karlan and List, 2007; Huck and Rasul, 2011). In the following, we address three of them. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. 2. Fundamentals of Matching Subsidies As charity receipts and checkbook giving are closely linked, we can use the price elasticity of the former to conclude about the latter: a price elasticity of charity receipts above –1 indicates that decreasing the price decreases MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 3. Experimental Evidence The methodological approaches used to study the effects of matching subsidies in the context of charitable giving comprise both laboratory and ﬁeld experiments. The former are mainly based on a modiﬁed dictator game, in which subjects allocate money between themselves and a chosen or exogenously determined charity (e.g. Eckel and Grossman, 2003). Usually individuals decide how much money to give to the charity net of the subsidy, but Davis (2006) deviates from this standard procedure: the matching subsidy is added to the individual’s endowment upfront and subsequently subtracted if the money is not allocated to the charity. In most laboratory experiments which we consider in this review, the comparison of a matching subsidy and a no subsidy control or different matching rates is based on a within-subjects design: Each individual faces several contribution decisions, differing in whether matching is offered and to which extent the experimenter matches the individual’s contribution. This does not exclude the possibility that other aspects are examined in a between-subjects design, like the comparison of matches to rebates in Eckel and Grossman (2006b). In contrast to most laboratory experiments, the ﬁeld experiments are based on a between-subjects design (each subject is assigned to a single treatment) and are commonly implemented together with a fundraiser via mail solicitations to a speciﬁc subject sample (see e.g. Karlan and List, 2007). The respective studies mainly focus on linear matching covering different matching rates, which are mostly not greater than one. The smallest matching rate considered is 0.1, while the largest amounts to 5. Another important methodological aspect is that most studies offer estimates of treatment effects relative to a no subsidy condition for each matching treatment. However, for some studies we are only able to infer the effect of matching from an estimated price elasticity, which also takes into account the change over several matching treatments with different rates (Eckel and Grossman, 2003, 2006b). In those cases, we are therefore unable to disentangle the effect of introducing a matching subsidy and changing the rate offered. Lack of information prevents researchers from determining the social optimum. Therefore, the four different outcome variables introduced in the previous section are used to judge the effectiveness of matching schemes. We classify the empirical evidence accordingly to increase comparability of results and infer general conclusions. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS ) g First, charitable giving can be seen as a public good, either if the population is altruistically inclined (Vesterlund, 2016) or if the potential donors directly beneﬁt from the good or service provided by the charity. In this case, the total amount of money received by the charity enters the individuals’ utility functions (e.g. Andreoni, 1989), as it translates into the respective goods or services provided by the charity. The matching subsidy decreases the price of giving, that is, it decreases the amount of money an individual needs to give so that the charity receives one dollar. Under the assumption of an ordinary good, this will increase charity receipts, whereas the impact on checkbook giving and both margins remains ambiguous. g Second, another commonly identiﬁed and discussed motive for giving is warm glow (Vesterlund, 2016). If people are purely motivated to give by warm glow, they only receive utility from what they give to the charity (and from private consumption), which implies that the level of checkbook giving and not the level of charity receipts directly contributes to the individual’s utility (Andreoni, 1989, 1990). As matching does not affect the marginal costs of checkbook giving (Huck and Rasul, 2011), in case of pure warm glow preferences matching will neither affect checkbook giving nor any of the two margins, leading to an increase in charity receipts by exactly the matching subsidy. If individuals are additionally motivated by altruism, referred to as impure altruism (Andreoni, 1989), predictions become qualitatively similar to those under pure altruism. Third, monetary incentives can crowd out intrinsic motivation (Frey and Jegen, 2001; B´enabou and Tirol, 2006). Thus, if matching is in place, giving of intrinsically motivated individuals might be crowed EPPERSON AND REIF 4 out (Huck and Rasul, 2011), resulting in a negative effect on checkbook giving and the extensive margin. If sufﬁciently strong, it might even lead to a decrease in charity receipts. This incomplete list of predictions on how matching might affect giving illustrates that empirical and especially experimental evidence is needed to assess matching subsidies. To the extent possible, we will relate the empirical evidence back to these predictions and highlight some other crucial aspects affecting the response to matching in Section 3.4. Note that an observed response might be consistent with various theoretical explanations (of which not all are necessarily presented here). Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS For example, crowding out of checkbook giving can result from altruistic preferences as well as crowding out of intrinsic motivation. 3.1 Does Linear Matching Affect Short-Term Giving? Our structured review on the effectiveness of matching subsidies in the short-term is supplemented by a detailed overview on the corresponding results in the matching literature, presented in Table A1 (laboratory experiments) and Table A2 (ﬁeld experiments) in the Appendix. For each study which we consider, the tables provide information on the authors, the year of publication, the matching rates applied, the limit of the matching gift if it is presented to the subjects, the number of subjects in each matching treatment, and the percentage of treated subjects donating. As key feature of the tables, we report the quantitative effects of matching on the four outcome variables analysed in the respective study. The effects on charity receipts, checkbook giving and the intensive margin are presented as relative change (in percent) using the control condition without matching subsidy as baseline. For example, the ﬁrst entry in the column of checkbook giving in Table A2 means that offering a match of 1, signiﬁcantly increased the level of checkbook giving by 2.4%. The effects on the extensive margin are presented as absolute difference in the likelihood to give between the matching treatment and the control condition (in percentage points). The tables help the reader to easily and separately access the results of different studies featuring either laboratory or ﬁeld experiments, to review their characteristics and draw comparisons. Furthermore, they allow to check which studies analyse a certain outcome variable or at least present data on it. The comment section is used to emphasize further important characteristics of a study and to report interesting results not ﬁtting into the standard form of the tables. The second column differs across tables: In Table A1 it reports whether a within- or between-subjects design is used, whereas in Table A2 it provides information on whether a lead donor is announced in the control condition. The reason for this difference is that in all laboratory experiments which are presented in Table A1 the control condition does not include a lead donor announcement and all ﬁeld experiments are based on a between-subjects design. We start our review on short-term effects with the most aggregated of the four outcome variables introduced above – charity receipts – and afterwards gradually move to more disaggregated levels – checkbook giving, the extensive and ﬁnally the intensive margin. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 5 short-term increase in giving due to matching might be driven by intertemporal substitution and thus come at the expense of future contributions. Finally, we discuss alternatives to linear matching (Section 3.6). 3.1 Does Linear Matching Affect Short-Term Giving? 3. Experimental Evidence In doing so, we ﬁrst focus on the effect of introducing matching compared to not offering any subsidy (Section 3.1). As a charity might also be able to use the large donation by a third party as lead donor gift (i.e. announcing that a large donation has already been made), we also address the comparison of matching subsidies to this alternative fundraising strategy. Afterwards we discuss the impact of the matching rate (Section 3.2) and take a closer look at small differences in settings across studies as well as aspects analysed within experiments to infer the impact of contextual factors on the effectiveness of matching subsidies (Section 3.3). Since the main motivation for matching is the resulting decrease in the effective price of giving, we discuss to which extent the empirical evidence supports that matching simply changes the price of giving (Section 3.4). Subsequently, we shift our focus to long-term effects (Section 3.5), which are crucial to judge the effectiveness on a more aggregate level. For example, a MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 3.1 Does Linear Matching Affect Short-Term Giving? (2011) are the only ones who identify a signiﬁcant negative effect for a subgroup of their sample, with the aggregate effect on the whole sample not being signiﬁcantly different from zero. p g g y With respect to the extensive margin, empirical evidence predominantly suggests a nonnegative effect. Huck and Rasul (2011), Karlan et al. (2011), Gee and Schreck (2018) and Helms McCarty et al. (2018) do not ﬁnd a signiﬁcant effect of linear matching on the aggregate, considering matching rates not greater than 1, while Karlan and List (2007) report a signiﬁcant increase in the extensive margin by 0.4 percentage points, when pooling data on matching rates of 1, 2 and 3. This effect is substantial since the likelihood of contributing in the control group is 1.8%. In Huck and Rasul (2011), Gee and Schreck (2018) and Helms McCarty et al. (2018) the differences in response rates between the control group and the treatment group, which receives a linear match of 1, amount to 0.5, 0.8 and 0.6 percentage points with baseline response rates of 3.7%, 1.6% and 3.0%, respectively. Since all three papers have a substantially lower number of observations than Karlan and List (2007), insigniﬁcant effects might result from a lack of power. Eckel and Grossman (2017) send out solicitations where individuals are asked to ﬁll out a survey and to donate. For each survey returned the researchers donate $5 to the charity. While the fraction of individuals completing the survey does not signiﬁcantly differ across treatments (including rebate treatments), the effect on the likelihood of donating among respondents is signiﬁcantly positive for a matching subsidy of 1/3 (8.4 percentage points) but insigniﬁcant for a matching subsidy of 0.25. Thus, it seems that a positive effect on the extensive margin might only arise if the matching rate is sufﬁciently large. Additional evidence for a positive impact is offered by Gneezy et al. (2014) for a matching rate of 1 and Knutsson et al. (2019) for matching rates of 1, 3 and 5. In both of these ﬁeld experiments, subjects face a discrete choice set, either because they are asked to choose from a predetermined set of monetary values or because they make a binary choice on whether to donate the deposit received when returning bottles and cans. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. 3.1 Does Linear Matching Affect Short-Term Giving? For now, we focus on the effect of introducing a matching subsidy compared to a baseline, in which neither a subsidy is offered nor a lead donor is announced. In line with the law of demand, laboratory experiments suggest a positive effect of matching subsidies on charity receipts (Eckel and Gossman, 2003; Davis et al., 2005; Davis, 2006; Eckel and Gossman, 2006b; Lukas et al., 2010). The increase is roughly proportional to the matching rate or higher. For example, Davis et al. (2005) and Davis (2006) observe an increase of 36.1–63.3% for a matching rate of 0.5 and of 113.3–134.0% for a matching rate of 1. Consistently, the price elasticity estimated by Eckel and Grossman (2003) including matching rates of 0.25, 0.33 and 1 amounts to –0.9, while Eckel and Grossman (2006b) ﬁnd a larger behavioural response resulting in a price elasticity of –2.6. The price elasticities of charity receipts around or below –1 already point towards a nonnegative effect on checkbook giving. In most ﬁeld experiments, linear matching signiﬁcantly increases the level of checkbook giving (Karlan and List, 2007; Gneezy et al., 2014; Huck et al., 2015; Eckel and Grossman, 2017). For example, Karlan and List (2007) implement matching rates of 1, 2, and 3 in a mail-out to former contributors of a nonproﬁt organization. The authors identify a signiﬁcant positive increase of 19% in the average checkbook giving across matching treatments relative to a baseline without subsidy. Some others ﬁnd larger effects despite considering smaller matching rates, for example, a 50% increase for a matching rate of 0.5 (Huck et al., 2015) or a 39% increase for a matching rate of 0.25 (Eckel and Grossman, 2017). Positive effects on checkbook giving are also found in laboratory experiments (Eckel et al., 2007), at least for some matching rates and endowments (Davis, 2006). However, Lukas et al. 6 EPPERSON AND REIF (2010) do not identify a signiﬁcant effect for a matching rate of 0.25 and in the laboratory experiment by Charness and Holder (2019) the increase due to a linear match of 1 is merely signiﬁcant in the case of a one-sided test. Similarly, Helms McCarty et al. (2018) do not ﬁnd a signiﬁcant impact on checkbook giving when considering matching rates of 0.1 and 1 in the ﬁeld. Karlan et al. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS donated in the year of the experiment, but a signiﬁcantly more positive one for those who have, which also holds true with respect to checkbook giving. Their ﬁndings emphasize that a lack of impact on the aggregate might be the result of substantial heterogeneous effects cancelling each other out. The donation history of individuals is not the only dimension at which heterogeneous treatment effects might occur. Karlan and List (2007) emphasize a spatial heterogeneity based on whether the majority of people in a state voted for George W. Bush in the 2004 presidential election. In the states that voted in favour of him, the match has a signiﬁcant positive impact on the likelihood of contributing, while for the others no signiﬁcant effect occurs. The importance of political aspects in this context might be particularly driven by the fact that the involved nonproﬁt organization is a political one. Based on the same data as Eckel and Grossman (2003), Eckel and Grossman (2004) investigate whether religious subjects are more or less responsive to matches than nonreligious subjects, but do not ﬁnd a signiﬁcant difference in implied price elasticities of charity receipts. A large pool of other individual characteristics is considered in Bekkers (2015). By including interaction effects in a multinomial logit model, he shows that the effect of matching on the likelihood of small donations (1–45% of the endowment) is more positive for females and ﬁrst increases with the level of education but eventually decreases when individuals hold at least a bachelor degree. These effects are not present for medium donations (45–55% of the endowment), while for large donations (55–100% of the endowment) only holding at least a bachelor degree has a weakly signiﬁcant negative inﬂuence on the impact of matching. A gender difference is also supported by Knutsson et al. (2019), who ﬁnd that the positive effect of matching on the extensive margin is primarily driven by females. The intensive margin exhibits a nonnegative response: Karlan and List (2007), Gee and Schreck (2018) and Helms McCarty et al. (2018) do not ﬁnd a signiﬁcant impact due to the introduction of linear matching, while Huck and Rasul (2011) ﬁnd a signiﬁcant increase of 35.9% due to a 0.5 match, but an insigniﬁcant one in case of a match of 1. Again, Eckel and Grossman (2008) emphasize the potential relevance of heterogeneity. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS They show that decreasing the effective price due to matching raises only the intensive margin of continuing members, with the corresponding price elasticity amounting to –0.1. However, it is important to note that (i) donors about whom the authors do not have complete information are excluded (about 1.1% of all donors), leading to the exclusion of some particularly high donations by continuing members in the control group4 and (ii) separate mean comparisons of each matching treatment to the baseline do not reveal signiﬁcant differences in the intensive margin for this subgroup. g g g p So far, we have focused on comparing a matching subsidy to a no subsidy control without a lead donor. If a generous gift by a third party is available, the charity might as well consider using the money as unconditional gift (if possible). Therefore, the comparison of matching to a baseline in which a lead donor is announced is of particular interest to tackle the question how a generous gift should be used. In Gneezy et al. (2014) as well as Huck et al. (2015), the level of checkbook giving in the lead donor control treatment exceeds that in the matching treatments, but this difference is not statistically signiﬁcant. Counterfactual simulations in Huck et al. (2015) suggest that a lead donor is preferable to linear matching, even when considering a larger range of linear matching rates. In contrast, Adena and Huck (2017) estimate a matching subsidy of 1 to increase checkbook giving by about 2.4% compared to a lead donor control. Similarly, Adena and Huck (2017) ﬁnd a signiﬁcant increase of 0.6 percentage points in the extensive margin whereas others do not ﬁnd any signiﬁcant difference (Huck and Rasul, 2011; Gneezy et al., 2014). Concerning the intensive margin, matching has no effect (Gneezy et al., 2014) or performs signiﬁcantly worse (Huck and Rasul 2011; Adena and Huck 2017). The decrease due to a match of 1 ranges from 24% in Adena and Huck (2017) to 35% in Huck and Rasul (2011). The insigniﬁcant result in Gneezy et al. (2014) might be caused by the design as potential donors can only choose between four different donation amounts. Overall, the comparison of linear matching to a baseline in which neither a matching subsidy is offered nor a lead donor is announced reveals a strictly positive impact on the amount the charity receives. 3.1 Does Linear Matching Affect Short-Term Giving? The result of the only laboratory experiment analysing the extensive margin generally ﬁts well into those of the presented ﬁeld experiments, but suggests a positive effect even for a matching rate as low as 0.25: Eckel et al. (2007) ﬁnd that matching with a rate of 0.25, 0.5 or 1 signiﬁcantly increases the likelihood to give by 2.0, 2.7 and 3.0 percentage points, respectively. g y p g p p y However, effects might be heterogeneous, also for outcome variables other than the extensive margin. For example, Eckel and Grossman (2008) send out mails to raise funds for a nonproﬁt organization, which include matching rates of either 0.25 or 1/3. Compared to a baseline without a lead donor, the extensive margin of regular contributors with a membership of the organization (continuing members) is signiﬁcantly lower when matching is in place. Such an effect cannot be identiﬁed for subjects who previously were members but are currently not and subjects who neither have been members nor have ever contributed.3 Although not tested for signiﬁcance, the descriptive statistics suggest that this negative impact might be strong enough to decrease the average checkbook giving of this particular subgroup. Karlan and List (2007) ﬁnd that matching is more effective in increasing the extensive margin of prior donors that have donated a small amount than of those that have donated a large amount. The authors also show that the effect of matching on the level of checkbook giving and the extensive margin is slightly larger for prior donors who have not provided funds in the year of the ﬁeld experiment yet than for those who have. Interestingly, the result that ‘more involved’ subjects react less positively to the match does not hold in general. Helms McCarty et al. (2018) categorize individuals into (i) those who are afﬁliated with the charity but have not donated so far, (ii) those that have donated regularly and (iii) those that have given their ﬁrst donation in the year of the ﬁeld experiment. The introduction of matching neither produced heterogeneous effects on the level of checkbook giving nor on the extensive margin. Furthermore, Karlan et al. (2011) detect a negative impact of a match on the extensive margin for prior donors who have not MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 7 3.2 Which Matching Rate to Choose? After discussing the effect of matching subsidies at different rates, we now focus on the relevance of the matching rate more explicitly. Since the matching rates considered in the literature range from 0.1 to 5, conclusions are generally limited to this interval. As in Section 3.1, we start with the most aggregate outcome variable and gradually move towards more disaggregated levels. We do not address the intensive margin as there is not sufﬁcient evidence to draw conclusions. The relative changes in charity receipts for different matching rates, which are presented in Tables A1 and A2, suggest a simple relationship: The level of charity receipts increases with the matching rate. This notion is supported by the negative price elasticity estimates for charity receipts by Eckel and Grossman (2003; 2006b) as well as the results on checkbook giving addressed in the following. We have already presented evidence that a matching subsidy can increase checkbook giving, but the relevance of the matching rate on this outcome variable seems to be rather limited. In Karlan and List (2007), levels of checkbook giving under the considered matching rates of 1, 2 and 3 do not differ signiﬁcantly.5 Such lack of responsiveness with regard to the magnitude of matching is supported by Bekkers (2015) for matching rates of 0.5 and 1, as well as Helms McCarty et al. (2018) for matching rates of 0.1 and 1. However, it is in contrast to the signiﬁcant positive impact of increases in the matching rate found in laboratory experiments (Eckel et al., 2007, for matching rates of 0.25, 0.5, and 1; Adena and Huck, 2017, for matching rates of 0.5, 1, 1.5). Lukas et al. (2010) conduct a laboratory experiment with matching rates of 0.25, 0.33 and 1, and two different endowment levels, in which only a matching rate increase from 0.33 to 1 with an endowment of $10 is found to create a signiﬁcant increase. In this study, the introduction of a 0.25 match does not affect checkbook giving either. y g g Additional evidence is offered by Kesternich et al. (2016) who study a special public good setting in which subjects make binary choices. Customers of a German long-distance bus operator have the opportunity to offset their carbon emissions at a predetermined price when purchasing tickets. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS Most studies observe a positive or at least nonnegative effect on checkbook giving, suggesting a low risk of a 8 EPPERSON AND REIF failed matching campaign in terms of crowded-out contributions in the short-term. Exclusively focusing on the aggregate might, however, leave aside crucial response patterns as heterogeneous treatment effects potentially lead to negative outcomes for subgroups. Furthermore, the empirical evidence suggests that the impact of matching is not limited to a speciﬁc margin. failed matching campaign in terms of crowded-out contributions in the short-term. Exclusively focusing on the aggregate might, however, leave aside crucial response patterns as heterogeneous treatment effects potentially lead to negative outcomes for subgroups. Furthermore, the empirical evidence suggests that the impact of matching is not limited to a speciﬁc margin. When evaluating the effectiveness of matching, it is important to consider the alternative of using the generous gift of a third party as lead donation. Although the empirical evidence on the comparison of a matching subsidy to a lead donor announcement is not distinct, a lead donor announcement can be an interesting alternative. An aspect in favour of the lead donor approach is that the gift is received by the charity for sure, whereas in case of a matching campaign the matching gift might not be exhausted, as happened in several ﬁeld experiments (e.g. Eckel and Grossman, 2008, 2017; Huck and Rasul, 2011; Huck et al., 2015; Adena and Huck, 2017). However, a charity might as well be able to receive the portion of the matching gift not exhausted during the matching campaign after the end of the campaign. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 9 checkbook giving in the ﬁeld, increasing the matching rate never resulted in a signiﬁcant further increase in checkbook giving. How to ensure that the chosen matching rate is sufﬁcient to create an effect? A matching rate of 1 has proven to be effective in crowding in donations in several settings (Davis, 2006; Gneezy et al., 2014, Huck et al., 2015; Kesternich et al., 2016). However, there are cases in which it did not work out (Davis, 2006; Karlan et al., 2011; Helms McCarty et al., 2018) and sometimes smaller matching rates have been sufﬁcient (Davis, 2006; Huck et al., 2015; Eckel and Grossman, 2017). g ( ) Findings on the extensive margin suggest that matching rates need to be large enough to create a positive impact. Eckel and Grossman (2017) do not ﬁnd an effect for a matching rate of 0.25 but for 1/3, whereas Kesternich et al. (2016) estimate a signiﬁcant impact for a matching rate of 1 but not for 1/3. Helms McCarty et al. (2018) neither ﬁnd a signiﬁcant effect for a low matching rate of 0.1 nor for a rate of 1. Meier (2007) investigates the donation behaviour of students in the ﬁeld, focusing on a slightly different version of the extensive margin. At the time of paying the tuition fees, each student has to decide whether to donate to no fund, a single fund, or two funds. Each decision is associated with a ﬁxed amount of money. Offering a matching rate of 0.25 or 0.5 for contributing to both funds, pooling the data for these into a single treatment and taking into account pretreatment differences reveals a signiﬁcant increase in the likelihood of contributing to both funds. However, if the two matching treatments are considered separately in a logit regression, the lower matching rate is not found to have a signiﬁcant coefﬁcient while the higher matching rate does. Nevertheless, the two coefﬁcients are not statistically different. There is additional laboratory evidence that increasing the matching rate up to 1 can increase the likelihood to give (Eckel et al., 2007). Despite this positive relation between the matching rate and the likelihood to give, increases above 1 have not been found to create an additional effect in the ﬁeld (Karlan and List, 2007; Kesternich et al., 2016). Yet, subgroups like females might remain responsive to increases of the matching rate above 1 (Knutsson et al., 2019). Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS To summarize, the choice of the matching rate depends on the objective. If a large gift can only be used as a matching subsidy and charity receipts in the short-term are to be maximized, a larger matching rate is beneﬁcial. If instead the focus is on checkbook giving or the likelihood to give, the level of the matching rate needs to be sufﬁciently large to create an effect, whereas further increases will do no harm but might as well not offer any additional advantage. There is some evidence that increasing the matching rate up to 1 can be beneﬁcial, while increases above 1 have not been found to be effective on the aggregate, at least in the ﬁeld. Furthermore, matching rates of 1 turned out to be effective across several studies, yet there are cases in which they were not. 3.2 Which Matching Rate to Choose? As the authors point out, the contribution decision is directly linked to a private consumption choice, which rather makes it an impure public good. If a match is in place, the company itself offsets an additional amount of carbon emissions determined by the matching rate offered. A matching rate of 1/3 creates no effect, but a matching rate of 1 is found to signiﬁcantly increase the likelihood of offsetting compared to the control group, which results in a signiﬁcant increase in checkbook giving. Interestingly, a further increase in the matching rate from 1 to 3 does not generate any additional impact, conﬁrming the ﬁnding of Karlan and List (2007) that an increase in the matching rate above 1 neither signiﬁcantly affects checkbook giving nor the extensive margin. The evidence on how the matching rate affects checkbook giving is rather mixed, but suggests that increasing the matching rate does no harm. If a matching subsidy at a given rate was sufﬁcient to increase MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 3.3 Does Context Matter? As a result the impact might be less positive than in settings without a threshold. Rondeau and List (2008) offer evidence on this, using a laboratory and a ﬁeld experiment, which are both based on a between-subjects design. Importantly, if the money raised exceeds the threshold, it is invested in additional provision. In the ﬁeld experiment, Sierra Club supporters are asked via mail to donate for the expansion of an environmental education program delivered to students and in each treatment subjects are informed about the threshold as well as the number of other supporters solicited. Holding the threshold constant at $5000, a matching subsidy of 1 has no signiﬁcant impact on the intensive margin compared to a no subsidy control. Furthermore, the level of checkbook giving is about 10% lower under the matching subsidy, though this difference is not tested for signiﬁcance. The laboratory experiment consists of a public good game, in which subjects form groups of 6 and allocate their endowment of $12 between a private and a group account. The money allocated to the group account only results in a payment if a certain threshold is reached. Keeping the threshold constant, matching is found to crowd out checkbook giving relative to a no subsidy condition without lead donor announcement. Hence, only the laboratory experiment offers evidence that in the presence of a threshold the matching impact might turn negative. Yet there is no direct comparison to a setting without a threshold and the fact that a public good game is used limits the comparability to other laboratory studies that rely on a modiﬁed dictator game with actual donations to a charity. In practice, the matching payment is often limited to a certain amount of money to eliminate risk for the party funding the subsidy. Subjects need to be informed about this, which might be a crucial aspect, since it offers information on the likelihood of being matched. In general, a smaller matching limit is exhausted more easily, which could decrease the perceived likelihood that the individual’s donation will be matched, and in turn increase the expected price of giving. An explicit test of different matching limits ($25,000; $50,000; $100,000; no limit) stated in a donation campaign did not impact the effectiveness of matches (Karlan and List, 2007). Yet, it is an open question whether these results apply to different fundraising campaigns. 3.3 Does Context Matter? The context in which a matching offer is made might alter its effectiveness, either by changing how the offer is perceived or by directly manipulating crucial aspects of decision making. An example for the former is the presentation of the matching subsidy. In most studies, matching is explained as a matching payment of a speciﬁc level for every dollar (euro) donated by the individual (see e.g. Davis et al., 2005; Eckel and Grossman, 2006b; Karlan and List, 2007; Lukas et al., 2010; Huck and Rasul, 2011). Karlan and List (2007) supplement the standard presentation by an illustrative example, which is based on one of three sample donations suggested to the individual. It turns out to be irrelevant for the effect of matching whether the highest, middle or lowest sample amount is used to illustrate the match. Nevertheless, the presentation of the match can be relevant. Karlan et al. (2011) ﬁnd that framing the matching rate of 1/3 as $25 for each $75 donated instead of $1 for each $3 donated negatively affects checkbook giving as well as the likelihood of donating. A possible reason put forward by the authors is that subjects might misunderstand the match, thinking they are required to donate $75 before any matching payment is made. A similar alternative representation of the match of 1 as $25 for each $25 donated does not create any signiﬁcant change in the impact of the match. 10 EPPERSON AND REIF Sometimes fundraising campaigns not only make use of suggested donation levels, but restrict the donor’s choice to certain amounts of money. In contrast to the previously discussed framing of the match, this has a direct impact on the nature of the decision problem faced by the individual. Five different studies analyse the effectiveness of matching under a discrete donation decision (Meier, 2007; Gneezy et al., 2014; Kesternich et al., 2016; Knutsson et al., 2019; Charness and Holder, 2019). Contrasting their results to other experiments suggests, however, that implementing a discrete choice set does not systematically alter the effectiveness of matching. In contrast to the experimental studies discussed so far, a fundraising campaign might aim at raising a particular amount of money while the money is returned if this threshold is not reached. In such a setting, the introduction of matching reduces the level of checkbook giving required to reach the threshold. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. 3.4 Is It All about the Price of Giving? As discussed in Section 2, there are several predictions on how matching might affect charitable giving, each associated with a set of assumptions about the underlying preferences. Overall, crowding out of the intrinsic motivation to give seems not to be a major aspect as matching mostly creates a nonnegative effect on checkbook giving. Furthermore, the number of cases in which checkbook giving is positively affected stands in contrast to the predictions based on pure warm glow. Instead, ﬁndings are in line with individuals being motivated by pure or impure altruism. Thus, one is tempted to conclude that all that matching does is changing the price of giving. In the following, we address three crucial aspects of why this might not be the case. First, several experimental studies comparing matching and rebate subsidies reveal higher charity receipts and a larger price elasticity under matching, although equivalent rebates imply the same price of giving (see e.g. Eckel and Grossman 2003; Davis et al., 2005; Eckel and Grossman, 2006a, 2006b; Davis, 2006; Eckel and Grossman, 2008; Lukas et al., 2010; Bekkers, 2015; Eckel and Grossman, 2017; or for an overview Vesterlund, 2016). Thus, it seems that either the matching subsidy, the rebate subsidy or both do not solely change the price of giving. It is not the goal of this paper to review the empirical evidence on how the difference between rebates and matches comes about, but matching might additionally encourage giving due to the cooperative element that somebody else is also contributing to the charity (Eckel and Grossman, 2003). However, Hungerman and Ottoni-Wilhelm (2018) offer an appealing explanation, which is in line with matching subsidies only changing the price of giving. In their model of charitable giving, referred to as impure impact giving model, subjects receive utility from the overall level of charity receipts (allowing that more weight is given to the charity receipts produced by the subject’s donation than to the charity receipts resulting from the donations of other subjects) as well as warm glow utility. As the warm glow utility is assumed to come from the level of checkbook giving (which for the rebate is the amount passed to the charity and not the amount actually sacriﬁced after accounting for the refund), a matching subsidy only changes the price of giving whereas a rebate subsidy additionally changes the price of the warm glow utility. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 11 substantial inﬂuence. While the literature offers some interesting insights into heterogeneous effects, more research is needed to analyse group speciﬁc responses. substantial inﬂuence. While the literature offers some interesting insights into heterogeneous effects, more research is needed to analyse group speciﬁc responses. 3.3 Does Context Matter? For example, a certain matching limit might appear to be more binding in case of a large international organization since a large pool of potential donors is involved. Finally, in Section 3.1, we discussed heterogeneous treatment effects. There is some evidence that females react more positively to matching subsidies. Furthermore, the donation history of individuals seems to inﬂuence the impact of matching, though the direction is ambiguous. Future research is needed to draw general conclusions and shed more light on heterogeneous treatment effects. At this point, it is also important to note that the experimental studies consider very different subject pools, like students (e.g. Eckel and Grossman, 2003; Davis et al., 2005; Eckel and Grossman, 2006b; Lukas et al., 2010), former contributors (Karlan and List, 2007; Karlan et al., 2011; Eckel and Grossman, 2017; Helms McCarty et al., 2018) or opera attendees (Huck and Rasul, 2011; Huck et al., 2015; Adena and Huck, 2017). While this might explain small differences, on the aggregate, empirical ﬁndings are relatively robust across different samples. There are several reasons why the effect of matching subsidies might be context-dependent. Nevertheless, most of the contextual factors on which empirical evidence is available do not exert a MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. 3.4 Is It All about the Price of Giving? One attempt to achieve this is by mentioning a lead donor who has already provided some funding for the project. If positive signalling matters, we should expect the pure price effect to be lower than the estimates presented so far. The discussion in Section 3.1 already suggests that the difference between a matching subsidy and a lead donor treatment is less positive compared to the difference of a matching subsidy and a no subsidy control treatment without lead donor announcement. Some studies offer direct evidence on this by considering both possible baselines (Rondeau and List, 2008; Huck and Rasul, 2011; Gneezy et al., 2014; Huck et al., 2015). Signiﬁcant positive effects on checkbook giving turn insigniﬁcant if a lead donor baseline is used (Gneezy et al., 2014; Huck et al. 2015). The same is found for the effect on the extensive margin (Huck and Rasul, 2011; Gneezy et al., 2014). On the intensive margin insigniﬁcant or signiﬁcant positive differences turn signiﬁcantly negative (Huck and Rasul, 2011), become insigniﬁcant (Gneezy et al., 2014) or remain insigniﬁcant (Rondeau and List, 2008). The empirical evidence shows that, in line with expectations, removing the signalling effect by using a lead donor baseline shifts estimates downwards. This is an important step in establishing the potential relevance of signalling embedded in matching subsidies. However, the approach hinges on the assumption that the signal sent by a lead donor baseline perfectly mirrors that of a matching subsidy. An intuitive example why this might not be the case is that a lead donor donation is unconditional on what others give, whereas the match is not. Despite the fact that most experimental evidence is consistent with the idea that the impact of matching solely results from a change in the price of giving, fully attributing the impact to the price change might be wrong. Yet the precise decision process and factors underlying the behavioural response to matches are not fully identiﬁed, which offers an interesting avenue for future research. 3.4 Is It All about the Price of Giving? The authors ﬁnd support for their theoretical model when exploring charitable giving data in the context of a real-world tax rebate policy and a matching subsidy offer which naturally occurred in the ﬁeld. Estimates of the price elasticity of charity receipts amount to 1.2 and 0.2 for the match and the rebate, respectively. p y Second, a change in the price of giving can only trigger a behavioural response if it is recognized in the ﬁrst place. While in a laboratory experiment it is unlikely that subjects are not aware of the subsidy offer, Eckel and Grossman (2017) show that this might not hold in the ﬁeld. In their ﬁeld experiment, donors are required to check a box to receive the matching payment offered in the solicitation letter. Slightly more than 25% of those that face a matching offer and donate fail to do so. When only those checking the box are treated as receiving the match and facing the reduced effective price, the price elasticity of charity receipts based on all subjects that answer an attached survey increases in absolute terms from 1.3 to 5.4. This suggests that not recognizing the offer might be a relevant factor. However, as the authors point out, they cannot verify why the box has not been checked, and thus it is not clear whether it is a lack of awareness or a conscious rejection of the matching offer which is driving the results. Furthermore, the design does not allow to draw conclusion about the extent to which missing the offer affects behaviour on the extensive margin. Third, matching possibly creates a signal for potential contributors, for example, that the nonproﬁt organization is worth supporting (Karlan and List, 2007; Huck and Rasul, 2011). If this is the case and subjects in the control group are simply asked for donations, the estimated effect on checkbook giving 12 EPPERSON AND REIF includes both a price and a signalling effect, at least in a between-subjects design. In a within-subjects design a signal rather affects all treatments equally as, if at all, it alters the belief about the true state of the world which is independent from the treatment condition. Still, most evidence on checkbook giving as well as the extensive and intensive margin is obtained in ﬁeld experiments with a between-subjects design. 3.4 Is It All about the Price of Giving? To isolate the part of the behavioural response caused by the decrease in the price of giving, it would be necessary to send a similar signal in the baseline treatment. One attempt to achieve this is by mentioning a lead donor who has already provided some funding for the project. If positive signalling matters, we should expect the pure price effect to be lower than the estimates presented so far. The discussion in Section 3.1 already suggests that the difference between a matching subsidy and a lead donor treatment is less positive compared to the difference of a matching subsidy and a no subsidy control treatment without lead donor announcement. Some studies offer direct evidence on this by considering both possible baselines (Rondeau and List, 2008; Huck and Rasul, 2011; Gneezy et al., 2014; Huck et al., 2015). Signiﬁcant positive effects on checkbook giving turn insigniﬁcant if a lead donor baseline is used (Gneezy et al., 2014; Huck et al. 2015). The same is found for the effect on the extensive margin (Huck and Rasul, 2011; Gneezy et al., 2014). On the intensive margin insigniﬁcant or signiﬁcant positive differences turn signiﬁcantly negative (Huck and Rasul, 2011), become insigniﬁcant (Gneezy et al., 2014) or remain insigniﬁcant (Rondeau and List, 2008). The empirical evidence shows that, in line with expectations, removing the signalling effect by using a lead donor baseline shifts estimates downwards. This is an important step in establishing the potential relevance of signalling embedded in matching subsidies. However, the approach hinges on the assumption that the signal sent by a lead donor baseline perfectly mirrors that of a matching subsidy. An intuitive example why this might not be the case is that a lead donor donation is unconditional on what others give, whereas the match is not. includes both a price and a signalling effect, at least in a between-subjects design. In a within-subjects design a signal rather affects all treatments equally as, if at all, it alters the belief about the true state of the world which is independent from the treatment condition. Still, most evidence on checkbook giving as well as the extensive and intensive margin is obtained in ﬁeld experiments with a between-subjects design. To isolate the part of the behavioural response caused by the decrease in the price of giving, it would be necessary to send a similar signal in the baseline treatment. 3.6 What about Alternative Matching Subsidies? Although the major part of the literature focuses on linear matching, alternative forms are considered as well. The mechanism in Meier (2007) can be interpreted as threshold matching since individuals’ checkbook giving is only matched if they donate the ﬁxed amount to both funds. Huck et al. (2015) introduce a different kind of threshold matching, in which individuals’ contributions are matched at a rate of 1 if they give at least €50. The performance of this matching scheme does not substantially differ from a simple linear match of 1, mainly because the threshold is below the amount potential donors give anyways. Another matching type investigated by Huck et al. (2015) consists of a ﬁxed gift of €20 for any positive donation. Unsurprisingly, this signiﬁcantly increases the extensive margin, but has no signiﬁcant effect on checkbook giving or the intensive margin when compared to a baseline without lead donor. As the impact of both nonlinear matching schemes is likely to heavily depend on the threshold at which the linear or ﬁxed gift match is offered, the authors estimate a structural model. Counterfactual simulations make them conclude that a charity is best off if it simply announces a lead donor. If the charity is forced to use the lead donor’s money in a matching scheme, they suggest using a ﬁxed matching gift with a strictly positive threshold. The comparison of the different matching schemes is based on the predicted level of checkbook giving. However, it is worth noting that the structural model used for the counterfactual simulations particularly overpredicts the intensive margin for ﬁxed gift matching. Gee and Schreck (2018) analyse a threshold matching gift for which the threshold is based on the number of donors. Potential donors are randomly and anonymously assigned to groups of 10 and a ﬁxed gift of $50 is additionally given to the charity if a certain number of people within the group donate. In their ﬁeld experiment, the potential donors are former attendees of an education program for exceptionally good students ﬁnanced by donations. Subjects receive a fundraising letter from the charity operating this program and either face a no subsidy condition, a linear matching subsidy at rate 1 or a threshold match gift varying in the number of donors within a group needed to reach the threshold (1, 2 or 3 donors). Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 13 match of 1/3 nor 3. While these studies focus on contributions which were previously matched, Bekkers (2015) investigates long-term consequences for contributions which are only related to those previously matched: Individuals complete an online survey and allocate their reward among themselves and one of three health charities. Looking at their giving behaviour in the context of a tsunami relief campaign nine months later reveals no signiﬁcant difference in contributions between individuals whose donations were matched and those whose donations were not. Nevertheless, there is weak evidence for motivational crowding out as altruistic values elicited in the survey seem to promote a negative long-term effect of matching subsidies on checkbook giving. In contrast, the interaction of joy of giving and having received a matching subsidy is signiﬁcantly positive. Overall, these ﬁndings show very mixed results of matching in the long-term, which might be driven by case speciﬁc characteristics. Yet, the limited empirical evidence available emphasizes that long-term consequences can occur and are a very relevant aspect to judge the effectiveness of matching subsidies as well as the risk associated with running matching campaigns. More research is needed to draw general conclusion, in particular about the donation behaviour after a match has been removed (see Meier, 2007; Karlan et al., 2011; Bekkers, 2015; Kesternich et al., 2016) as well as the dynamics of repeatedly applying a matching subsidy (see Kesternich et al., 2016). Future research should also try to identify under which conditions and through which channels long-term effects are likely to occur. Can matches form habits and keep contributions on a higher level or does matching cause harm due to intertemporal substitution or motivational crowding out? There is some evidence of motivational crowding out (Bekkers, 2015) as well as evidence that matching subsidies, which are successful in increasing the extensive margin, can create a long-term effect on the extensive margin and checkbook giving (Kesternich et al., 2016). 3.5 Do Matches Work in the Long-Term? So far, we have focused on the effect of matching on immediate contribution decisions. What about long-term consequences, especially if the matching offer is subsequently removed? Few studies take the long-term perspective into account and it is still unclear what channels are driving long-term effects if they arise. A negative long-term effect might arise from intertemporal substitution or a persistent reduction of intrinsic motivation, whereas habit formation potentially keeps contributions at a high level if they have been increased by matching in the ﬁrst place (Meier, 2007). Furthermore, a matching campaign which successfully increases the extensive margin might create a positive long-term impact as previous donors are more likely to give in the future than nondonors (Landry et al., 2010; Adena and Huck, 2019). y g y Studying the donation decision students regularly face when paying their tuition fees, Meier (2007) ﬁnds a negative effect of matching on checkbook giving after the matching offer has been removed. This impact is strong enough to outweigh any positive effect observed in the matching period, leading to an overall negative but insigniﬁcant point estimate for the net effect of matching on checkbook giving. In contrast, Karlan et al. (2011) ﬁnd no effect of matching on checkbook giving during the treatment period but an increase in checkbook giving in the six subsequent months. As pointed out by the authors, this result should be treated with caution, since signiﬁcance diminishes as soon as a logarithmic speciﬁcation is used and the timing of the study might be relevant. Kesternich et al. (2016) identify a persistent effect of matching compared to the control group. For repeated bookings with the match still in place as well as for subsequent bookings in a period where the match is removed, the treatment group with a match of 1 has a signiﬁcantly higher extensive margin and checkbook giving. However, this neither holds for a MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 3.6 What about Alternative Matching Subsidies? Subjects are aware that their group consists of program attendees of the same year. Only the matching gift 14 EPPERSON AND REIF with a threshold of three donors is sufﬁcient to signiﬁcantly increase the extensive margin compared to the no subsidy condition by about 2 percentage points. Yet, this estimated effect is not signiﬁcantly different from those of the other matching treatments. No treatment effects are found on the intensive margin. In a within-subject design laboratory experiment with binary donation decisions of giving $5 or not, the authors investigate a wider range of thresholds (1, 3, 5, 7 or 10) but do not offer a comparison to linear matching. Again, the threshold of 3 performs best with respect to the extensive margin, outperforming the lower threshold and a no subsidy control while being signiﬁcantly indistinguishable from higher thresholds. Furthermore, the authors show that the belief of being pivotal to secure the matching gift is one important channel to explain differences between the different threshold treatments. p p Another matching alternative is presented in Meer (2017), who investigates daily data from an online fundraising platform used by teachers to request funding for school materials. Projects that satisfy certain observable criteria receive a match of 1 from partners of the website or an ‘Almost Home’ match, in which all of the needed funds are provided by a partner organization as long as the last $100 are raised by private donors. This represents a cumulative matching threshold for all potential donors. If a project’s goal is not reached, donors can decide to get back their donations or to have it reallocated to a different project. In a pooled analysis of both schemes, matching is found to signiﬁcantly increase the likelihood of receiving a donation on a given day (by 0.76 percentage points, with a baseline of 3 percentage points). However, conditional on receiving a positive amount, the pooled analysis shows that matching has a slightly negative effect on the checkbook giving received per day. As a result, average checkbook giving is raised by 2.8%. Charness and Holder (2019) investigate two alternative matching schemes which contain an element of competition. In the ﬁrst scheme, a subject’s donation is matched at a rate of 1 if it is at least as large as the median donation. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. 3.6 What about Alternative Matching Subsidies? In the second scheme, subjects are anonymously assigned to groups of three and the total amount donated by the group is matched at rate 1 if it does not fall below the median donation of all groups. Both schemes are compared to a no subsidy baseline as well as a classic linear match of 1 in a laboratory experiment, which is based on a between-subjects design. The group competition scheme raises a signiﬁcantly higher level of checkbook giving than any other treatment, while the level of checkbook giving does not signiﬁcantly differ between the individual competition scheme and the classic linear match. As has been shown in Section 3.1, a matching subsidy has a rather negative impact on the intensive margin when compared to a lead donor baseline.6 Adena and Huck (2017) investigate a matching scheme designed to eliminate such a negative effect: In contrast to previously discussed matching schemes, the matching payment is not given to the same project the contributor supports, but to a different one. In particular, opera visitors are asked to donate for a project aimed to offer children at the age of 2–4 their ﬁrst experience with classical music. In case of the alternative matching scheme, the matching payment supports the visits of an opera bus at certain locations like schools. Whereas the classical linear match is found to signiﬁcantly decrease the intensive margin compared to a lead donor baseline, the alternative matching scheme does not. However, both matching treatments increase the level of checkbook giving compared to the lead donor control and their effects do not signiﬁcantly differ. The theoretical model in the paper suggests that the alternative matching scheme should become more effective when complementary projects are used. This is supported by the results of the within-subjects design laboratory experiment: If the two projects are complements, the alternative matching scheme outperforms the classic linear matching subsidy with respect to the level of checkbook giving as well as the intensive margin. No such difference is found if the projects are substitutes. Although linear matching schemes are simple to explain to potential contributors and seem to be a natural choice, the empirical evidence shows that small deviations from these linear matching schemes can offer attractive alternatives, especially if they are designed to target particular shortcomings of linear schemes. 4. Conclusion Matching schemes are applied frequently in fundraising campaigns, which makes their effectiveness an object of particular interest. To offer a comprehensive review and allow a more diversiﬁed assessment of matching subsidies, we classiﬁed results according to four different outcome variables and discussed (i) short-term effects of linear matching, (ii) the choice of the matching rate, (iii) context-dependence, (iv) the relevance of the price of giving, (v) long-term effects and (vi) nonlinear matching schemes. Furthermore, we supplemented our review with detailed tables on the short-term effects of linear matching in laboratory and ﬁeld experiments (Tables A1 and A2). A crucial remark is that although matching subsidies manage to increase charity receipts and can even increase checkbook giving, they are not necessarily the best strategy at hand. If possible, the money of the third party could be used in a different way, for example, as an unconditional and announced lead donor gift. As pointed out in Sections 3.1 and 3.4, comparing the match to a lead donor treatment generally shifts estimated effects downwards. Checkbook giving in the matching condition is once estimated to signiﬁcantly exceed (Adena and Huck, 2017) and never falls signiﬁcantly below that of the lead donor treatment (Gneezy et al., 2014; Huck et al., 2015), but point estimates speak in favour of a lead donor in two of three studies (Gneezy et al., 2014; Huck et al., 2015). Another promising alternative strategy might be to use the money of the third party to cover a charity’s overhead costs, which reduces the price of giving with respect to the output produced by the charity. There is evidence that the level of overhead costs negatively affects giving (e.g. Bowman, 2006; Meer, 2014). Furthermore, Gneezy et al. (2014) ﬁnd that using the money of a third party to cover overhead costs results in higher checkbook giving and a higher extensive margin than using it as a matching subsidy of 1. It might even be possible to incorporate this ﬁnding in the design of matching by framing the matching payment as being used to cover overhead costs. We have already emphasized that focusing on short-term effects can be problematic as important long- term consequences are neglected. When assessing matching subsidies from a social planner’s perspective, there is another crucial aspect we need to take into account: potential business stealing due to reallocation of donations towards charities that implement matching. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 15 15 4. Conclusion For example, a charity might increase checkbook giving by attracting donors which otherwise would have given the same amount of money to a different charity. Meer (2017) does not ﬁnd any support for business stealing but shows that the number of matched competitors on an online fundraising platform positively affects checkbook giving to projects, although the magnitude is very small. Even an intertemporal cannibalization is rejected since the average daily number of matched competitors over the previous sixty days has a small but signiﬁcantly positive effect on checkbook giving. Null (2011) shows in a within-subjects design experiment that the matching rate affects the allocation of a limited amount of money between three similar charities. However, different matching rates across these charities rarely result in subjects shifting the entire money to the charity with the highest matching rate (considering only the charities that receive a positive donation when matching rates across charities are equal). Although further recent studies analyse inter- and intracharity spillover effects of fundraising campaigns (e.g. Krieg and Samek, 2017), more evidence on how particularly matching affects giving to competing charities at the same point in time or over time is needed. g g p g p We have learned a lot about matching from the experimental literature, but some crucial questions remain open. Closing these research gaps will not only elevate our level of understanding but will also enable practitioners to improve the design of fundraising schemes and subsidies to foster voluntary giving. 3.6 What about Alternative Matching Subsidies? It will be interesting to observe whether such alternative schemes ﬁnd their way into fundraising campaigns and support the ﬁndings obtained so far. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS Notes 1. While we refer to matching as a situation in which some party matches the individuals’ contributions, it is also possible to allow each individual to match the contributions of others. However, such mutual matching has not been investigated in the charitable giving literature so far and is difﬁcult to apply in practice if the number of players is large. In the context of public goods, there is a large amount of theoretical literature on mutual matching (e.g. Guttman, 1978; Danziger and Schnytzer, 1991; Varian, 1994; Boadway et al., 2007; Buchholz et al., 2011, 2012; Guttman, 2013) but empirical evidence is rare, even when we consider settings in which the matching payment is directed to the other agent and not to the public good (Andreoni and Varian, 1999; Charness et al., 2007; Bracht et al., 2008; Kass et al., 2015). 2. In general, matching does not need to be explicit. For example, Guttman (1978) subsumes all forms of voluntary cost sharing of public good provision as implicit matching, including conditional cooperation behavior. In such a case, an individual can theoretically identify the implicit matching rate offered by another party by observing that party’s reaction to changes in the own contribution level. However, this is a highly complex task and experiments are by and large designed to make matching explicit. 3. The response rates for the different subject types are presented in the paper but it is unclear whether the reported test results are based on pairwise comparisons or comparing the control to the pooled subsidy treatments including rebates. Therefore, we conduct Pearson’s χ2 tests, comparing the response rate of each matching treatment to that of the control for each subject type (p < 0.01 for each matching treatment considering regular contributors with a membership, referred to as continuing members in the paper, and p > 0.2 in all other cases considered). 4. The information on the number of mailed solicitations, the revenue per solicitation and the mean checkbook donation (intensive margin of donors on which the authors have complete information), presented in Table 1 of Eckel and Grossman (2008), allows to calculate that the average donation of those 55 continuing members excluded from the control group amounts to about $1871 (compared to $85.15 of those considered). 5. Acknowledgements We are grateful to Wolfgang Habla, Martin Kesternich, Florian Landis, Dirk R¨ubbelke, an anonymous editor and two anonymous referees for their helpful comments. The ﬁnancial support by the German Federal Ministry 16 EPPERSON AND REIF of Education and Research (FKZ 01UT1411A) is gratefully acknowledged. Further details can be obtained from http://kooperationen.zew.de/en/intrans/home.html. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. Adena, M. and Huck, S. (2017) Matching donations without crowding out? Some theoretical considerations, a ﬁeld, and a lab experiment. Journal of Public Economics 148: 32–42. Adena, M. and Huck, S. (2019) Giving once, giving twice: a two-period ﬁeld experiment on intertemporal crowding in charitable giving. Journal of Public Economics 172: 127–134. Notes If the treatments with different matching rates are not pooled in the regression, the match of 1 is not estimated to signiﬁcantly affect checkbook giving in the ﬁrst place. If instead they are pooled, matching is estimated to have a signiﬁcant effect on checkbook giving. The same holds for the results on the extensive margin. 6. Some authors refer to this as crowding out (Huck and Rasul, 2011; Huck et al., 2015; Adena and Huck, 2017). To avoid confusion we exclusively use this term to refer to a decrease in checkbook giving compared to a baseline without lead donor. 6. Some authors refer to this as crowding out (Huck and Rasul, 2011; Huck et al., 2015; Adena and Huck, 2017). To avoid confusion we exclusively use this term to refer to a decrease in checkbook giving compared to a baseline without lead donor. Data sharing is not applicable to this article as no new data were created or analysed in this study. Data sharing is not applicable to this article as no new data were created or analysed in this study. ﬁeld, and a lab experiment. Journal of Public Economics 148: 32–42. Adena, M. and Huck, S. (2019) Giving once, giving twice: a two-period ﬁeld experiment on intertemporal crowding in charitable giving. Journal of Public Economics 172: 127–134. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS Andreoni, J. (1989) Giving with impure altruism: applications to charity and Ricardian equivalence. The Journal of Political Economy 97(6): 1447–1458. f y ( ) Andreoni, J. (1990) Impure altruism and donations to public goods: a theory of warm-glow giving. The Economic Journal 100(401): 464–477. f y ( ) Andreoni, J. (1990) Impure altruism and donations to public goods: a theory of warm-glow giving. The Economic Journal 100(401): 464–477. ( ) Andreoni, J. and Varian, H. (1999) Preplay contracting in the prisoners’ dilemma. Proceedings of the National Academy of Sciences 96(19): 10933–10938. Bekkers, R. (2015) When and why matches are more effective subsidies than rebates. In C.A. Deck, E. Fatas, and T. Rosenblat (eds.), Replication in Experimental Economics (pp. 183–211). Bingley, UK: Emerald Group Publishing Limited. p g Bekkers, R. and Wiepking, P. (2011) A literature review of empirical studies of philanthropy: eight mechanisms that drive charitable giving. Nonproﬁt and Voluntary Sector Quarterly 40(5): 924–973. B´enabou, R. and Tirole, J. (2006) Incentives and prosocial behavior. American Economic Review 96(5): 1652– 1678. Boadway, R., Song, Z. and Tremblay, J.F. (2007) Commitment and matching contributions to public goods. Journal of Public Economics 91: 1664–1683. Bowman, W. (2006) Should donors care about overhead costs? Do they care? Nonproﬁt and Voluntary Sector Quarterly 35(2): 288–310. Bracht, J., Figui`eres, C. and Ratto, M. (2008) Relative performance of two simple incentive mechanisms in a public goods experiment. Journal of Public Economics 92(1–2): 54–90. Buchholz, W., Cornes, R. and R¨ubbelke, D. (2011) Interior matching equilibria in a public good economy: An aggregative game approach. Journal of Public Economics 95: 639–645. Buchholz, W., Cornes, R. and R¨ubbelke, D. (2011) Interior matching equilibria in a public good economy: An aggregative game approach. Journal of Public Economics 95: 639–645. Buchholz, W., Cornes, R. and R¨ubbelke, D. (2012) Matching as a cure for underprovision of voluntary public Buchholz, W., Cornes, R. and R¨ubbelke, D. (2012) Matching as a cure for underprovision of volun good supply. Economics Letters 117(3): 727–729. Charness, G., Fr´echette, G.R. and Qin, C.-Z. (2007) Endogenous transfers in the Prisoner’s Dilemma game: an experimental test of cooperation and coordination. Games and Economic Behavior 60(2): 287–306. Charness, G. and Holder, P. (2019) Charity in the laboratory: matching, competition, and group identity. Management Science 65(3): 1398–1407. Danziger, L. and Schnytzer, A. (1991) Implementing the Lindahl voluntary-exchange mechanism. European Journal of Political Economy 7(1): 55–64. Davis, D.D. References MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 17 Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS (2006) Rebate subsidies, matching subsidies and isolation effects. Judgment and Decision making 1(1): 13–22. Davis, D.D., Millner, E.L. and Reilly, R.J. (2005) Subsidy schemes and charitable contributions: a closer look. Experimental Economics 8(2): 85–106. . and Grossman, P.J. (2003) Rebate versus matching: does how we subsidize charitable contribution r? Journal of Public Economics 87(3–4): 681–701. Eckel, C.C. and Grossman, P.J. (2004) Giving to secular causes by the religious and nonreligious: an experimental test of the responsiveness of giving to subsidies. Nonproﬁt and Voluntary Sector Quarterly 33(2): 271–289. Eckel, C.C. and Grossman, P.J. (2006a) Do donors care about subsidy type? An experimental study. In R.M. Isaac and D.D. Davis (eds.), Experiments Investigating Fundraising and Charitable Contributors (pp. 157–175). Bingley, UK: Emerald Group Publishing Limited. ) g y p g Eckel, C. C. and Grossman, P.J. (2006b) Subsidizing charitable giving with rebates or matching: further laboratory evidence. Southern Economic Journal 72(4): 794–807. Eckel, C.C. and Grossman, P.J. (2008) Subsidizing charitable contributions: a natural ﬁeld experiment comparing matching and rebate subsidies. Experimental Economics 11(3): 234–252. Eckel, C.C. and Grossman, P.J. (2017) Comparing rebate and matching subsidies controlling for donors’ awareness: evidence from the ﬁeld. Journal of Behavioral and Experimental Economics 66: 88–95. Eckel, C.C., Grossman, P.J. and Milano, A. (2007) Is more information always better? An experimental study of charitable giving and hurricane Katrina. Southern Economic Journal 74(2): 388–411. F B S d J R (2001) M ti ti di th J l f E i S 15(5) 589 611 y p y of charitable giving and hurricane Katrina. Southern Economic Journal 74(2): 388–411. Frey, B.S. and Jegen, R. (2001) Motivation crowding theory. Journal of Economic Surveys 15(5): 589–611. Frey, B.S. and Jegen, R. (2001) Motivation crowding theory. Journal of Economic Surveys 15(5): 5 Gee, L.K. and Schreck, M.J. (2018) Do beliefs about peers matter for donation matching? Experiments in the ﬁeld and laboratory. Games and Economic Behavior 107: 282–297. 18 EPPERSON AND REIF Gneezy, U., Keenan, E.A. and Gneezy, A. (2014) Avoiding overhead aversion in charity. Science 346(6209): 632–635. Guttman, J.M. (1978) Understanding collective action: matching behavior. The American Economic Review 68(2): 251–255. ( ) Guttman, J.M. (2013) On the evolution of conditional cooperation. European Journal of Political Economy 30: 15–34. Helms McCarty, S., Diette, T.M. and Bugg Holloway, B. (2018) How low can you go? An investigation into matching gifts in fundraising. Review of Behavioral Economics 5: 23–37. Huck, S. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS and Rasul, I. (2011) Matched fundraising: evidence from a natural ﬁeld experiment. Journal of Public Economics 95(5–6): 351–362. Huck, S., Rasul, I. and Shephard, A. (2015) Comparing charitable fundraising schemes: evidence from a natural ﬁeld experiment. American Economic Journal: Economic Policy 7(2): 326–369. Hungerman, D. M. and Ottoni-Wilhelm, M. (2018) Impure impact giving: theory and evidence. NBER Working Paper No. 24940. Cambridge, MA: National Bureau of Economic Research. arlan, D. and List, J.A. (2007) Does price matter in charitable giving? Evidence from a large-sca ﬁeld experiment. American Economic Review 97(5): 1774–1793. p Karlan, D., List, J.A. and Shaﬁr, E. (2011) Small matches and charitable giving: evidence from a natural ﬁeld experiment. Journal of Public Economics 95(5–6): 344–350. Kass, M., Fatas, E., Eckel, C. and Arce, D. (2015) The UN in the lab. Social Choice and Welfare 45(3): 625–651. Kesternich M L¨oschel A and R¨omer D (2016) The long term impact of matching and rebate subsidies Kass, M., Fatas, E., Eckel, C. and Arce, D. (2015) The UN in the lab. Social Choice and Welfare 45(3): 625–651. Kesternich, M., L¨oschel, A. and R¨omer, D. (2016) The long-term impact of matching and rebate subsidies when public goods are impure: ﬁeld experimental evidence from the carbon offsetting market. Journal of Public Economics 137: 70–78. Knutsson, M., Martinsson, P., Persson, E. and Wollbrant, C. (2019) Gender differences in altruism: evidence from a natural ﬁeld experiment on matched donations. Economics Letters 176: 47–50. Krieg, J. and Samek, A. (2017) When charities compete: a laboratory experiment with simultaneous public goods. Journal of Behavioral and Experimental Economics 66: 40–57. Landry, C.E., Lange, A., List, J.A., Price, M.K. and Rupp, N.G. (2010) Is a donor in hand bette the bush? Evidence from a natural ﬁeld experiment. American Economic Review 100(3): 95 the bush? Evidence from a natural ﬁeld experiment. American Economic Review 100(3): 958 983. Lukas, I., Grossman, P.J. and Eckel, C.C. (2010) Preference or confusion: understanding the differential impact of rebate and matching subsidies Working Paper Indiana University Indianapolis IN Lukas, I., Grossman, P.J. and Eckel, C.C. (2010) Preference or confusion: understanding the differ of rebate and matching subsidies. Working Paper. Indiana University, Indianapolis, IN. Meer, J. (2014) Effects of the price of charitable giving: evidence from an online crowdfunding platform. Journal of Economic Behavior and Organization 103: 113–124. Meer, J. (2017) Does fundraising create new giving? Journal of Public Economics 145: 82–93. Meier, S. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS (2007) Do subsidies increase charitable giving in the long run? Matching donatio experiment. Journal of the European Economic Association 5(6): 1203–1222. C. (2011) Warm glow, information, and inefﬁcient charitable giving. Journal of Public Economics 95 455–465. Rondeau, D. and List, J.A. (2008) Matching and challenge gifts to charity: evidence from laboratory and natural ﬁeld experiments. Experimental Economics 11(3): 253–267. p p Varian, H.R. (1994) Sequential contributions to public goods. Journal of Public Economics 53: 165–186. Vesterlund, L. (2016) Using experimental methods to understand why and how we give to charity. In J.H. Kagel and A.E. Roth (eds.), Handbook of Experimental Economics, Volume 2 (pp. 91–152). Princeton: Princeton University Press. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 19 MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 19 Appendix Table A1. Short-Term Effects of Matching in Laboratory Experiments (Relative to Baseline without Lead Donor). Betw. Stated Number of Donors in Charity Checkbook Extensive Intensive subjects Matching matching subjects treatment receipts giving margin margin Paper design rate limit in treatment (%) (%) (%) (pp) (%) Comments Charness and Holder (2019) Yes 1 48 29.2 186.6d Not sig. –4.2d Subjects can donate nothing, $4, $8, $12, or $16. Subjects’ donations are either not matched, matched at a linear rate of 1, matched at a linear rate of 1 if they are not below the median donation, or matched at a rate of 1 if the amount donated by their group of 3 (to which they are anonymously allocated) is not below the median group donation. 1 if not below median 40 32.5 174.0d Not sig. –0.8d 1 if group not below median 42 28.6 240.3*d 102.1d –4.8d Davis et al. (2005) No 0.5 43 62.2d/ 8.0d/ Endowment $8/$12. Tested for signiﬁcance in Davis (2006). Treatments in which subjects make their decision by circling a row in an outcome table are not reported. 36.1d −8.3d 1 43 134.0d/ 17.0d/ 113.3d 6.7d Davis (2006) No 0.5 61 63.3*d/ 8.2a/ Endowment $8/$12. Values in squared brackets are based on data from Davis et al. (2005). Rephrases decision problem to shift focus towards amount the charity receives. Using one-sided t-tests testing whether the effect is positive. 57.1*d Not sig. [62.2d/ [8.0a/ 36.1*d] Not sig.] 1 61 132.7d/ 17.0a/ 124.5d 12.2a [134.0*d/ [17.2a/ 113.3*d] Not sig.] Eckel and Grossman (2003) No 0.25 168 Primary focus on comparing rebates and matches. Reported result is for endowment level $6. Signiﬁcantb (one-sided test) negative price elasticity of charity receipts in random effects Tobit model (−0.94). 0.33 168 32.4d 1 168 Eckel and Grossman (2006b) No 0.25 46 107.9d/ Endowment $4/$6/$7.5. Signiﬁcantb negative price elasticity of charity receipts in Tobit model (−2.59). 123.7d/ 87.2d 0.33 46 115.8d/ 142.7d/ 86.3d 1 46 347.5d/ 366.4d/ 287.7d (Continued) Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. (Continued) pp C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. 20 EPPERSON AND REIF Table A1. Continued. Betw. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS Stated Number of Donors in Charity Checkbook Extensive Intensive subjects Matching matching subjects treatment receipts giving margin margin Paper design rate limit in treatment (%) (%) (%) (pp) (%) Comments Eckel et al. (2007) No 0.25 256 34.9d/ 7.9d/ Endowment $10/$20/$50. Signiﬁcant positive effect of each matching treatment on extensive margin in random effects logit model (2.0pp,* 2.7pp* and 3.0pp* for matching rate 0.25, 0.5 and 1, respectively). Signiﬁcant positive effect of matching on checkbook giving in random effects Tobit model. 38.0d/ 10.4d/ 37.5d 10.0d 0.5 256 68.1d/ 11.9d/ 73.7d/ 15.9d/ 75.6d 17.0d 1 256 146.8d/ 23.3d/ 155.7d/ 27.9d/ 157.4d 28.7d Gee and Schreck (2018) No $50 if 1 of 10 give 100 39 15.0* Endowment $16. Binary decision to give $5 or nothing. $50 if 3 of 10 give 100 50 26.0* $50 if 5 of 10 give 100 43 19.0* $50 if 7 of 10 give 100 45 21.0* $50 if 10 of 10 give 100 42 18.0* Lukas et al. (2010) No 0.25 55 37.2d/ Not sig./ Endowment $10/$20. Effect of each matching treatment on charity receipts is signiﬁcantly positive in Tobit regression in which the matching effect is not allowed to differ by endowment. 33.1d Not sig. 0.33 55 40.0d/ 5.2d/ 54.2d 15.9d 1 55 157.1d/ 28.5d/ 163.0d 31.5d The effects on charity receipts, checkbook giving and the intensive margin are presented as relative change (in percent) using the control condition without matching subsidy as baseline. The effects on the extensive margin are presented as absolute difference in the likelihood to give between the matching treatment and the control condition (in percentage points). All experimental results which are presented in this table are based on a control condition without lead donor announcement. Matching rates of 0.33 and 1/3 are sometimes used interchangeably. If instructions are provided, we use the corresponding matching rate, otherwise, we rely on the rate stated in the paper. p < 0.01, p < 0.05, and p < 0.10 in conducted test, two-sided if not otherwise stated in the comment section. Values without an asterisk are not tested for signiﬁcance. Using regression results (including covariates) whenever applicable. aSince the tables in Davis et al. (2005) and Davis (2006) showed inconsistencies, values are based on own calculations using the data retrieved October 10, 2017, from http://www.people.vcu.edu/˜dddavis/vita.htm. bRefers to the coefﬁcient and not directly to the reported marginal effect. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS The coefﬁcient is signiﬁcant at the 5% level or better. cWe thank the author(s) for providing this information upon request. dValue is calculated by using descriptive statistics, e.g. reported means or response rates. eNot considering undelivered solicitations. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors Journal of Economic Surveys published by John Wiley & Sons Ltd C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 21 Table A2. Short-Term Effects of Matching in Field Experiments (Between-Subjects Design). Lead Stated Number of Donors in Charity Checkbook Extensive Intensive donor Matching matching subjects treatment receipts giving margin margin Paper control rate limit in treatment (%) (%) (%) (pp) (%) Comments Adena and Huck (2017) Yes 1 €30,000 6143 2.1 2.4 0.6 −23.7** 1 to diff. project €30,000 6144 1.9 1.8* 0.4* Not sig. Bekkers (2015) No 0.5 184 51.1 57.5d 15.9d 8.4d Survey based ﬁeld experiment. Checkbook giving and intensive margin are based on the fraction of the endowment passed, since endowment varies across subjects (depends on the time spend to answer the survey). 1 173 57.2 89.7d 22.0d 16.5d Eckel and Grossman (2008) No 0.25 $7500 1200/ 19.2/ −33.4d/ −46.7d/ −9.4d/ Not sig./ Continuing/lapsed/ prospect members. Intensive margin based on donors for which authors receive complete information on a set of covariates (about 1.1% of donors excluded). OLS regression results show a signiﬁcant negative price elasticity of the intensive margin for continuing members (−0.099) but not for others. 15,000/ 0.9/ 32.7d/ 5.5d/ 0.0d/ Not sig./ 30,000 0.6 56.5d 26.1d 0.1d Not sig. 1/3 $15,000 1200/ 19.8/ −28.5d/ −46.4d/ −8.8d/ Not sig./ 15,000/ 1.0/ 50.9d/ 12.7d/ 0.1d/ Not sig./ 30,000 0.4 13.0d −13.0d −0.1d Not sig. Eckel and Grossman (2017) No 0.25 $10,000 4462 3.4 38.6d Not sig. Additional $5 donated by the researchers if the survey attached to the solicitation was completed. Only individuals who returned the complete survey are considered (completion rate does not differ across all treatments). Signiﬁcant positive effect on checkbook giving, but signiﬁcance level not stated. Signiﬁcant negative price elasticity of charity receipts in Tobit model (−1.25 and −5.43 if corrected for donors’ awareness). 1/3 $15,000 4834 4.0 47.4d 8.4 (Continued) onomic Surveys (2019) Vol. 00, No. 0, pp. 1–24 Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. 22 EPPERSON AND REIF Table A2. Continued. Lead Stated Number of Donors in Charity Checkbook Extensive Intensive donor Matching matching subjects treatment receipts giving margin margin Paper control rate limit in treatment (%) (%) (%) (pp) (%) Comments Gneezy et al. (2014) Yes 1 $10,000 10,000 4.4 Not sig. Not sig. Not sig. Individuals can donate nothing, $20, $50 or $100. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS No 1 $10,000 10,000 4.4 51.9*d 1.05d 15.7*d Individuals can donate nothing, $20, $50 or $100. Gee and Schreck (2018) No 1 $3000 599 2.3 83.0d −8.6d Not sig. Not sig. Subjects attended a program operated by the organization in the past. In all nonlinear treatments, subjects are allocated to groups of 10 and additional $50 are given to the charity if at least 1, 2 or 3 subjects of the group donate. All group members attended the program in the same year, and groups are anonymous and randomly assigned. $50 if 1 of 10 give 300 2.0 17.0d −3.4d Not sig. Not sig. $50 if 2 of 10 give 298 2.4 −45.1d −45.1d Not sig. Not sig. $50 if 3 of 10 give 299 3.7 190.4d 190.4d 1.9 Not sig. Helms McCarty et al. (2018) No 0.1 2119 2.8 Not sig. Not sig. Not sig. 1 €100 per donor 2122 3.6 Not sig. Not sig. Not sig. Huck and Rasul (2011) Yes 0.5 €60,000 3745 4.2 Not sig. Not sig. −30.1 Relative effects for charity receipts are based on absolute effects in OLS regression divided by average charity receipts in lead donor control obtained from the descriptive statistics. In a corresponding Tobit model both estimated treatment coefﬁcients are signiﬁcant at the 10% level. 1 €60,000 3718 4.2 66.5 Not sig. −35.4*** No 0.5 €60,000 3745 4.2 125.8d Not sig. 35.9d 1 €60,000 3718 4.2 176.3d Not sig. Not sig. mic Surveys (2019) Vol. 00, No. 0, pp. 1–24 ors. Journal of Economic Surveys published by John Wiley & Sons Ltd. C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS 23 Table A2. Continued. Lead Stated Number of Donors in Charity Checkbook Extensive Intensive donor Matching matching subjects treatment receipts giving margin margin Paper control rate limit in treatment (%) (%) (%) (pp) (%) Comments Huck et al. (2015) Yes 0.5 €60,000 3745 4.2 Not sig. Results for matching rates 0.5 and 1 are based on same data as Huck and Rasul (2011). Only new insights reported here. €20 threshold gift means that additional €20 are given to the charity if the subject donates. 1 €60,000 3718 4.2 Not sig. 1 above €50 €60,000 3746 4.3 Not sig. 0.8d −25.8*d €20 threshold gift €60,000 3746 4.7 −29.2d 1.2*d −47.6d No 0.5 €60,000 3745 4.2 50.2d Results for matching rates 0.5 and 1 are based on the same data as Huck and Rasul (2011). Only new insights are reported here. €20 threshold gift means that additional €20 are given to the charity if the subject donates. 1 €60,000 3718 4.2 37.6d 1 above €50 €60,000 3746 4.3 49.8d Not sig. 31.8d €20 threshold gift €60,000 3746 4.7 Not sig. 1.0*d Not sig. Karlan and List (2007) No 1 Varying 11,133 2.1 130.9d Not sig. Not sig. Not sig. Pooled analysis of matching vs. control: 18.9% increase of checkbook giving and 0.4pp* increase of the extensive margin. No signiﬁcant effect on the intensive margin. Maximum matching amount is either not stated, $25,000, $50,000 or $100,000. 2 Varying 11,134 2.3 280.2d Not sig. Not sig. Not sig. 3 Varying 11,129 2.3 363.9d Not sig. Not sig. Not sig. Karlan et al. (2011) No 1/3 6714 Not sig. Not sig. Signiﬁcant positive effect on checkbook giving in post-experiment period. 1 6438 Not sig. Not sig. Kesternich et al. (2016) No 1/3 1342 26.4 Not sig. Not sig. Not sig. Binary decision of whether or not to offset carbon emissions from bus travel (in ﬁrst booking). 1 1282 31.0 126.2d 13.1d 4.9 3 1364 29.3 327.9d Not sig. 3.1 Knutsson et al. (2019) No 1 541c 14.4 4.6c Individuals can decide whether to donate the deposit received when returning cans and bottles. 3 544 16.5 6.7c 5 541 18.9 9.1c (Continued) onomic Surveys (2019) Vol. 00, No. 0, pp. 1–24 Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd. 24 EPPERSON AND REIF Table A2. Continued. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS Lead Stated Number of Donors in Charity Checkbook Extensive Intensive donor Matching matching subjects treatment receipts giving margin margin Paper control rate limit in treatment (%) (%) (%) (pp) (%) Comments Meer (2017) 1 or threshold gift Project speciﬁc 2.8* 0.8*** −1.5* Units of analysis are fundraising results of different projects on different days. Results are based on pooled analysis of a match at rate 1 and a threshold gift match, where the full project is funded if the last $100 are provided by donors. 20.8% of the more than 27 million observations include one of the two matches. The intensive margin is thus the average effect on checkbook giving, considering only those day-project observations with a positive amount. Contributions returned or reallocated to another project if project’s goal not reached. Meier (2007) No 0.25 265 Not sig.c Students face decision whether to contribute ﬁxed amounts to no, one or two funds. Only in the latter case a match is received if offered. The extensive margin refers to the likelihood of contributing to both funds. 67.9% of those receiving a match donate to both funds. 0.5 267 13.5c Rondeau and List (2008) Yes 1 $2500 750 4.8 −23.9d,e −0.1d,e Not sig. Contributions returned if threshold is not reached. No 1 $2500 750 4.8 −10.4d,e 0.8d,e Not sig. Contributions returned if threshold is not reached. Include additional control with different threshold than matching treatment, but results are not reported here. The effects on charity receipts, checkbook giving and the intensive margin are presented as relative change (in percent) using the control condition without matching subsidy as baseline. In a baseline with a lead donor, charity receipts do not include the lead donation. The effects on the extensive margin are presented as absolute difference in the likelihood to give between the matching treatment and the control condition (in percentage points). All experimental results, which are presented in this table, are based on a between-subjects design. Matching rates of 0.33 and 1/3 are sometimes used interchangeably. If instructions are provided, we use the corresponding matching rate; otherwise, we rely on the rate stated in the paper. p < 0.01, p < 0.05 and p < 0.10 in conducted test, two-sided if not otherwise stated in the comment section. Values without an asterisk are not tested for signiﬁcance. Using regression results (including covariates) whenever applicable. aSince the tables in Davis et al. MATCHING SUBSIDIES AND VOLUNTARY CONTRIBUTIONS (2005) and Davis (2006) showed inconsistencies, values are based on own calculations using the data retrieved October 10, 2017, from http://www.people.vcu.edu/˜dddavis/vita.htm. bRefers to the coefﬁcient and not directly to the reported marginal effect. The coefﬁcient is signiﬁcant at the 5% level or better. cWe thank the author(s) for providing this information upon request. dValue is calculated by using descriptive statistics, e.g. reported means or response rates. eNot considering undelivered solicitations. Journal of Economic Surveys (2019) Vol. 00, No. 0, pp. 1–24 C⃝2019 The Authors. Journal of Economic Surveys published by John Wiley & Sons Ltd.
https://openalex.org/W4327812076	https://zenodo.org/records/7747366/files/1116%20shiva%2047-50.pdf	English	null	Effects of pesticide use in agriculture and its impact on human health	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,886	ABSTRACT Going by the characterization of pesticides, they are chemical substances that are made use of to eliminate animals, insects, plant and fungal bugs in cultivation, at home as well as in institutions. Therefore, the objective of this study is to evaluate the various aspects of pesticide use in agriculture and to study the effect of pesticides use in agriculture on human health. Agricultural community was taken for this study and areas were randomly chosen. All farmers, both men and women were interviewed if they were working in an agricultural field. The most commonly used pesticides according to the sprayers were Rogar/Dimethoate (55%), Ekalux/Qunalphos (49.4 %), Endosulphan (48.5%) and Monocrotophos (45.9%). While pesticides were sprayed, non sprayers (40.1%), including women (19.3%) continued to work in the same field, which exposed them to pesticides (Table 5). Time of reference for the non-sprayers was while working in the field during or after pesticide spraying. These signs and symptoms were reported by a large number of sprayers. 373 sprayers (86.1%) and 156 (78.8%) of non-sprayers reported at least one of these signs and symptoms. Key words: Pesticides, Monocrotophos, Endosulphan, Health, Ekalux, Agriculture. Shailaja K Shailaja K 1Department of Zoology, Govt. Degree College for Women, Hussaianialam-500002, Hyderabad, TS Email : raju.matoori@gmail.com 1Department of Zoology, Govt. Degree College for Women, Hussaianialam-500002, Hyderabad, TS Email : raju.matoori@gmail.com 1Department of Zoology, Govt. Degree College for Women, Hussaianialam-500002, Hyderabad, TS Email : raju.matoori@gmail.com ISSN (online): 2320-4257 www.biolifejournals.com ISSN (online): 2320-4257 www.biolifejournals.com ISSN (online): 2320-4257 www.biolifejournals.com Effects of pesticide use in agriculture and its impact on human health Shailaja K INTRODUCTION It is important to note that the growth of the fetus is also impinged on owing to exposure to pesticides. In addition, the limits set by the regulatory bodies for the use of different pesticides too are impractical as well as dangerous [5]. Moreover, a child’s nature or behavior, like frequently putting the hand in mouth and the time they spend playing on the ground, put them at a greater danger of being exposed to the poisonous chemicals contained in the pesticides. According to a report on poisoning through insecticides, approximately 56 per cent of such cases take place in children below the age of six [6]. Exposure to pesticides both occupationally and environmentally causes a range of human health problems. It is estimated that nearly 10,000 deaths annually to use of chemical pesticides worldwide, with about three-fourths of these occurring in developing countries [1]. At present, India is the largest producer of pesticides in Asia and ranks twelfth in the world for the use of pesticides with an annual production of 90,000 tons. A vast majority of the population in India (56.7 percent) are engaged in agriculture and are therefore exposed to the pesticides used in agriculture [2, 3]. Pesticides being used in agricultural tracts are released into the environment and come into human contact directly or indirectly. Humans are exposed to pesticides found in environmental media (soil, water, air and food) by different routes of exposure such as inhalation, ingestion and dermal contact. Exposure to pesticides results in acute and chronic health problems. These range from temporary acute effects like irritation of eyes, excessive salivation to chronic diseases like cancer, reproductive and developmental disorders etc [4]. How to Cite this Article: Shailaja K (2023). Effects of pesticide use in agriculture and its impact on human health. Biolife, 11(1), 47-50. DOI: https://dx.doi.org/10.5281/zenodo.7747366 Received: 21 January 2022; Accepted: 27 February 2023; Published online: 17 March 2023. Biolife \| 2023 \| Vol 11 \| Issue 1 Biolife \| 2023 \| Vol 11 \| Issue 1 47\| © http://airaacademy.com/ Shailaja Copyright@2023 and the interviewers obtained verbal consent before proceeding with the interviews. and the interviewers obtained verbal consent before proceeding with the interviews. Specific studies dealing with the agricultural practices of the farmers regarding pesticide use and its health impacts is needed to make informed policy decisions to bring about changes in the agricultural practices in India. Table-1. Characteristics of the sample population Age group s Male Female Tota l Sprayer s Non sprayer s Sprayer s Non sprayer s <20 21-30 31-40 41-50 51-60 >60 Total 21 122 140 73 50 23 429 12 17 18 21 21 19 108 1 0 1 2 0 0 4 6 24 32 15 15 6 90 40 163 191 111 78 48 631 INTRODUCTION Therefore, we undertook a study among the farmers of Nalgonda district, Telangana to look into the various aspects of pesticide use in agriculture and its impact on human health. The main objectives of the study are to evaluate the various aspects of pesticide use in agriculture and to study the effect of pesticides use in agriculture on human health. The interviews were conducted in the local language, namely Telugu. Each interview took about 30- 45 minutes to be completed. The completed interviews were collected at the end of each day, checked, coded and stored for data entry. Data analysis The data was entered in Excel (version 4), cleaned and further data analysis was done in statistical software SPSS (version 11). Univariate and bivariate analyses were carried out. Relative Risk (RR) values were calculated followed by Chi- square test for proportions (where appropriate) and a p-value of less than 0.05 was considered statistically significant. Study area The study targeted mainly the agricultural community of Nalgonda district of the State of Telangana State. Nalgonda is ranked first in the production of rice in the state. The other crops grown in the area include betel leaf cultivation, sugarcane, cotton, some vegetables. Data collection It was decided that for the convenience of the farmers, interviews should be taken in the field, in the fore noon hours. With the help of the local government agricultural authorities, areas with higher agricultural activity (i.e. extended land areas with year round agriculture) were identified. Six areas (Chintapally, Marriguda, Chandur, Munugode, Chityal, Kanagal) were randomly chosen for conducting the survey. All farmers, both men and women were interviewed if they were working in an agricultural field. On encountering farmer/s working in a particular field, the farmers were informed about the purpose of the study Commonly used pesticides Interview questionnaire The primary exposure status to pesticides was ascertained based on whether or not the farmer sprayed pesticides. Out of the 631 farmers interviewed, 433 reported that they sprayed pesticides by themselves (we refer to them as “sprayers”). The remaining 198 were involved in other agricultural activities (like weeding, replanting, watering etc) (we refer them as “non-sprayers”). Majority of sprayers were men (429, 79.9%). The average age of farmers was 38.9 years (Table 1). 23.5% of farmers received no formal schooling. 207 farmers (32.8%) were currently working on betel leaf plantation; 143 (22.6%) were working on paddy fields and the rest were involved in cultivation of other agricultural products such as brinjal, banana and sugarcane etc (Table-1). The interview questionnaire was designed to elicit details on land ownership, plantation where the farmer is currently working, exposure to pesticides, the use of pesticides, the commonly used pesticides, precautions taken, the source of information, signs and symptoms of illnesses related to pesticide exposure etc. Some signs like, tremors, skin lesions, wheezing which can be chronic in nature were observed at the time of the interview. Being a cross- sectional survey, details on signs and symptoms were collected as self reported by the farmers. The questionnaire was pilot tested in an adjacent panchayat the feedback from the interviewers and some respondents were considered and the questionnaire was revised to improve the data gathering ability of the same. Data collected was with reference to the time of pesticide spraying. Information source for the farmers Sprayers used mainly the retail shop owner as the information source for knowledge regarding the pesticides they used (56.1%). 35.3% of farmers consulted other farmers and only 9.5% of sprayers considered government or any other agricultural authorities as their source of information. Duration of spraying pesticides Other farming activities during pesticide spraying 37.3% farmers (156 sprayers and 77 non sprayers) reported that other farming activities continued in the farm while pesticides were sprayed. While pesticides were sprayed, non sprayers (40.1%), including women (19.3%) continued to work in the same field, which exposed them to pesticides (Table 2). The period of exposure ranged from 10 year to 15 years with an average duration of 11.8 years. A majority of them, 255 (58.9%), have been spraying pesticides for the past ten years. 203 farmers (46.9%) had sprayed some kind of pesticides within a month. The characteristic features of samples population is described in Table-1. Commonly used pesticides The most commonly used pesticides according to the sprayers were Rogar/Dimethoate (55%), Ekalux/Qunalphos (49.4 %), Endosulphan (48.5%) and Monocrotophos (45.9%). 7.7% of the sprayers used pesticides which are extremely hazardous. Rogar, Ekalux 8 \|© 2023 AIRA PUBLISHERS Biolife \| 2023 \| Vol 11 \| Issue 1 48 Shailaja Copyright@2023 and Endosulfan is classified as moderately hazardous by WHO (2004), while Monocrotophos is classified as highly hazardous. Only a very few farmers (43 ie. 0.2%) used the pesticides in the class III (Slightly hazardous). The retail shop owners were the major source of information regarding usage of pesticides (56.1%). using bare hands. The common alternative to this practice was to use a stick. Use of gloves was 2.7%, while a few of them also used old cotton cloths as masks. While spraying, 147 sprayers (33.9%) chewed either tobacco or gum and 16 (3.7%) of them smoked. Table-3: Signs and symptoms of illness among study population (as percentage) Signs and Symptoms Sprayers (n-433) Non sprayers (n=198) Overall (n=631) Excessive sweating Burning/Stinging/ Itching eyes Sore throat Fatigue Dizziness Skin redness/white patches Numbness/muscle Weakness Blurred vision Chest pain Cough Excessive salivation Nausea/vomiting Stomach pain Diarrhoea 38.6 37.6 27.3 26.3 25.6 21.9 21.7 18 16.2 15.7 15 14.8 7.4 6 5.3 31.8 31.8 21.7 39.4 34.3 1.7 28.8 13.1 29..3 28.8 23.2 12.6 11.6 16.7 10.1 36.5 35.7 25.5 30.4 28.4 20.6 23.9 16.5 20.3 19.8 17.6 14.1 8.7 9.4 6.8 Factors affecting indirect exposure to pesticides Table-3: Signs and symptoms of illness among study population (as percentage) Table-2. Formers and continuation of farming activities while spraying pesticides Sprayers (n=433) No. (%) Non sprayers (n=192) N0. (%) Total (n=625) No. (%) Male 155 (35.8) 40 (20.8) 533 (85.3) Female 1 (0.2) 37 (19.3) 92 (14.7) Total 156 (36) 77 (40.1) 625 (100) Information source for the farmers Table-2. Formers and continuation of farming activities while spraying pesticides References [1] Horrigan L, Lawrence RS, Walker P. How sustainable agriculture can address the environmental and human heath harms of industrial agriculture. Environ Health Perspect. 2002. 110(5): 445-456. p ( ) [2] Gupta P K. Pesticide exposure – Indian scene. Toxicology. 2004. 198: 3-90. gy [3] Goverment of India. Tenth five-year plan: 2002-2007. Planning Commission of India, New Delhi . 2001. 513 – 566. Most farmers in our study were not aware of the health hazards caused by the inappropriate handling of pesticides. The use of cotton apparel as protective clothing was common among them. Studies show that wet cotton clothing and cotton cloth masks in fact increase the person’s personal absorption rate of pesticides [8]. The practise of chewing or smoking while spraying “to reduce the nauseating feeling” is also hazardous to health. This may also indicate that the farmers were symptomatic enough to self-medicate during a pesticide spraying session. But many are unwilling to follow the necessary precautions attributing non-availability and high cost of personal protection products, and the prevailing hot and humid weather conditions. [4] Yassi, A, Kjellstrom. T, Kok.T.K., Gudotli.T.L. Basic Environmental Health, World Health Organozation. Oxford University Press. 2001. [5] Ware, G.W.1994. The Pesticide Book, 4th edition. Fresno, CA: Thomson Publications. [6] Papendick RI, Elliott LF, and Dahlgren RB, Environmental consequences of modern production agriculture: How can alternative agriculture address these issues and concerns? American Journal of Alternative Agriculture, 1986. 1(1): 3-10. [7] FAO. Guidelines for personal protection when working with pesticides in tropical climate. 1990. Food and Agriculture Organization of the United Nations, Rome. [8] [8] Kishi M, Hirschhorn N, Djajadisastra M, Satterlee LN, Strowman S, Dilts R. Relationship of pesticide spraying to signs and symptoms in Indonesian farmers. Scand J Work Environ Health 1995. 21:124-133. These reasons were similar to experiences of other developing countries like Indonesia {9, 10]. Combining more than one pesticide together, many of which are duplicates (different trade names but the same common name and thus the same active ingredient) should be discouraged. This could be a dangerous concoction, because mixing of pesticides can alter their chemical properties, thereby increasing its detrimental effects. The combination of use of hazardous pesticides and the absence of appropriate precautions are detrimental to the farmers’ health [11, 12, 13]. [9] Antonella F, Bent Iverseb, Manuela Tiramani, Sara Visentin, Marco Maroni. Preventing health risks from the use of pesticides in agriculture. 2001. Signs and symptoms of illness among farmers Handling of pesticide concentrations and application of diluted formulation requires the use of appropriate personal protection equipment as a precaution against pesticide exposure. This would include the use of gloves, masks, protective clothes, personal hygiene, appropriate footwear, head gear etc., as indicated in the respective pesticide labels [7]. The sprayers in our study did not take necessary personal protective measures while handling pesticides. The signs and symptoms related to pesticide exposure was included in the questionnaire. The sprayers were asked whether they experienced these signs and symptoms during or immediately after pesticide spraying. Time of reference for the non-sprayers was while working in the field during or after pesticide spraying. These signs and symptoms were reported by a large number of sprayers. 373 sprayers (86.1%) and 156 (78.8%) of non-sprayers reported at least one of these signs and symptoms (Table- 2). 382 sprayers (88%) reported that they took no precaution while handling and spraying pesticides. 244 (56.4%) farmers made a cocktail of different kinds of pesticides before spraying. The farmers mixed the different pesticides in a vessel with water or they poured it directly into the spraying can and then mixed the pesticides in the spraying can itself. 117 (27%) mixed or diluted pesticides Some of the signs and symptoms with a higher prevalence were excessive sweating (36.5%), eyes/stinging eyes/itching eyes (35.7%), fatigue (30.4%), dizziness (28.4%), dry/sore throat (25.5%) and numbness/ muscle weakness/ muscle cramps (23.9) (Table-3). As these signs and symptoms are self reported by the farmers, it was 9 \|© 2023 AIRA PUBLISHERS 49 Biolife \| 2023 \| Vol 11 \| Issue 1 Shailaja Copyright@2023 from synthetic pesticides to bio pesticides and organic farming. from synthetic pesticides to bio pesticides and organic farming. difficult to discriminate and confirm the occurrence of a specific sign or symptom, like excessive sweating, to pesticide exposure or to other environmental factors like hot weather. Conflicts of Interest The other signs and symptoms with Relative Risk values were fatigue (1.02), dizziness (1.1), skin redness/white patches on skin/skin scaling (1.46), runny/ burning nose (1.64), shortness of breath/cough (1.06), excessive salivation (1.46), nausea/ vomiting (1.34) and wheezing (1.38). 75% (473) farmers were free from any chronic diseases like diabetes, hypertension, asthma, tuberculosis etc. 14 (2.2%) farmers suffered from asthma. 10 (1.5%) farmers suffered from diabetes, 16 (2.5%) from hypertension, 5 farmers (0.8%) from tuberculosis and 88 (13.9%) reported reduced vision. The other signs and symptoms with Relative Risk values were fatigue (1.02), dizziness (1.1), skin redness/white patches on skin/skin scaling (1.46), runny/ burning nose (1.64), shortness of breath/cough (1.06), excessive salivation (1.46), nausea/ vomiting (1.34) and wheezing (1.38). 75% (473) farmers were free from any chronic diseases like diabetes, hypertension, asthma, tuberculosis etc. 14 (2.2%) farmers suffered from asthma. 10 (1.5%) farmers suffered from diabetes, 16 (2.5%) from hypertension, 5 farmers (0.8%) from tuberculosis and 88 (13.9%) reported reduced vision. Authors declare that there is no conflict of interests regarding the publication of this paper. References Protecting Workers’ health series. Edited by International Centre for Pesticide Safety, WHO, Geneva. [10] Amer M, Metwelli M, Abu El- Magd Y. Skin diseases and enzymatic antioxidant activity among workers exposed to pesticides. Eastern Mediterranean Health Journal. 2002; 2, 241. [11] Salameh R P, Baldi I, Brochard P and Saleh B A. Pesticides in Lebanon: a knowledge, attitude and practise study. Environmental Research. 2004. 94:1-6. CONCLUSION [12] WHO. 2004. The WHO recommended classification of pesticides by hazard and guidelines to classification: 2004. World Health Organization. This study concludes that, the farmers experienced a variety the signs and symptoms related to pesticide. Among men, the prevalence of signs and symptoms related to pesticide exposure was higher among the sprayers. In contrast, the higher percentage of some signs and symptoms among the non-sprayers could be due to their “direct” exposure to pesticides or due to previous exposure to pesticides. Awareness needs to be created on use of personal protective measures among farmers, while handling pesticides. Farmers needs to be encouraged to reduce, if not eliminate the use of pesticides, with the introduction of incentives to the farmers to help them shift g [13] Gurrapu, S., & Mamidala, E. (2016). Medicinal plants used by traditional medicine practitioners in the management of HIV/AIDS-related diseases in tribal areas of Adilabad district, Telangana region. The Ame J Sci & Med Res, 2(1), 239-245. Biolife \| 2023 \| Vol 11 \| Issue 1 Biolife \| 2023 \| Vol 11 \| Issue 1 50 \|© 2023 AIRA PUBLISHERS 50
W946167242.txt	https://periodicos.uem.br/ojs/index.php/ActaSciLangCult/article/download/6562/6562	pt		As dinâmicas do poder	Acta Scientiarum. Language and Culture	2,010	cc-by	1,010	DOI: 10.4025/actascilangcult.v32i1.6562 As dinâmicas do poder KING-ARIBISALA, Karen. The Hangman’s Game. Leeds: Peepal Tree Press, 2007. 192 p. ISBN 9781845230463. Thomas Bonnici Universidade Estadual de Maringá, Avenida Colombo 5790, 87020-900, Maringá, Paraná, Brasil. E-mail: bonnici@wnet.com.br Uma das características contemporâneas da literatura em língua inglesa é o grande número de obras literárias escritas por autoras e autores oriundos das ex-colônias britânicas, as quais reinterpretam o relacionamento colonizadorcolonizado a partir de uma perspectiva de desconstrução e de resistência. O Caribe, incluindo a Guiana, mostra-se muito profícuo na literatura e na teoria pós-coloniais e se destaca mais de que outras regiões e outros países que foram parte do Império britânico. A publicação de The Hangman’s Game [O jogo do carrasco], da guianense Karen King-Aribisala (outras obras, Our Wife and Other Stories e Kicking Tongues), ainda sem tradução para o português, mostra o potencial literário da Guiana após os sucessos de Wilson Harris, David Dabydeen, Mark McWatt e Pauline Melville. Indagando as razões da escravidão africana na Guiana, a jovem narradora negra guianense em The Hangman’s Game vai à Nigéria, país de onde milhões de africanos foram arrancados de suas aldeias para trabalharem nas fazendas europeias de cana-deaçúcar no Novo Mundo, e escreve um romance hipodiegético intitulado Three Blind Mice, enquanto está narrando as vicissitudes dela e de sua família na Nigéria sob o regime ditatorial (de Sani Abacha) nos anos 1990. Three Blind Mice (cantiga tradicional inglesa) reescreve a ‘história’ da última rebelião dos escravos na Demerara, Guiana, em 1823, antes da Emancipação, analisada, entre outros, por Emília Viotti da Costa em Coroas de Glória, Lágrimas de Sangue. A ligação entre estes dois períodos (1823 e a década de 1990), os dois países (Guiana e Nigéria) e os dois eventos (a resistência dos escravos e a resistência contra a ditadura nigeriana) revela o anseio histórico das populações colonizadas para tornar-se livres de qualquer forma de tirania. Na camada metaficcional, a narradora mostra como a escritora tenta controlar (CONTROL é a palavra-chave no romance cujo título alude a uma brincadeira infantil de formação de palavras) as suas personagens e a rebeldia destas para executarem o papel que ela quer lhes atribuir no romance Acta Scientiarum. Language and Culture hipodiegético. As sete personagens da ‘história’ guianense (Auntie Lou, a curandeira; Quamina, o chefe da rebelião; Rosita, a escrava; o pastor John; sua esposa Mary, a narradora da história hipodiegética; o governador inglês Murrain; o capataz McTurkeyen) são emparelhadas a sete personagens na ‘história real’ da narradora. Além de The Hangman’s Game ser um romance pós-moderno que aprofunda a autorreflexividade sobre a escrita da ficção, que envolve o absurdo, a frivolidade, a gravidade das situações e as condições ambíguas da rebelião e dos sujeitos fragmentados, a permeabilidade entre eventos históricos e contemporâneos constituem uma metonímia do funcionamento do poder. Contrariando a história oficial na qual se destacam, sem rival, os negros rebeldes contra o colonialismo britânico, King-Aribisala manipula as personagens para mostrar o poder feminino no contexto da rebelião escrava de Demerara. Se Auntie Lou percebe o lado sórdido do governador britânico e subverte a sua posição aparentemente intocável, Rosita fragiliza o pastor John Smithers pela sedução. Os ‘assaltos’ dissimulados contra a autoridade civil e religiosa revelam-se muito mais devastadores em seu efeito moral da colônia do que a violência física deflagrada por Quamina e os escravos rebeldes. Esta dinâmica do poder se repete na Nigéria contemporânea onde a dissimulação subversiva da população solapa o poder de ‘Butcher Boy’ e do regime militar instalado. King-Aribisala mostra, por outro lado, que o poder feminino pode ser também ambíguo e facilmente aniquilado por fatores mesquinhos. The Hangman’s Game retoma várias discussões que remetem principalmente a Fanon, Arendt e Ashcroft sobre a viabilidade da violência e o exercício do poder pelos subalternos. Parece que King-Aribisala controla melhor a ficcionalidade da subversão do poder na Demerara do que o faz no caso do regime ditatorial nas ex-colônias africanas. Neste caso, verifica-se que a estratégia de dissimulação para enfrentar o ‘neocolonizador’ torna-se menos eficiente, enquanto a Maringá, v. 32, n. 1, p. 145-146, 2010 146 construção de comunidades, tipicamente feminina, é anulada pela truculência masculina. Esta dinâmica do poder se manifesta na fragilidade do controle do narrador sobre suas personagens. Além disso, pelas condições descritas na Guiana e na Nigéria, fica subliminar (o que não quer dizer inútil ou supérfluo) o propósito do romance hipodiegético, ou seja, as razões da escravidão e do tráfico negreiro. A condição quase velada não tolhe, porém, a sua força, visto que todo o contexto ficcional cria um jogo de sujeitos transformados em objetos, o qual revela a resposta à pergunta inicial. De fato, o mérito do romance multiestruturado de King-Aribisala se encontra no leitmotiv da polaridade sujeito-objeto no qual metonimicamente se verifica que qualquer hierarquização é fadada ao fracasso. Diferente da ficção sul-africana contra o regime de apartheid em que os autores, geralmente brancos, se aliam aos colonizados, o romance guianense revela sinais de reviravolta esperançosa no meio da universalidade da violência. Numa recente entrevista, King-Aribisala afirmou: “Percebo que é muito importante dialogar com a história, com um período, lugar e Acta Scientiarum. Language and Culture Bonnici comunidade específicos, para que possamos investigar os problemas que, mais do que nos divide, nos unem, ou seja, o sofrimento da mente, do corpo, da psique, que constitui todas as formas de escravidão. Este diálogo favorece uma espécie de processo simpático de sofrimento compartilhado e revela que, como comunidade humana, temos os recursos para aliviar, ou até acabar com, nosso sofrimento; e que temos a capacidade de assegurar a liberdade em qualquer nível se percebermos nós mesmos como um poder coletivo”. Como The Hangman’s Game é um romance multinível, a sua leitura é uma experiência do inter-relacionamento nem sempre harmonioso entre o pós-colonial e o pós-moderno. Received on March 10, 2009. Accepted on March 13, 2009. License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Maringá, v. 32, n. 1, p. 145-146, 2010
https://openalex.org/W4205738737	https://essuir.sumdu.edu.ua/bitstream/123456789/86688/1/Aljaloudi_Evaluation_Of_The.pdf	English	null	Evaluation Of The Decentralization Experience In Jordan By Local Stakeholders	Socioeconomic challenges	2,021	cc-by	12,678	Evaluation Of The Decentralization Experience In Jordan By Local Stakeholders https://doi.org/10.21272/sec.5(4).55 73.2021 Prof. Dr. Aljaloudi, Jameel, ORCID ID: https://orcid.org/0000-0002-2924-4119 Department of Planning and Project Management, College of Business, Al-Balqa Applied University, Jordan Prof. Dr. Abu Zaid, Mohammad, ORCID ID: https://orcid.org/0000-0002-6320-9878 Department of Planning and Project Management, College of Business, Al-Balqa Applied University, Jordan Mr. Alfauri, Moath, ORCID ID: https://orcid.org/0000-0003-0021-8264 Arab medical Center Amman Jordan SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 Abstract The study aims to evaluate Jordan’s experience in decentralization at the regional and local levels by heads and members of provincial councils, municipal councils and opinion leaders. This evaluation includes the three dimensions of decentralization (political, financial and administrative). For evaluation purposes, two questionnaires were designed. The first one for the governorates and the second one for the municipalities. The questionnaires were judged by academic experts and verified for credibility. The sample size was (578) and distributed as follows (141) at the governorate level, (370) at the municipality level, and the rest belong to opinion leaders in the communities. The statistical program SPSS was used to enter and analyze the data. The descriptive statistical method was used, represented by the arithmetic mean and standard deviation. The t-test was used to test the validity of the null hypotheses at a level of significance (0.05). The finding indicates that there is a moderate extent of the implementation of political, fiscal, and decentralization in Jordan at regional with a mean variable (3.13), (2.70), and (3.10), and with the estimated standard division was (1.10), (1.08), and (0.98) respectively. It indicates also a moderate extent of decentralization in all dimensions (political, fiscal, and administrative) at Municipality level with a mean variable (2.62), (2.98), and (2.84), and with the estimated standard division was (1.20), (1.026), and (1.037 ) respectively . The statistical tests for all the main and sub- hypotheses confirmed the acceptance of the null hypothesis, which means that the degree of decentralization in all its three dimensions (political, financial and administrative) is low in Jordan. The study recommended that decision-makers should review the experience and amend the laws governing decentralization in order to ensure more decentralization in its political, financial and administrative dimension. Keywords: state and local government, public administration, decentralization in Jordan. Cite as: Aljaloudi, Jameel, Abu Zaid, Mohammad, Alfauri, Moath (2021). Evaluation Of The Decentralization Experience In Jordan By Local Stakeholders. SocioEconomic Challenges, 5(4), 55-73. Cite as: Aljaloudi, Jameel, Abu Zaid, Mohammad, Alfauri, Moath (2021). Evaluation Of T Experience In Jordan By Local Stakeholders. SocioEconomic Challenges, 5(4), 55-73. h //d i /10 21272/ 5(4) 55 73 2021 4).55-73.2021. Accepted: 30.11.2021 https://doi.org/10.21272/sec.5(4).55-73.2021. Received: 04.10.2021 Accepted: 30.11.2021 Published: 30.12.2021 Received: 04.10.2021 Copyright: © 2021 by the authors. Licensee Sumy State University, Ukraine. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). 55 55 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 1. Introduction 1. Jordan is a unified state based on a high degree of political, administrative and financial centralization with a two-tiered system of subnational government (the 12 governorates, and the 100 municipalities). Each governorate is headed by a governor appointed by the king through the Ministry of Interior, who works alongside the decentralized directorates of the executive ministries as an extension of the central government. They are administrative units directly linked to the Ministry of Interior. As for the municipalities, they enjoy some financial and administrative independence (Municipality Law, 2015). It is subject to the supervision of the Ministry of Municipal Affairs, except for the Greater Amman Municipality and the Aqaba Special Economic Zone. The central level still provides all basic services to the citizen including water, electricity, gas, sewage, primary education, health care, among others. As for the municipalities, they have traditionally played a marginal role in providing local services to the citizen due to the weakness of the political authority, the lack of qualified human and non-human resources, and the insufficiency of funding resources. As a result, a number of the municipal functions stipulated in the Municipal Law 2015 are performed by central government units rather than by municipalities. The application of this central model led to the following: The application of this central model led to the following: ➢ The failure of successive governments to provide public services to citizens in an unfair manner (increasing regional disparity). regional disparity). ➢ The inadequacy of development projects to the needs and desires of citizens in the governorates. ➢ The inadequacy of development projects to the needs and desires of citizens in the g equacy of development projects to the needs and desires of citizens in the governorates. pular participation in decision-making and contribution to regional and local development ➢ Weak popular participation in decision-making and contribution to regional and loca ➢ Increasing the regional variation in unemployment and poverty rates. ➢ Increasing the public financial burden of the central government (such as an increase in the public budget deficit, an increase in public indebtedness and an increase in tax burden). ➢ Increasing the public financial burden of the central government (such as an increase in the public budget deficit, an increase in public indebtedness and an increase in tax burden). 1. Introduction 56 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 As well as the answer to the sub-questions related to the three dimensions of decentralization, namely: As well as the answer to the sub-questions related to the three dimensions of decentralization, namely: hat extent do stakeholders evaluate the experience of political decentralization in Jordan? 1. To what extent do stakeholders evaluate the experience of political decentralization in Jordan? 2. To what extent do stakeholders evaluate the experience of administrative decentralization in Jordan? 3. To what extent do stakeholders evaluate the experience of financial decentralization in Jordan? 2. To what extent do stakeholders evaluate the experience of administrative decentralization in Jordan? o what extent do stakeholders evaluate the experience of administrative decentralization in o what extent do stakeholders evaluate the experience of financial decentralization in Jorda p hat extent do stakeholders evaluate the experience of financial decentralization in Jordan? 3. To what extent do stakeholders evaluate the experience of financial decentralization in The importance of this study is that it deals with a very important issue in Jordanian society, and its findings and recommendations are believed to contribute to assisting Jordanian legislators and decision-makers when amending the 2015 decentralization law. Its importance from an academic point of view is that it helps researchers understand and develop the Jordanian experience . This study consists of the introduction, the theoretical framework and previous studies in the second part, the methodology in the third part, the decentralized model according to the 2015 decentralization law in the fourth part, the analysis of the output and recommendations in the fifth part, and the conclusion and recommendation in sixth section. 1. Political Will: There must be a strong political will from all leaders at all levels in society that supports decentralization reform. 1. Theoretical Framework and Field Studies 1.1 Theoretical Framework 1. (The World Bank, 2013) defines Decentralization as “transfers authority and responsibility of major government functions from central to sub-national governments — including local governments, civil society, and the private sector”. The United Nations Development Program ( UNDP, 1997) defines decentralization also as following: Decentralization, or decentralizing governance, refers to the restructuring or reorganization of authority so that there is a system of co-responsibility between institutions of governance at the central, regional and local levels according to the principle of subsidiarity, thus increasing the overall quality and effectiveness of the system of governance, while increasing the authority and capacities of sub-national levels”. Decentralization could also be expected to contribute to key elements of good governance, such as increasing people's opportunities for participation in economic, social and political decisions; assisting in developing people's capacities; and enhancing government responsiveness, transparency and accountability. For researchers in their scientific papers, decentralization usually means the transfer of powers from centralization government to lower levels in the political, administrative, and territorial hierarchy ( Crook and Manor 1998, Agrawal and Ribot 1999). Some believe that the transfer of power takes two main forms: administrative decentralization and political or democratic decentralization (the transfer of power to representative actors and downward accountability, such as Elected Local Governments (Rebot, 2002). Decentralization can take many forms, such as: Devolution, Delegation, Deconcentration, or Divestment/Privatization. Researchers postulate three basic dimensions to the concept of decentralization. They are: political decentralization, administrative decentralization and financial decentralization. Adopting of a decentralization model (Schneider, A., 2003) (financial, administrative and political) mostly leads to: improving efficiency (or economic benefits), improving political and financial accountability and improving effectiveness. At the same time, it is likely to lead to the following negative effects: macroeconomic instability, a decline in investment in social infrastructure, - an increase in inequality and horizontal conflicts, a breakdown of the safety net (poverty), and any increase in corruption in regional and local bodies. The sum of these effects depends on choosing a model of decentralization that is commensurate with the political, economic and social conditions in the state. Continuous work on evaluating and updating it in a way that maximizes the positives and avoids the negatives resulting from its application. The application of a decentralized model with its dimensions (political, administrative and financial) that ensures maximizing the positive effects and minimizing the negative effects requires verifying the availability of the following prerequisites: 1. 1. Introduction The issuance of the decentralization law in 2015 was the result of the following: ➢ Royal directives in 2005 on the need for political and administrative reform. Implementation of Jordan's vision – 2025. p ➢ Assisting foreign donor countries in building an appropriate decentralization model and benefiting from their experiences in this field. ➢ Assisting foreign donor countries in building an appropriate decentralization model and benefiting from their experiences in this field. According to this law, an elected council was formed for each governorate and granted development, investment and oversight powers. Giving governors additional powers in the field of regional planning and development. It is believed that this decentralized model contributes to a better fight against unemployment and poverty. To ensure the quality of public services and their suitability to the needs of citizens. Stimulate the regional economy, and achieve regional development based on strong popular participation. After the provincial elections were held in 2017, and these councils exercised their duties, the urgent need to amend the current model emerged after the royal meeting with the heads of the provincial councils and the royal directive of the central government to amend the Decentralization Law 2015 and the Municipal Law 2015. As a result, the central government reformed through the preparation of a draft local administration law. The draft of Local Administration Law has been prepared by the central government to replace the 2015 Decentralization Law and the current Municipalities Law 2015. It presented by central government to the legislative authority for discussion and approval in summer 2021. This urged without any participation of members of the elected councils and opinion leaders in the governorates to express their opinion on this draft law. This study aims to evaluate the decentralization model in Jordan by the heads and members of the elected councils, and the opinion leaders in the governorates, by conducting a sample survey. The study will answer the following main question: To what extent do stakeholders evaluate the decentralization experience in Jordan? To what extent do stakeholders evaluate the decentralization experience in Jordan? 2.2 Field Studies Al-Jaloudi (2021) measured the degree of fiscal decentralization in Jordan by estimating the indicators used by the World Bank and the International Monetary Fund. It is represented by the share of local units in public revenue and public spending share and share Compensation of workers in local units from the total Compensation for public sector workers. The study uses data on public revenue and central expenditures government and independent government units, as well as municipal budget numbers. These data covered the period 2016-2018 and were published electronically by the Ministry of Finance and the Ministry of Local Administration in Jordan. He found that the degree of financial decentralization is very low in Jordan, especially when compared to other countries that have implemented a decentralization reform. (Abu Jabal ,2020) dealt with the study of the extent to which the administrative independence of the elected governorate councils in Jordan has been achieved, which was stipulated in the Decentralization Law 2015 on the one hand and in light of the method used in its formation on the other hand. He used the descriptive analytical approach, by reviewing the opinions of the jurists regarding administrative decentralization and its pillars and elements, and dropping the element related to the method of forming regional decentralized administrative units on the status of the governorate councils in Jordan, and then analyzing the legal texts related to this topic that were contained in the Jordanian legislation. With the possibility of presenting some comparative models of this idea with other legal systems, but within a specific scope. He concluded that these councils do not enjoy financial and administrative independence, as they remain under the influence of central government intervention through its authority to appoint (15%) of its members. Accordingly, the researcher recommended the necessity of amending the law of decentralization to ensure the direct election of all council members. (Al-Sarhan, 2020) analyzed the results of the elections at the governorate level that were held in 2017, based on the decentralization law. He concluded that the percentage of participation in the elections was very low, estimated at (31.7%), and to much lower in the predominantly urban governorates such as (Amman and Zarqa), where it was estimated (17.6% and 20.1%), respectively. The share of women was (13.9%) of the total number of seats in those councils. 1. Theoretical Framework and Field Studies 1.1 Theoretical Framework Political Will: There must be a strong political will from all leaders at all levels in society that supports decentralization reform. here must be a strong political will from all leaders at all levels in society that support eform. 57 57 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 2. Legal framework: Preparing an appropriate legal framework for decentralization, which clearly defines the tasks of each of the regional and local bodies and their financial sources. It also describes their rights and responsibilities to central authorities, local communities, courts and other important stakeholder. 2. Legal framework: Preparing an appropriate legal framework for decentralization, which clearly defines the tasks of each of the regional and local bodies and their financial sources. It also describes their rights and responsibilities to central authorities, local communities, courts and other important stakeholder. 3. Institutional structures: The organizational structures of official bodies (central, regional and local) must be aligned with their new functions. In addition to the need to carry out training and continuous technical support for the employees of these institutions to enable them to carry out their new responsibilities efficiently and effectively. 4. Ensuring the oversight of the central government and people’s organizations: Finding mechanisms and systems that guarantee the oversight of the central government and others that guarantee the oversight of civil society institutions and private sector institutions. These would ensure that regional and local institutions provide subnational public services to citizens and investors in a satisfactory, effective and low- cost manner. 4. Ensuring the oversight of the central government and people’s organizations: Finding mechanisms and systems that guarantee the oversight of the central government and others that guarantee the oversight of civil society institutions and private sector institutions. These would ensure that regional and local institutions provide subnational public services to citizens and investors in a satisfactory, effective and low- cost manner. 2.2 Field Studies The researcher concluded that the hypothesis of the study was not accepted: that the decentralization elections enhance the level of political participation in the management of local affairs. The decline in popular participation was attributed to the poor economic conditions suffered by the citizen and the lack of citizen confidence in the elected national parliaments in the past years. (Al-Tarwana, 2020), conducted a study on the participation of women in provincial and municipal councils, using the descriptive analytical method. It became clear to him that the participation of women in the regional and municipal elections that took place in 2017 was significant and important. The reason for this was attributed to the fact that the law included a certain share (25%) in those councils. He recommended maintaining the percentage of women's representation in councils and the need for governments to continue educating communities in governorates and municipalities of the importance of the role of women in political participation. Taamneh.M et al. 2019 evaluated the decentralization experience in Jordan by providing an empirical test of the triple model of decentralization (political, administrative and financial) developed by Schneider (2003) and by using descriptive and inferential analyses. Sample survey was adopted. The sample size was (502) employees working in official departments at the governorate level, Al-Jaloudi (2021) measured the degree of fiscal decentralization in Jordan by estimating the indicators used by the World Bank and the International Monetary Fund. It is represented by the share of local units in public revenue and public spending share and share Compensation of workers in local units from the total Compensation for public sector workers. The study uses data on public revenue and central expenditures government and independent government units, as well as municipal budget numbers. These data covered the period 2016-2018 and were published electronically by the Ministry of Finance and the Ministry of Local 58 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 municipalities and civil society organizations in the northern governorates of Jordan (Irbid, Ajloun, Jerash and Mafraq). municipalities and civil society organizations in the northern governorates of Jordan (Irbid, Ajloun, Jerash and Mafraq). The researchers found that the degree of decentralization in Jordan was estimated at a mean (3.12) and a standard deviation of 0.581. 2.2 Field Studies As for each indicator, the average score was estimated at (3.33) for the political indicator, (3.11) for the financial indicator, and (2.95) for the administrative indicator. As a result, researchers believe that the local administration in Jordan still depends mainly on the local arms of the central authority. The researchers found that the degree of decentralization in Jordan was estimated at a mean (3.12) and a standard deviation of 0.581. As for each indicator, the average score was estimated at (3.33) for the political indicator, (3.11) for the financial indicator, and (2.95) for the administrative indicator. As a result, researchers believe that the local administration in Jordan still depends mainly on the local arms of the central authority. (Jordan Economic and Social Council 2019), worked on reviewing the governance and management system at the governorate level, which was prepared in accordance with the Decentralization Law No. 49 for the year 2105. It also evaluated its implementation in its first phase after the election of the governorate councils in 2017. This council called for the necessity of exerting efforts to formulate a vision and a coherent strategic policy on the decentralization process in Jordan. In light of this, it presented three possible options for structuring the governance and public administration system at the governorate level: • The first option is to keep the system. • The first option is to keep the system. he second option: establishing a local authority with full conditions at the governorate level • The second option: establishing a local authority with full conditions at the gover • The third option: the establishment of a joint authority between the municipalities. The Council recommended the necessity of preparing a national program for the implementation of decentralization under the umbrella of the Prime Ministry, in order to review the legislation governing decentralization, institutional development and capacity building at the local level. (Al-Khawaldeh, S., 2019) dealt with identifying the nature of the constitutional and legal framework regulating the work of the provincial councils (their function and the method of electing and appointing its members). This study concluded that the constitutional and legal framework regulating the work of the governorate councils in Jordan is represented by the Jordanian Constitution of 1952 and its amendments, and the Decentralization Law No. 49 of 2015 AD. 2.2 Field Studies The governorate council is an elected council, (85%) of its members are directly elected, provided that 10% is for women) and the rest (15%) is appointed by the central government (5%) of which is for females. In 2017, the provincial councils were elected for the first time. The study concluded that the governorate councils worked to expand the base of popular participation in local governance. The researcher recommended that all members of governorate councils should be elected directly by citizens, and that the powers and tasks of governorate councils should be increased. Karak Castle Center for Consultation and Training in cooperation with Friedrich Ebert Office / Amman (2018), conducted a review of the decentralization law 2015. In this research, the focus group methodology was used, where 98 elected male members in 12 governorates participated in the discussions, and the main media interview methodology with all the heads of the 12 centers, and with 32 women out of 36 elected females in the governorate councils. The results of this study indicated a negative evaluation of the decentralization experience, which can be explained as follows: The results of this study indicated a negative evaluation of the decentralization experience, which can be explained as follows: ➢ The biggest challenge facing the councils in the governorates is the law itself. Therefore, the councils did not achieve the expected goals and objectives of the decentralization system. Lack of sufficient powers to ensure control over the implementation of projects. Demanding the need to amend the decentralization law. ➢ The biggest challenge facing the councils in the governorates is the law itself. Therefore, the councils did not achieve the expected goals and objectives of the decentralization system. Lack of sufficient powers to ensure control over the implementation of projects. Demanding the need to amend the decentralization law. ➢ The government did not provide support to these councils with qualified administrative cadres Ministries ➢ The biggest challenge facing the councils in the governorates is the law itself. Therefore, the councils did not achieve the expected goals and objectives of the decentralization system. Lack of sufficient powers to ensure control over the implementation of projects. Demanding the need to amend the decentralization law. ➢ The government did not provide support to these councils with qualified administrative cadres. Ministries do not cooperate with them effectively. 2.2 Field Studies independence of the local councils, so that they can take their decisions and carry out their responsibilities easily. )Abu Hazeem, ( 2016 has demonstrated the importance of implementing decentralization in the political reform process in Jordan from the viewpoint of the members of the seventeenth parliament. The descriptive survey method was adopted, and the study sample amounted to (51) members of the total number of members of the House of Representatives. The researcher concluded that there is a high degree of approval among the members of the seventeenth parliament on the importance of implementing decentralization in the political reform process in Jordan. He recommended the necessity of implementing decentralization, which guarantees the real . independence of the local councils, so that they can take their decisions and carry out their responsibilities easily. Our review of previous field studies shows that most of them focused on the political dimension only, and some of them on the financial dimension only, to evaluate the decentralization experience in Jordan (except Taamneh study). Also, the target parties in these studies to know their views on the decentralization experience did not include all stakeholders involved in decentralization at the level of governorates and municipalities who represent the communities in these areas. Some of them were restricted to members of the House of Representatives and others to the women's sector or to the elected presidents and members of the provincial councils. Although the study of Tamannah included the three dimensions of decentralization, the study sample was limited to the interrogation of employees working in government departments in the governorates and municipalities in the northern region only. The study sample did not include the targeted members of the elected councils in the governorates and municipalities and opinion leaders in society and in all twelve governorates who represent citizens through direct election. The sample was limited to the northern governorates only and did not include the rest of the governorates in the center and south. Therefore, this study complements the previous studies, and its originality lies in the fact that it includes the three dimensions of decentralization (political, administrative and financial) and that the study population in it are the heads and members of councils in the governorates and municipalities and opinion leaders in all twelve governorates and in all municipalities in Jordan. 2.2 Field Studies The sample will be selected from Al-Balqa Governorate and Amman Governorate (because of its political importance as the capital of Jordan, 42.o% of the total population, and 43.8% of total economic establishment), Irbid governorate is from the northern region, and Karak governorate is from the southern province. 2.2 Field Studies It did not provide financial sources to guarantee the financial independence of these councils ➢ The government did not provide support to these councils with qualified administrative cadres. Ministries do not cooperate with them effectively. It did not provide financial sources to guarantee the financial independence of these councils. 59 w and audit budgets, includ s for following up and mo needs and prioritize the com the work of executive coun powers and the functions o sing the local administration f provincial councils. ng decentralization in the po SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 ➢ The necessity of training provincial council members to review and audit budgets, including gender- sensitive budgets. They are also trained in effective methods for following up and monitoring the implementation process. ➢ The necessity of training provincial council members to review and audit budgets, including gender- sensitive budgets. They are also trained in effective methods for following up and monitoring the implementation process. p p ➢ The necessity of training provincial council members to identify needs and priori which they work. ➢ Other laws, such as the Municipal Law, and the laws relating to the work of executi amended. ➢ Many members of the Council lacked an understanding of their powers and the functions of the various official bodies. ➢ Creating a new ministry to take over the responsibility of supervising the local administration in Jordan, to replace the Ministry of Municipal Affairs. ➢ Increasing the share of women's participation in the membership of provincial counc ➢ Increasing the share of women's participation in the membership of provincial councils. )Abu Hazeem, ( 2016 has demonstrated the importance of implementing decentralization in the political reform process in Jordan from the viewpoint of the members of the seventeenth parliament. The descriptive survey method was adopted, and the study sample amounted to (51) members of the total number of members of the House of Representatives. The researcher concluded that there is a high degree of approval among the members of the seventeenth parliament on the importance of implementing decentralization in the political reform process in Jordan. He recommended the necessity of implementing decentralization, which guarantees the real . 2. The Local Administrative System in Jordan The local administration system in Jordan consists of (12) governorates and (100) municipalities, including the Greater Amman Municipality, which has a special status as the capital of Jordan. It is directly linked to the central authorities (see Figure 1). In order to clarify the extent of decentralization in the Jordanian system of governance and administration, the parties involved in the local administration will be identified, the mechanism of its formation analyzed, and their tasks determined in order to provide public services to citizens, as follows : 60 60 SocioEconomic Challenges, Volume 5, Issue 4, ISSN (print) – 2520-6621, ISSN (online) – 2520 Figure 1. The Structure of Government in Jordan S d b th A th SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 SocioEconomic Challenges, Volume 5, Issue 4, 2021 Figure 1. The Structure of Government in Jordan Sources: prepared by the Authors. Sources: prepared by the Authors. 3.1.1 The Appointed Governor He is the public servant appointed by the king based on the recommendation of the Minister of Interior, who represents the king in the governorate. He chairs the Executive Council, of which the directors of the official departments of the various ministries are its members. According to Article 3 of the Decentralization Law 2015 the Governor shall undertake the following tasks: g rvise and ensure that the official bodies in the governorate are carrying out their tasks. g 1. Lead, supervise and ensure that the official bodies in the governorate are carrying o 2. Follow up on the execution of state public policy in the governorate; take whatever necessary action needed to ensure these policies are heeded so that state departments and institutions in the governorate are performing their duties. 3. Coordinate among the council and municipalities of the governorate, the ministries, government departments and public institutions. p p 4. Supervise development and service plans and the preparation of the annual governorate 5. Take the necessary measures to ensure the execution of the council’s decisions; and refer these to the competent body. p y 6. Avail the best services to the citizens in coordination with the council. p y 6. Avail the best services to the citizens in coordination with the council. 7. Work with the council and the executive council to avail the appropriate environment to encourage investment at the governorate; avail socio-economic development requirements; and take the necessary measures to realize this in coordination with the stakeholders. 8. Protect state property, work on developing and utilizing it, and take necessary measures to guarantee this. 9 T k h h l h bli f d h i f l i i d 9. Take the necessary measures to protect health, public safety and the environment; formulate monitoring and inspection committees with the authority to temporarily close shops, enterprises and sites in violation; and seizing assets until these violations are referred to the competent court. g p 10. Take necessary measures in cases of emergency and coordinate efforts of all competent authorities. 11. Conduct periodic meetings for the council and committees that he/she presides over and take the measur p sary measures in cases of emergency and coordinate efforts of all competent authorities. g p 10. Take necessary measures in cases of emergency and coordinate efforts of all competent authorities. 11. 3.1.2.1 The Board membership uncil is formed in each governorate (chaired by the Governor and with membership of: An executive council is formed in each governorate (chaired by the Governor and with mem 1. The deputy governor, the administrators who preside over the districts, and two district administrators who they head the district directorates in the governorate and the assistant governor for development affairs. 2. Directors of the executive directorates and service departments in the governorate, and if there are more from the director of a directorate or department affiliated to a specific ministry named the competent minister or official The first for the management of the sector is one of the directors of the directorates or departments, as the case may be, as a membervin the Executive Council. The first for the management of the sector is one of the directors of the directorates or departments, as the case may be, as a membervin the Executive Council. 3. Managers of development zones and industrial cities in the governorate, if any. 4. Three of the executive directors of municipalities in the governorate, a maximum, to be named by a minister of Municipal affairs. 5. One of the commissioners of the Aqaba Special Economic Zone Authority, to be named by its head in relation to Aqaba Governorate, and one of the commissioners of the Petra Development and Tourism Region Authority, named by its president, relates to Ma'an Governorate. 3.1.2.2 The functions of the executive council The Executive Council shall assume the following duties and powers in a manner that does not conflict with the provisions of the law: Aqaba Special Economic Zone in force and the Petra Tourism Development Region Authority Law and the powers of the Board of Commissioners in each of them: The Executive Council shall assume the following duties and powers in a manner that does not conflict with the provisions of the law: Aqaba Special Economic Zone in force and the Petra Tourism Development Region Authority Law and the powers of the Board of Commissioners in each of them: 1. Preparing draft strategic and executive plans for the governorate and aligning them with the plans. The strategy prepared by the municipal councils and other official bodies, and to ensure that Its consistency with national strategies and plans and referring it to the Council for decision making appropriate about it. 1. 3.1.1 The Appointed Governor Conduct periodic meetings for the council and committees that he/she presides over and take the measure necessary to execute their decisions and recommendations. ny powers or duties as delegated by the cabinet, the prime minister or the competent minist 12. Exercise any powers or duties as delegated by the cabinet, the prime minister or the co 61 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 3.1.2.1 The Board membership Preparing draft strategic and executive plans for the governorate and aligning them with the plans. The strategy prepared by the municipal councils and other official bodies, and to ensure that Its consistency with national strategies and plans and referring it to the Council for decision making appropriate about it. 2. Preparing a guide to the governorate’s needs of development and service projects, including a guide The needs received from the municipalities and other official bodies and referred to the Council. 2. Preparing a guide to the governorate’s needs of development and service projects, including a guide The needs received from the municipalities and other official bodies and referred to the Council. 3. Preparing the governorate's budget draft within the ceilings specified by the Ministry of Finance / Department The general budget and referral to the Council. 3. Preparing the governorate's budget draft within the ceilings specified by the Ministry of Finance / Department The general budget and referral to the Council. 4. Reviewing the general conditions in the governorate and discussing matters related to public services and to consider any proposal submitted by any member and take the necessary decisions in this regard. Review the reports it receives from the municipal councils, and take appropriate decisions in this regard. 4. Reviewing the general conditions in the governorate and discussing matters related to public services and to consider any proposal submitted by any member and take the necessary decisions in this regard. Review the reports it receives from the municipal councils, and take appropriate decisions in this regard. 5. Laying the foundations that ensure the proper functioning of the administrative and executive bodies in the governorate. 5. Laying the foundations that ensure the proper functioning of the administrative and executive bodies in the governorate. 6. Submitting the necessary recommendations regarding investment in the governorate and referring them to the Council unless It conflicts with any other legislation. 6. Submitting the necessary recommendations regarding investment in the governorate and referring them to the Council unless It conflicts with any other legislation. 7. Preparing reports on the progress of work in projects and services and referring them to the Board each six months. 7. Preparing reports on the progress of work in projects and services and referring them to the Board each six months. 8. Take the necessary measures regarding the decisions and recommendations issued by 9. 3.1.2 The Executive Council 3.1.2.1 The Board membership 3.1.3 The elective Governor He is the member who is elected from among the members of the council (in its first meeting after the elections) as the president of this council 3.1.2.1 The Board membership Coordination with official and public bodies and institutions that have jurisdiction over plans and the programs it implements. 9. Coordination with official and public bodies and institutions that have jurisdiction over plans and the programs it implements. 10. Study the issues referred to it by the governor or the council. 10. Study the issues referred to it by the governor or the council. 62 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 11. Preparing the necessary emergency plans at the governorate level to face emergency cases Disasters such as floods, torrential rains, snow, fires and others, and submitting them to the Council. 11. Preparing the necessary emergency plans at the governorate level to face emergency cases Disasters such as floods, torrential rains, snow, fires and others, and submitting them to the Council. 3.1.4.1 The Members Each governorate shall have a council called the governorate council, and it shall enjoy a legal personality with financial and administrative independence. 85% of the council members are directly elected (75% for males and 10% for females) and the rest of the members (15%) are appointed by the Council of Ministers upon the recommendation of the Minister of Interior provided that one third of this percentage is allocated to women. The term of the Council is four years. The Council of Ministers has the right to dissolve it upon the recommendation of the Minister of Interior before the end of the term of this Council 3.1.4.2 The Responsibilities This Council shall undertake the following tasks ( in a manner that does not conflict with the provisions of the Aqaba Region Law, and the effective special economic law ): 1. Approving the projects of the strategic and executive plans related to the governorate and referred to it from the Executive Council and ensure its implementation. 2. Approval of the governorate’s draft budget within the ceilings set by the Ministry of Finance, the general budget referred to by the Executive Council for inclusion in the general budget in accordance with Procedures for preparing the state's general budget. 3. See how to implement the annual budgets of all the governorate's municipalities. 4. Approval of a guide to the governorate’s needs for development and service projects referred to it from Executive Board and prioritize those needs. 5. Approval of the service and investment projects referred to it by the Executive Council after Complete the necessary procedures in accordance with the applicable legislation. 6. Approval of development projects that are of public interest to the preservation taking into account the development projects proposed by municipal councils, departments and institutions official within the governorate and submit it to the governor to take the necessary measures in this regard. 7. Discussing the reports of the implementation of projects, plans and programs undertaken by the department’s governmental authorities in the governorate to implement them in a manner that does not conflict with the work of government oversight bodies concerned with following up and evaluating the progress of development projects. 8. Suggesting the establishment of investment projects and carrying out joint projects with other governorates with the approval of the competent authorities. 9. Develop recommendations and proposals for the competent authorities to ensure the improvement of the performance of the departments Governmental and public institutions operating within the governorate to ensure the provision of the best services. 9. Develop recommendations and proposals for the competent authorities to ensure the improvement of the performance of the departments Governmental and public institutions operating within the governorate to ensure the provision of the best services. 10. Determine the areas within the borders of the governorate that suffer from a lack of services and development or emergency problems and propose solutions to them from the relevant authorities and approve County contingency plan. 11. Discussion of any of the Executive Council members on the topics within his specialty. 3.1.4.2 The Responsibilities 63 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 3.2.1 The Mayer The mayor is a person who is directly elected by the citizens of the municipality. He is the head of the municipal council and exercises the following duties: 1. Inviting the Municipal Council to convene and presiding over its sessions 2. Representing the municipality in external meetings and conferences, subject to the approval of the Minister of Local Administration. 3. Preserving the rights of the municipality and defending its interests legally 4. Signing agreements, memoranda of understanding, protocols and twinning with the relevant authorities approved by the Municipal Council inside Jordan. If these agreements are with parties outside Jordan, the approval of the Minister of Local Administration is required. 5. Presenting the draft of (municipal budget, strategic plan, and local needs guide) to the municipal council for discussion and approval. 6. Presenting the various issues sent by the municipal executive director to the municipal council for discussion and approval. 3.2.2 The Town council 3.2.2.1 The formation of the municipal council 2. Representing the municipality in external meetings and conferences, subject to the approval of the Minister of Local Administration. 4. Signing agreements, memoranda of understanding, protocols and twinning with the relevant authorities approved by the Municipal Council inside Jordan. If these agreements are with parties outside Jordan, the approval of the Minister of Local Administration is required. pp f f q 5. Presenting the draft of (municipal budget, strategic plan, and local needs guide) to the municipal council for discussion and approval. 5. Presenting the draft of (municipal budget, strategic plan, and local needs guide) to the municipal council for discussion and approval. 6. Presenting the various issues sent by the municipal executive director to the municipal council for discussion and approval. 3.2.2.1 The formation of the municipal council The municipality is a civil institution that enjoys a legal personality with financial and administrative independence. The municipality is managed by a council consisting of the president and a number of members (all of them were elected directly by the citizens of the municipality). The number of council members is determined by the Minister of Local Administration, and that the number of council members is not less than seven, including the president in any case. An exception is made to the Greater Amman Municipality Council, whose members are determined by the Council of Ministers. It is composed as follows: (75%) are the directly elected heads of the local councils of the Greater Amman Municipality, and (25%) of the members are members appointed by the Council of Ministers and upon the recommendation of the Minister of Local Administration. 3.2.2.2 Municipal council tasks The Municipal Council exercises the following powers: 1. Approval of the estimated annual draft budget as well as the actual budget 2. Approval of the local development strategic plan and a guide to the needs of the town 3. Follow up and monitor the implementation of development programs. Which can be implemented by the municipality itself or with the participation of other municipalities or with the private sector 4. Surveillance of open lands and protection of streets and roads 5. Coordination with the central official authorities in us regarding the determination of the locations of schools, health centers, and places of worship 6. Coordination with the central official authorities with regard to determining the distribution of electricity and water to citizens 7. Coordination with the central official authorities regarding the construction of sewage networks. And the establishment and operation of public toilets. 8. Establishment and organization of public markets 64 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 9. Organizing crafts and industries and establishing industrial zones 10. Construction of squares, gardens, parks and tourist places 11. Coordination with the Civil Defense Foundation to prevent and extinguish fires 12. Take the necessary measures to prevent natural disasters and establish public shelters 13. In coordination with the central official authorities, the establishment of museums, public libraries, sports and social clubs, and slaughterhouses 14. Construction, management and maintenance of cemeteries 15. 3.2.2.1 The formation of the municipal council Investing the movable and immovable property of the municipality SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 9. Organizing crafts and industries and establishing industrial zones 10. Construction of squares, gardens, parks and tourist places 11. Coordination with the Civil Defense Foundation to prevent and extinguish fires 12. Take the necessary measures to prevent natural disasters and establish public shelters 13. In coordination with the central official authorities, the establishment of museums, public libraries, sports and social clubs, and slaughterhouses 14. Construction, management and maintenance of cemeteries 15. Investing the movable and immovable property of the municipality 3.2.3 Municipal Manager SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 In each municipality, an executive director is appointed by a decision of the Minister of Local Administration and upon the recommendation of the municipal council. He is the head of the administrative body and is responsible for its proper functioning. He is responsible to the mayor and for preparing and implementing the annual budget for the municipality 4. The Methodology The study community consists of opinion leaders and all members of the elected councils in all 12 governorates, and in all municipalities, a choice of (100). These categories were chosen because the researchers believe that these categories represent the concerned citizens and their satisfaction with the implementation of decentralization . The study sample was determined to represent the entire study population, by selecting one governorate at random from each of the three regions. In addition to choosing the governorate of Amman due to its political, economic and demographic importance. It is the capital of Jordan and the seat of the central government, 42% of the total population resides in it, and 43.6% of the total economic establishments in Jordan reside there. Table No. 1 shows the sample size, which numbered 578, and their distribution. Table 1. The Sample distribution Region Governorate Member of governorate council Member of municipality council Opinion Leaders Meddle Amman 53 75 28 Al-Balqa 23 72 10 North Irbid 41 142 19 South Al-Karak 24 81 10 Total 578 141 370 67 Source: prepared by the Authors. Source: prepared by the Authors. Two forms of the questionnaire were designed, the first to assess the decentralization experience at the governorate level and the second to evaluate the decentralization experience at the municipality level. Each questionnaire includes two sections. The first relates to the social characteristics of the respondents (gender, age, qualification, years of experience and job title). The second contains 24 questions to assess the political, administrative and financial decentralization at the governorate level. The other contains 28 questions to assess political, administrative and financial decentralization at the municipal level. The five-point Likert scale was used to evaluate the study questions, as follows: I never agree I don't agree neutral I agree I totally agree 1 2 3 4 5 65 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 The researcher used this scale to evaluate the extent of decentralization experience in Jordan: (1- < 2.33) low, (2.33 - < 3.66) moderate, and (3.66 – 5) high. The mean and standard deviation were calculated for each question and a t-test was used to test the study hypotheses. The researcher used this scale to evaluate the extent of decentralization experience in Jordan: (1- < 2.33) low, (2.33 - < 3.66) moderate, and (3.66 – 5) high. 4. The Methodology The mean and standard deviation were calculated for each question and a t-test was used to test the study hypotheses. 4.1 Governorate Level The data contained in Table No. 2 indicate that the degree of decentralization assessment at the governorate level was moderate. The arithmetic mean value was estimated at (3.13, 2.70 and 3.1) for the political, financial and administrative dimension, respectively. and standard deviation of (1.18, 1.08, and 0.98), respectively. Table 2. Means and standard deviations of the extent of the implementation of decentralization in Jordan: Political, administrative, and fiscal indicators no Decentration Dimension Mean Standard Deviation Extent 1 Political 3.13 1.18 Neutral 2 Administrative 2.7 1.08 Neutral 3 Financial 3.1 0.98 Neutral Source: prepared by the Authors. Source: prepared by the Authors. 5.1.2 Fiscal decentralization The mean values of the five questions evaluating fiscal decentralization ranged between (4.38) and (1.73), and the standard deviation ranged between (0.619) and (1.205) (see Table 4). The mean values of the five questions evaluating fiscal decentralization ranged between (4.38) and (1.73), and the standard deviation ranged between (0.619) and (1.205) (see Table 4). Table 4 also shows that the respondents strongly believe in the necessity of obtaining sufficient self-financing sources for the governorate, similar to the municipalities, to ensure the financial independence stipulated in the decentralization law. But this law never mentioned what these financial sources are. They also do not believe in the fairness of the basis for distributing the capital expenditure ceilings of the central government among the provinces, and it does not grant the provinces financial independence. They also believe that the provincial councils do not participate with the central government in the distribution of capital spending amount among the governorates. Table 4. Means, Standard deviation, and Extent of financial decentralization no Rank The Question Mean Standard Deviation Extent 5 1 I feel that there is an urgent need to provide financial resources to the governorates, like the municipalities 4.38 0.619 high 2 2 The mechanism for determining financial ceilings for capital spending and distributing it to governorates enhances their financial independence and ability to carry out their tasks 3.78 1.064 high 3 3 The provincial councils participate with the central government in determining the bases for distributing investment spending among the provinces 2.84 1.205 Moderate 1 4 The Decentralization Law 2015 specified financial resources for the governorate to ensure its financial independence 2.76 1.157 Moderate 4 5 Feel the fairness of the standards adopted in the distribution of financial ceilings between governorates by the central government 1.73 0.890 Moderate TOTAL 3.10 0.980 Moderate Table 4. Means, Standard deviation, and Extent of financial decentralization Source: prepared by the Authors. 5.1.1 Political decentralization The mean values of the eight questions evaluating political decentralization ranged between 4.4 and 2.36), and the standard deviation value ranged between 0.69 and 1.436. There is a strong belief among the respondents that the success of political decentralization depends on the availability of transparency and integrity in the elections of provincial council members. Citizens do not trust the central government to achieve these conditions. This explains the low rate of citizen participation in the 2017 elections, as this percentage reached (32%). Among the other reasons that the respondents see as negatively affecting political decentralization are: 1. The central government intervenes by appointing 15% of the total number of provincial council members. 2 D i i h ' b h l r reasons that the respondents see as negatively affecting political decentralization are: 3. The lack of political will for a number of central leaders, which would ensu decentralization experience at the governorate level. Table 3. Means, Standard deviation, and Extent of political decentralization no Rank The Question Mean Standard Deviation Extent 5 1 The success of decentralization depends on the extent of transparency and integrity in the provincial council elections 4.4 0.69 high 6 2 The appointed members participate in choosing the head of the provincial council 3.38 1.216 Moderate 2 3 The percentage allotted to elected members and the majority (85%) in the provincial council is considered appropriate and guarantees the implementation of political decentralization 3.27 1.193 Moderate 3 4 The application of the women's quota provided for in the decentralization law contributes to an appropriate representation of the women sector in the provincial council 3.14 1.265 Moderate 8 5 I believe that the application of political decentralization has contributed to increasing the participation of citizens in formulating the development decision at the governorate level 3.09 1.339 Moderate 4 6 The provincial council represents all segments of society 2.87 1.436 Moderate 1 7 There is the necessary political will in Jordan to implement decentralization successfully at the governorate level 2.54 1.119 Moderate 7 8 Within the decentralization law 2015 an active role for civil society organizations and professional unions 2.35 1.137 Moderate TOTAL 3.13 1.184 Moderate S d b h A h Table 3. Means, Standard deviation, and Extent of political decentralization 66 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 5.1.2 Fiscal decentralization 5.1.3 Administrative decentralization The mean values of the eleven questions evaluating fiscal decentralization ranged between (3.86) and (2.17), and the standard deviation ranged between (0.944) and (1.222) (see Table 5). The respondents believe that the central government strongly controls the decisions of the governorate councils through the Ministry of Interior, and that the implementation of tasks in the governorate is carried out by the branches of the service ministries. They also believe that the decentralization law 2015 did not grant the governorate councils the guarantor powers to achieve their administrative independence. They also pointed out that the law did not guarantee that there would be no duplication of powers between the official institutions in the governorate, especially between the elected president of the provincial council and the appointed governor and the head of the executive council. The finding of our study is consistent with a number of local studies (Taamneh et al., 2019). Table 5. Means, Standard deviation, and Extent of administrative decentralization No Rank The Question Mean Standard Deviation Extent 2 1 The central government controls the decisions and actions of the provincial councils 3.86 1.130 High 1 2 There is harmony between the elected provincial council members and members of the executive council 3.04 1.222 Moderate Table 5. Means, Standard deviation, and Extent of administrative decentralization 67 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 Table 5 (cont.). Means, Standard deviation, and Extent of administrative decentralization No Rank The Question Mean Standard Deviation Extent 9 3 The decentralization law clearly ensured that the disputes between the two councils in the governorate were resolved 3.04 1.004 Moderate 8 4 The decentralization law clearly ensured the limitation of duplication of powers between the elected provincial council and the central government agencies operating in the province 2.75 1.078 Moderate 7 5 The councils in the governorate have the technical and administrative capabilities to carry out their tasks stipulated in the decentralization law 2.67 1.134 Moderate 11 6 The supervisory role that the elected provincial council plays over the municipalities does not conflict with the council’s oversight role over government agencies operating in the province. 5.1.3 Administrative decentralization 2.63 1.084 Moderate 3 7 The powers contained in the Decentralization Law are sufficient to implement administrative decentralization at the governorate level 2.52 1.013 Moderate 10 8 The decentralization law defines the oversight role of the elected governorate council over the municipalities 2.40 0.944 Moderate 4 9 Decisions issued by the elected provincial council are binding on the central government agencies operating in the province 2.38 1.137 Moderate 5 10 The oversight role of the elected provincial council is very effective 2.21 1.092 Moderate 6 11 The decentralization law grants the governor and the directors of government departments operating in the governorate more powers, so that they cannot return to their relevant ministries to carry out their work. 2.17 1.008 Moderate TOTAL 2.70 1.084 Moderate Table 5 (cont.). Means, Standard deviation, and Extent of administrative decentralizat Source: prepared by the Authors. 5.1 The data contained in Table No. 7 indicate that the degree of decentralization assessment at the municipality level was moderate. The arithmetic mean value was estimated at (2.62, 2.98 and 2.84) for the political, financial and administrative dimension, respectively. and standard deviation of (1.120, 1.026, and 1.037), respectively. Table 7. Means and standard deviations of the extent of the implementation of decentralization in Jordan: Political, administrative, and fiscal indicators Decentration Dimension Mean Standard Deviation Extent 1 Political 2.62 1.120 Neutral 2 Administrative 2.98 1.026 Neutral 3 Financial 2.84 1.037 Neutral Source: prepared by the Authors. 5.1.4 Testing Hypotheses H01.1: The political decentralization experience at governorate level is characterized by a low degree of decentralization (0.05). H01.2: The financial decentralization experience at governorate level is characterized by a low degree of decentralization (0.05). H01.3: The administrative decentralization experience at governorate level is characterized by a low degree of decentralization (0.05). Given the estimated values of (T( , and the level of significance contained in Table No. 6, all the null hypotheses mentioned above are accepted. In other words, the degree of decentralization is low, with its three dimensions together: political, financial and administrative. 68 68 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 Table 6. Testing research hypothesis Table 6. Testing research hypothesis Variable Mean Standard Deviation T-Value calculated df Sig. Decentralization at Governorate level 2.92 0.638 - 1.265 267 0.00 Political decentralization 3.13 0.731 1.858 267 0.00 Financial decentralization 3.10 0.623 1.669 267 0.00 Administrative decentralization 2.70 0.723 -4. 443 267 0.00 Source: prepared by the Authors. Source: prepared by the Authors. 5.2.2 Fiscal decentralization The mean values of the eight questions evaluating fiscal decentralization ranged between 4.32 and 2.12), and the standard deviation value ranged between 0.748 and 1.628 (see Table 9). The respondents strongly emphasize the need for the central government to grant municipalities more self-financial resources that would enable them to achieve financial independence and enable them to better provide local public services to citizens. They moderately believe that the municipality has qualified human and technical capabilities that ensure the efficient and effective collection of the municipality's financial resources. The mean values of the eight questions evaluating fiscal decentralization ranged between 4.32 and 2.12), and the standard deviation value ranged between 0.748 and 1.628 (see Table 9). The respondents strongly emphasize the need for the central government to grant municipalities more self-financial resources that would enable them to achieve financial independence and enable them to better provide local public services to citizens. They moderately believe that the municipality has qualified human and technical capabilities that ensure the efficient and effective collection of the municipality's financial resources. Table 9. 5.2.2 Fiscal decentralization Means, Standard deviation, and Extent of fiscal decentralization no Rank The Question Mean Standard Deviation Extent 8 1 I think that one of the duties of the CEO is to prepare and implement the municipal budget and prepare the final accounts 4.32 0.767 high 7 2 I think that one of the duties of the municipal council is to approve the municipal budget and follow up its implementation 4.25 0.847 high 6 3 There is an urgent need to provide additional funding sources to enable the municipality to carry out its functions efficiently and effectively 4.04 0.888 high 6 4 I think that the financial independence of the municipality enhances the Implementation of financial decentralization at the local level 3.69 0.983 high 4 5 Municipalities possess the technical and human capacities qualified to collect the self-financial resources efficiently and effectively 2.52 1.215 moderate 5 6 The central government is obligated to implement the distribution mechanism stipulated in the Municipalities Law in distributing the money that the government collects on behalf of the municipalities 2.46 1.071 moderate 1 7 I believe that the municipalities' share of taxes and fees collected by the state is sufficient to enhance the financial independence of the municipalities 2.37 1.37 moderate 2 8 I think that there is fairness in distributing the money collected by the central government in favor of the municipalities from taxes and fees between the municipalities 2.33 0.965 moderate Total 2.98 1.026 moderate Table 9. Means, Standard deviation, and Extent of fiscal decentralization Source: prepared by the Authors. 5.2.1 Political decentralization The mean values of the eight questions evaluating political decentralization ranged between 4.33 and 2.12), and the standard deviation value ranged between 0.748 and 1.628 (see Table 8). The respondents strongly believe that the 2015 Municipalities Law guarantees political representation for all segments of society and professional organizations. But the success of political decentralization, in their opinion, depends on the extent to which transparency and integrity are taken in conducting local elections. This explained the low rate of citizen participation in the voting process in the 2017 elections. They believe, to a very low degree, that the appointment of an executive director of the municipality strengthened the oversight role of the elected municipal council. Table 8. Means, Standard deviation, and Extent of political decentralization no Rank The Question Mean Standard Deviation Extent 5 1 Within the Municipal Law 2015 an active role for civil society organizations 4.33 0.874 high 1 2 The division of the municipality into local areas, each headed by an elected local council, led to an increase in political participation 3.02 1.628 moderate 4 3 The success of political decentralization depends on the extent of transparency and fairness in municipal elections 2.42 1.145 moderate 3 4 The elected municipal council represents all segments of the local community 2.29 1.080 low 6 5 The application of political decentralization has contributed to increasing citizen participation in the formulation and implementation of local development decisions 2.26 1.075 low 2 7 The application of the women's quota contributes to the representation of all spectrums of the local community 2.22 1.102 low 7 8 The appointment of an executive director for each municipality strengthened political decentralization and activate the oversight role of the elected municipal council 2.12 1.022 low Total 2.62 1.120 moderate Table 8. Means, Standard deviation, and Extent of political decentralization 69 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 5.2.4 Testing Hypotheses The main null hypothesis at the municipality level states the following: H01.1: The political decentralization experience at municipality level is characterized by a low degree of decentralization (0.05). H01.2: The financial decentralization experience at municipality level is characterized by a low degree of decentralization (0.05). H01.3: The administrative decentralization experience at municipality level is characterized by a low degree of decentralization (0.05). H01.3: The administrative decentralization experience at municipality level is characterized by a low degree of decentralization (0.05). Given the estimated values of (T(, and the level of significance contained in Table No. 6, all the null hypotheses mentioned above are accepted. In other words, the degree of decentralization is low, with its three dimensions together: political, financial and administrative. Given the estimated values of (T(, and the level of significance contained in Table No. 6, all the null hypotheses mentioned above are accepted. In other words, the degree of decentralization is low, with its three dimensions together: political, financial and administrative. Table 6. Testing research hypothesis Variable Mean Standard Deviation T-Value calculated df Sig. Decentralization at municipality level 2.84 0.588 -3.319 267 0.00 Political decentralization 2.62 0.710 - 6.650 267 0.00 Financial decentralization 2.98 0.585 - 0.372 267 0.00 Administrative decentralization 2.92 0.609 - 1.703 267 0.00 Source: prepared by the Authors. 5.2.3 Administrative decentralization The mean values of the seven questions evaluating administrative decentralization ranged between 4.06 and 2.31), and the standard deviation value ranged between 0.837 and 1234 (see Table1 ). Respondents highly believe that the 2017 Municipalities Law has given local authorities more local tasks, and reduced duplication of powers. However, they moderately believe that the municipalities do not have the human, technical and qualified capabilities that would enhance administrative decentralization. Table 10. Means, Standard deviation, and Extent of administrative decentralization no Rank The Question Mean Standard Deviation Extent 1 1 The amended Municipalities Law 2015 includes more powers for the municipality 4.06 0.837 high 4 2 The amended Municipalities Law 2015 limited the duplication of powers between the Municipal Council and the central government agencies at the local level 3.97 0.892 high 5 3 The formulation of the powers in the amended law was clear and free from duplication that enables the municipal council and the municipality director to implement them without hindrances 2.45 1.141 moderate 7 4 I think that the experience of administrative decentralization at the local level was successful 2.37 1.234 moderate Table 10. Means, Standard deviation, and Extent of administrative decentralization 70 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 Table 10 (cont.). Means, Standard deviation, and Extent of administrative decentralization no Rank The Question Mean Standard Deviation Extent 6 5 Municipalities possess the technical and human capabilities that are able to carry out the tasks of the municipality efficiently and effectively 2.36 1.116 moderate 2 6 I think that the amended Municipalities Law 2015 gave the municipalities more tasks, which in turn promote more administrative decentralization 2.35 1.041 moderate 3 7 There is a need to separate the oversight role of the council from the executive role of the municipality director in order to promote decentralization 2.31 0.997 moderate Total 2.84 1.037 moderate 0 (cont.). Means, Standard deviation, and Extent of administrative decentralization Source: prepared by the Authors. 5.2.4 Testing Hypotheses 6. Conclusion and Recommendation The respondents believed that the amended Municipalities Law 2015 granted municipalities additional powers and reduced duplication of powers. Assign the executive tasks in the municipality to the municipality manager instead of the mayor and the municipal councilز The researchers recommend the following: - Demanding the amendment of the Municipalities Law and the Decentralization Law by the central government, with the need to consider the results of studies conducted in the past period and to involve opinion leaders in regional and local communities in the expected amendment. - Demanding the amendment of the Municipalities Law and the Decentralization Law by the central government, with the need to consider the results of studies conducted in the past period and to involve opinion leaders in regional and local communities in the expected amendment. Recommending the need for the central government to delegate more powers to the governorate’s organs, especially that the decisions of the elected governorate council become executive rather than advisory. Recommending the need for the central government to delegate more powers to the governorate’s organs, especially that the decisions of the elected governorate council become executive rather than advisory. - Recommending the necessity of directly electing all members of the provincial council, similar to the House of Representatives at the national level and municipal councils at the municipal level. - Recommending the necessity of directly electing all members of the provincial council, similar to the House of Representatives at the national level and municipal councils at the municipal level. We recommend the necessity of ensuring the financial independence of the governorates, like the municipalities, by allocating the proceeds of some taxes and fees from one side to them and sharing the proceeds of some of them, such as the oil derivatives tax, vehicle licensing fees, and violations. We recommend the necessity of ensuring the financial independence of the governorates, like the municipalities, by allocating the proceeds of some taxes and fees from one side to them and sharing the proceeds of some of them, such as the oil derivatives tax, vehicle licensing fees, and violations. We recommend that the central government should continue to prepare and implement plans, in cooperation with donor countries and agencies, to supply and rehabilitate human cadres and technical resources in order to enable them to carry out their planning and executive responsibilities in the political, financial and administrative fields efficiently and effectively. 6. Conclusion and Recommendation 6. Conclusion and Recommendation This study concluded that the respondents evaluated the Jordanian decentralization experience at the two levels (governorates and municipalities) as moderate. This moderate evaluation (from their point of view as well) applies to all three dimensions of decentralization (political, financial and administrative). The statistical tests used in the study showed the acceptance of all the main hypotheses of the study, which confirm that the degree of decentralization in general at the level of the governorate and municipalities is low. The result does not differ for the sub-hypotheses of the following dimensions: political, financial and administrative. (estimated low). 71 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 The study found the respondents' lack of conviction and confidence in the integrity and transparency of the local electoral process that took place in 2017. This was reflected in the low participation in those elections, as it was estimated at (31%) according to the official reports of the Jordanian government. The study found the respondents' lack of conviction and confidence in the integrity and transparency of the local electoral process that took place in 2017. This was reflected in the low participation in those elections, as it was estimated at (31%) according to the official reports of the Jordanian government. The respondents believed that the decentralization law did not guarantee the financial independence of the provincial council, despite its affirmation of that. This is because financial resources (such as taxes and fees) are not the respondents believe that the provincial councils are unable to influence the determination and distribution of financial ceilings for the capital expenditure of the central government among the provinces. As well as the spending mechanism, which is the responsibility of the branches of ministries operating in the governorates. They also believe in the unfair distribution of public capital spending among the governorates. allocated to the governorate or shared with the central government . From an administrative point of view, the respondents believe that the central government does not grant the agencies in the governorates powers to enable them to carry out their duties without consulting and waiting for the approval of the central authorities. The lack of qualified human cadres, financial and technical resources in the governorates and in the municipalities weakens their ability to carry out their duties towards citizens and to implement decentralization successfully. 6. Conclusion and Recommendation In a manner that ensures an increase in the degree of decentralization in order to achieve the royal directives, which King Abdullah II has consistently emphasized. Funding: self-funded. Funding: self-funded. Author contribution: conceptualization, Aljaloudi, Jameel and Abu Zaid, Mohammad; data curation, Abu Zaid, Mohammad and Alfauri, Moath; formal analysis, Abu Zaid, Mohammad and Alfauri, Moath; funding acquisition, Aljaloudi, Jameel, Abu Zaid, Mohammad, Alfauri, Moath; investigation, Aljaloudi, Jameel, Abu Zaid, Mohammad, Alfauri, Moath; methodology, Abu Zaid, Mohammad and Alfauri, Moath; project administration, Aljaloudi, Jameel, Abu Zaid, Mohammad, Alfauri, Moath; Aljaloudi, Jameel; resources, Alfauri, Moath; software, Abu Zaid, Mohammad and Alfauri, Moath; supervision, Aljaloudi, Jameel; validation, Aljaloudi, Jameel; visualization, Alfauri, Moath; writing – original draft, Abu Zaid, Mohammad and Alfauri, Moath; writing – review & editing Aljaloudi, Jameel. 72 SocioEconomic Challenges, Volume 5, Issue 4, 2021 ISSN (print) – 2520-6621, ISSN (online) – 2520-6214 References 1. Abu Jabal Yazan (2020). The extent to which the administrative independence of the governorate councils in Jordan has been achieved in light of the method used in their formation. Journal of the Islamic University of Sharia and Legal Studies, 28(2). [Link]. h 1. Abu Jabal Yazan (2020). The extent to which the administrative independence of the governorate councils in Jordan has been achieved in light of the method used in their formation. Journal of the Islamic University of Sharia and Legal Studies, 28(2). [Link]. 2. Abu-Hazeem, Ghaith (2016). Decentralization and political reform in Jordan; the 17th Parliament Memebrs point of view, Abo-Hazeem, Ghaith, Master Thesis, Middele East University Jordan. [Link]. 2. Abu-Hazeem, Ghaith (2016). Decentralization and political reform in Jordan; the 17th Parliament Memebrs point of view, Abo-Hazeem, Ghaith, Master Thesis, Middele East University Jordan. [Link]. 3. Agrawal, A. and Ribot J. (1999). Accountability in Decentralization: A Framework with South Asian and West African Environmental Cases. The Journal of Developing Areas, 33, 473-502. [Link]. 3. Agrawal, A. and Ribot J. (1999). Accountability in Decentralization: A Framework with South Asian and West African Environmental Cases. The Journal of Developing Areas, 33, 473-502. [Link]. 4. Al-Khwaldeh, Saleh (2019). Local Governance In Jordan: Governorate Councils Under The Law Of Decentralization No. (49) Of 2015: An Analytical Study”. In Journal of legal and political science, 9(3). [Link]. 4. Al-Khwaldeh, Saleh (2019). Local Governance In Jordan: Governorate Councils Under The Law Of Decentralization No. (49) Of 2015: An Analytical Study”. In Journal of legal and political science, 9(3). [Link]. 5. Aljaloudi, Jameel (2021). Fiscal decentralization in Jordan. In Journal of Governance and Regulation, 9(4). [Link]. 5. Aljaloudi, Jameel (2021). Fiscal decentralization in Jordan. In Journal of Governance and Regulation, 9(4). [Link]. 6. Al Sarhan, Attallah (2020). Decentralization election in Jordan (2017). in Journal of Humanities and Social Sciences, 47(3). [GoogleScholar]. 6. Al Sarhan, Attallah (2020). Decentralization election in Jordan (2017). in Journal of Humanities and Social Sciences, 47(3). [GoogleScholar]. 7. Al Tarawneh , Bashar (2020). Jordanian women’s participation in elected local councils: A study of municipal and decentralization elections. In Journal of Humanities and Social Sciences, 47(3). [Link]. 7. Al Tarawneh , Bashar (2020). Jordanian women’s participation in elected local councils: A study of municipal and decentralization elections. In Journal of Humanities and Social Sciences, 47(3). [Link]. p 8. Crook, R. and Manor, J. (1998). Democracy and Descentralisation in South Asia and West Africa. References Cambridge University Press, Cambridge. [Link]. 9 D li i L (2015) Mi i f I i A J d [Li k] 8. Crook, R. and Manor, J. (1998). Democracy and Descentralisation in South As Cambridge University Press, Cambridge. [Link]. g y g 9. Decentralization Law (2015). Ministry of Interior, Amman. Jordan. [Link]. 10. Economic & Social Council of Jordan (2019). Decentralization: Diagnosis and Suggested Scenario: ESC- report 2019. [Link]. 10. Economic & Social Council of Jordan (2019). Decentralization: Diagnosis and Suggested Scenario: ESC- report 2019. [Link]. 11. Karak Castle Center for consultation and training in cooperation with Friedrich Ebert Stiftung (2018). Legal review of the Jordanian decentralization law. [Link]. 11. Karak Castle Center for consultation and training in cooperation with Friedrich Ebert Stiftung (2018). Legal review of the Jordanian decentralization law. [Link]. 12. Municipalities Law (2015). Ministry of Interior, Amman, Jordan. [Link]. unicipalities Law (2015). Ministry of Interior, Amman, Jordan. [Link]. 13. Ribot, J. (2002). Democratic Decentralization of Natural Resources: Institutionalizing Popular Participation. World Resources Institute, Washington, DC. [GoogleScholar]. 13. Ribot, J. (2002). Democratic Decentralization of Natural Resources: Institutionalizing Popular Participation. World Resources Institute, Washington, DC. [GoogleScholar]. 14. Schneider, A. (2003). Decentralization, conceptualization, and measurerment. Studio in Comparative International Development, 38(3), 35–55. [GoogleScholar]. 14. Schneider, A. (2003). Decentralization, conceptualization, and measurerment. Studio in Comparative International Development, 38(3), 35–55. [GoogleScholar]. 15. Taamneh, M., Rawabdeh, M., and Abu-Hummour, A. (2019). Evaluation of decentralization experience through political, administrative, and fiscal: the case of Jordan”. In Journal of Public Affairs, 20(4). [Link]. 16. The World Bank” Decentralization” (2013). [Link]. g p f ff 16. The World Bank” Decentralization” (2013). [Link]. 16. The World Bank” Decentralization” (2013). [Link]. 17. UNDP, Decentralized Governance Programme: Strengthening Capacity for People -Centered Development, Management Development and Governance Division, Bureau for Development Policy (September 1997). [Link] 17. UNDP, Decentralized Governance Programme: Strengthening Capacity for People -Centered Development, Management Development and Governance Division, Bureau for Development Policy (September 1997). [Li k] 73 73
https://openalex.org/W2014974509	https://periodicos.utfpr.edu.br/revistagi/article/download/413/312	Portuguese	null	UMA PROPOSTA DE BASE DE DADOS SOCIOAMBIENTAIS: DIAGNÓSTICO DE SUSTENTABILIDADE DOS DIFERENTES TIPOS DE EMPRESAS DO CONTEXTO BRASILEIRO	Revista Gestão Industrial	2,009	cc-by	6,831	Resumo O trabalho apresenta uma arquitetura de dados socioambientais para diagnóstico das organizações em termos de sustentabilidade econômica. É proposto um modelo de base de dados de indicadores socioambientais, fundamentado em pesquisa empírica, desenvolvida pelo método grounded theory. A ênfase da grounded theory é o aprendizado a partir dos dados (interativa e indutiva), e não a partir de uma visão teórica existente (dedutiva). Tais indicadores, além de refletir o estágio de sustentabilidade em que se encontra a empresa analisada, subsidiariam o mapeamento socioambiental dos diferentes segmentos econômicos do universo empresarial brasileiro. A arquitetura, estruturada com o suporte de recursos das tecnologias da informação possibilitaria o planejamento de decisões inerentes à gestão socioambiental de sua cadeia produtiva. Palavras-chave: sustentabilidade; gestão socioambiental; indicador de desenvolvimento socioambiental. SUSTAINABILITY AND ACCOUNTABILITY: PROPOSAL OF SOCIAL AND ENVIRONMENTAL DIAGNOSTIC MODEL BASED IN EMPIRICAL SURVEY Takeshy Tachizawa1; José Henrique Souza2 1Faculdade Campo Limpo Paulista – FACCAMP – Campo Limpo Paulista – Brasil usptakes@uol.com.br 2Pontificia Universidade Católica de Campinas – PUCCAMP – Campinas – Brasil josehenriquesouza@yahoo.com.br Universidade Tecnológica Federal do Paraná - UTFPR Campus Ponta Grossa - Paraná - Brasil ISSN 1808-0448 / v. 05, n. 03: p. 55-77, 2009 D.O.I.: 10.3895/S1808-04482009000300004 Universidade Tecnológica Federal do Paraná - UTFPR Campus Ponta Grossa - Paraná - Brasil ISSN 1808-0448 / v. 05, n. 03: p. 55-77, 2009 D.O.I.: 10.3895/S1808-04482009000300004 Revista Gestão Industrial Revista Gestão Industrial 1. Introdução A responsabilidade socioambiental corporativa, na trilha de desastres socioambientais envolvendo organizações globalizadas, ganha importância como instrumento de gestão da controladoria para evitar riscos econômicos. Os clientes, neste novo cenário empresarial, têm expectativas de interagir com organizações que sejam éticas, tenham boa imagem institucional no mercado, e que atuem de forma ecologicamente responsável. Neste ambiente, emerge a questão da sustentabilidade, que enfatiza o compromisso dos gestores junto aos stakeholders, como instrumento de gestão para a otimização econômica da organização. Neste contexto, foram analisados os fatores de influência da sustentabilidade nas empresas brasileiras e as possibilidades do uso de indicadores de diferenciação de custos socioambientais, proposta do presente trabalho. Foram abordadas as atividades econômicas exercidas pelas organizações, em seus diferentes ramos de negócios, relacionando-as com suas características de sustentabilidade. Como decorrência desta análise, estabeleceu-se uma metodologia para o diagnóstico socioambiental com enfoque diferenciado para cada tipo de organização do cenário empresarial brasileiro. Este diagnóstico permitiu estruturar um indicador de desenvolvimento socioambiental (IDS) que levasse em conta as peculiaridades existentes nas empresas em função do seu setor econômico de atuação. Com isso ele pode-se constituir em uma referência para análise dos custos socioambientais, que seriam singulares aos efeitos da sustentabilidade, intrínsecos a cada setor econômico. O princípio que serviu de base para a composição do IDS, foi fundamentado na avaliação do desenvolvimento na empresa, não do ponto de vista do crescimento econômico, mas pelo prisma de outras dimensões capazes, de interpretar a sustentabilidade de suas ações empresariais. Estas dimensões, em sintonia com o potencial crescimento profissional dos colaboradores da empresa, na medida em que convivem com saudáveis práticas de qualidade de vida, constituiu o embasamento filosófico deste trabalho, quais sejam: cidadania corporativa; transparência; governança corporativa; e capital humano. O IDS, portanto, objetivou o desenvolvimento de uma métrica que espelhasse o estágio evolutivo da organização em termos socioambientais, em seu contexto interno e externo. As razões que justificariam seu uso como instrumento de gestão pelas empresas não são apenas decorrentes de aderência à legislação vigente, mas, principalmente, em função da possibilidade de direcionar as decisões de gestão de custos e da controladoria de forma convergente com os objetivos da sustentabilidade empresarial. Os indicadores a serem configurados pela arquitetura de dados socioambientais, além de refletir o estágio de sustentabilidade em que se encontra a empresa analisada, subsidiariam o mapeamento, em termos de responsabilidade social e ambiental, dos diferentes segmentos econômicos do universo empresarial brasileiro. 1. Introdução A arquitetura, estruturada com o suporte de recursos das tecnologias da informação possibilitaria o planejamento de decisões inerentes à gestão socioambiental de sua cadeia produtiva. 2. Fundamentação teórica 56 Revista Gestão Industrial Problemas como poluição, falta de segurança, corrupção, má gestão dos recursos públicos, aética, e tantos outros, perpassam toda a história da humanidade desde o surgimento dos primeiros agrupamentos urbanos, ainda na Antiguidade (TACHIZAWA e ANDRADE, 2008). Para Henderson (2001), tal perspectiva tem se acentuado no mundo contemporâneo, como decorrência das mutações ocorridas na escala de valores e crenças do ser humano, delineadas pela Revolução Industrial. Essas mutações evoluíram para os tempos atuais como responsabilidade social. Esta é entendida como a forma de gestão que se define pela relação ética e transparente da empresa com os públicos com os quais ela se relaciona e pelo estabelecimento de metas empresariais compatíveis com o desenvolvimento sustentável, preservando recursos ambientais e culturais para as gerações futuras (INSTITUTO ETHOS, 2008). O envolvimento empresarial em ações sociais na comunidade começou na forma de ações voluntárias das empresas, focando problemas sociais (BRONN e VRIONI, 2001). Uma das principais características presentes nesse cenário é a mudança no conceito “daquilo que é importante, daquilo que tem valor, das metas a serem atingidas e dos meios de medir (indicadores) o progresso coletivo em direção a essas metas” (HENDERSON, 2001). Estes instrumentos de mensuração irão desempenhar diferentes papéis sob vários aspectos, pois orientam o campo econômico e social, sendo indispensáveis aos pesquisadores e empresários, além de contribuir para que o cidadão possa ter diferentes visões do que está ocorrendo na sociedade (BESSERMAN, 2003). Para a Organization for Economic Co-operation and Development – OECD (2008), um indicador deve ser entendido como um parâmetro, ou valor derivado de parâmetro, que aponta e fornece informações sobre o estado de um fenômeno com uma extensão significativa. Evans e Würster (2000) prescreveram a importância estratégica da informação que deixou de ser apenas uma ferramenta de controle para os gestores e tomou posição de um recurso básico que justifica sua utilização como base para a formulação de indicadores, principalmente para suporte ao processo decisório (LAUDON e LAUDON, 2000; RAYPORT e JAWORSKI, 2001). O Programa das Nações Unidas para o Desenvolvimento (PNUD, 2008) publicou o Índice de Desenvolvimento Humano (IDH) com propósito de desafiar as estreitas definições econômicas de progresso que compõem o Produto Interno Bruto (PIB). O IDH incorpora, além dos indicadores econômicos, os sociais, as estatísticas sobre os índices de alfabetização, a média da expectativa de vida e o poder de compra. 2. Fundamentação teórica O IDH tornou-se, pois, o mais importante instrumento de medida e de comparação da condição de vida não só de países, mas também de espaços menores como estados e municípios (BESSERMAN, 2003). A Comissão de Desenvolvimento Sustentável da ONU publicou, em 1996, Revista Gestão Industrial 57 57 Revista Gestão Industrial o documento Indicadores de Desenvolvimento Sustentável – estrutura e metodologia, apresentando um conjunto de indicadores econômicos, sociais e ambientais (IBGE, 2008). 3. Metodologia da pesquisa Os dados da pesquisa empírica, desenvolvida ao longo do ano de 2008, e obtidos segundo uma perspectiva indutiva, foram base do presente estudo. Nesta pesquisa, foi utilizado o método grounded theory (GLASER e STRAUSS, 1967) que é uma modalidade de pesquisa que busca gerar novas teorias através de conceitos, categorias e propriedades. A ênfase da grounded theory é o aprendizado a partir dos dados (interativa e indutiva), e não a partir de uma visão teórica existente (dedutiva). A maior diferença entre grounded theory e outros métodos de pesquisa é seu foco específico no desenvolvimento da teoria, através de uma contínua interdependência entre a coleta de dados e a análise. É um método que provê uma estrutura metodológica freqüentemente ausente em outras abordagens, tanto qualitativa como quantitativa, sem sacrificar a flexibilidade ou o rigor. A grounded theory foi desenvolvida no âmbito da pesquisa em ciências sociais, enfatizando a descoberta indutiva de teorias a partir dos dados analisados sistematicamente. Outros autores desenvolveram e debateram o método (GLASER, 1998; GLASER e HOLTON, 2004; STRAUSS e CORBIN, 1994; STRAUSS e CORBIN, 1997) reafirmando que: (a) a proposta principal do método é a construção de teoria, e não somente a codificação e análise de dados; (b) regra geral, o pesquisador não deve definir um quadro conceitual que antecede ao início da pesquisa, como premissa, para garantir que os conceitos possam emergir sem vieses conceituais pré-definidos; (c) a análise e a conceituação são obtidas através do processo de coleta de dados e comparação constante, no qual cada segmento de dados é comparado com construtos existentes, visando enriquecer uma categoria existente, formar uma nova ou estabelecer novos pontos de relação entre categorias. O universo desta pesquisa foi o conjunto das maiores organizações do ramo industrial, comercial e de prestação de serviços que atuam na economia nacional (REVISTA EXAME, 2008). O universo desta pesquisa foi o conjunto das maiores organizações do ramo industrial, comercial e de prestação de serviços que atuam na economia nacional (REVISTA EXAME, 2008). A pesquisa, foi desenvolvida ao longo do ano de 2008, com a coleta de dados, através de questionários eletrônicos, encaminhados diretamente aos executivos das 1.000 maiores empresas brasileiras, critério da publicação Melhores e Maiores e das 150 melhores empresas em sustentabilidade (REVISTA EXAME, 2008). Complementando as respostas dos questionários preenchidos, foram obtidas informações disponibilizadas diretamente nos sites corporativos das mesmas empresas da amostra pesquisada. o documento Indicadores de Desenvolvimento Sustentável – estrutura e metodologia, apresentando um conjunto de indicadores econômicos, sociais e ambientais (IBGE, 2008). Neste cenário se insere a ISO14000, para meio ambiente e, mais recentemente, a ISO16000, AA1000 e SA8000 (TACHIZAWA, 2009), como certificações sociais com o objetivo de atestar que a organização, além de ter procedimentos internos corretos, participa de ações não lucrativas. A Organização para a Cooperação e o Desenvolvimento Econômico – OCDE recomenda que as organizações, públicas e privadas, adotem princípios focados na transparência de suas ações. O balanço social se insere neste cenário de transparência e disseminação de informações junto aos seus diferentes públicos (RAYNARD e FORSTARTER, 2002). O Instituto Ethos de Responsabilidade Social (ETHOS, 2008) sugere um padrão de balanço social, que explicita os impactos da atividade da empresa na sociedade e evidencia o relacionamento com os seus diferentes públicos. O modelo Ethos considera, ainda, a apresentação do relatório sugerido pelo Instituto Brasileiro de Análises Sociais e Econômicas (IBASE, 2008), e constitui uma alternativa de evidenciação das atividades empresariais através de balanço social. A Bolsa de Valores de São Paulo (BOVESPA, 2008), face ao crescente interesse dos investidores em migrar para os portfólios verdes, e em resposta à demanda de bancos, fundos de pensão e gestores de recursos, organizou um índice de responsabilidade social e sustentabilidade (índice de sustentabilidade empresarial - ISE), baseado no Dow Jones Sustainability Indexes da Bolsa de Nova York - DJSI. Este indicador global de sustentabilidade monitora o desempenho financeiro das companhias de capital aberto. O DJ-SI, na sua composição, pondera a importância da integração dos fatores econômicos, ambientais e sociais na estratégia da empresa e avalia aspectos como inovação tecnológica, governança corporativa, interesse dos investidores, expectativas dos públicos de interesse, liderança e capacidade de resposta às mudanças sociais (TACHIZAWA e ANDRADE, 2008). Nesse sentido, torna-se fundamental aprimorar o processo de armazenamento e recuperação de informações, razão pela qual Draper e Dunlop (2008), procuraram desenvolver métodos de identificar e acessar informações relevantes segundo a percepção dos usuários de nível estratégico nas empresas. Vindo ao encontro disso, emerge o conceito de gerenciamento de desempenho corporativo (corporate performance management – CPM), cuja questão-chave não é só medir, mas projetar um processo que contemple decidir quais necessidades podem ser medidas, como e quando (BUYTENDIJK, WOOD e GEISHECKER, 2004). Motivado pela existência de uma lacuna entre o que os gestores necessitam em termos de sustentabilidade e o que ocorre no dia-a-dia das operações empresariais, foi desenvolvido o presente trabalho. 58 Revista Gestão Industrial 3. Metodologia da pesquisa Foram consideradas, a título de informações adicionais, informações relacionadas a balanços sociais e relatórios de sustentabilidade foram acessados, via Internet, junto às empresas da amostra, para fins de análise adicional. Foram consideradas, também, informações específicas obtidas de sites do Instituto Ethos, Ibase, Bovespa, IBGC, Revista Exame, Época e publicações especializadas em negócios. 59 59 Revista Gestão Industrial 4. Análise dos resultados 4. Análise dos resultados Para a concepção da arquitetura de dados socioambientais, procurou-se analisar as respostas das 458 empresas que responderam a pesquisa (42% de respostas em relação ao total das 1.150 empresas pré-selecionadas), o que permitiu estabelecer uma compreensão da sustentabilidade das organizações da economia nacional. Pela aplicação do questionário eletrônico e acesso aos sites das empresas, obtiveram-se dados relativos aos setores de atuação, constatando-se uma predominância das empresas industriais. Pelos dados primários coletados, ficou evidenciado que 65,7% do total das empresas da amostra que responderam ao questionário são indústrias, seguido das empresas de serviços com 21,6% e, complementarmente, 12,7% de empresas comerciais. Para entender metodologicamente a proposta de classificação das organizações em termos de sustentabilidade, considerou-se, inicialmente, uma classificação simples (organizações industriais, comerciais e de serviços), para posteriormente adotar uma tipologia mais completa de organizações que são interligadas entre si no ambiente empresarial brasileiro. Como empresas do setor industrial, enquadraram-se as organizações relacionadas a atividades vinculadas à siderurgia, ao cimento, ao papel e celulose, ao segmento metal-mecânico, à metalurgia, ao segmento automotivo, e a assemelhadas (bens duráveis e de consumo). São aquelas empresas que transformam insumos produtivos (matérias primas em geral) em produtos acabados. Já as empresas prestadoras de serviços foram enquadradas como tal, aquelas prestadoras de serviços financeiros (bancos, financeiras e corretoras valores e seguros), engenharia, publicidade e propaganda, hospitais, hotelaria e afins. Como empresas comerciais foram consideradas aquelas dedicadas ao ramo atacadista e varejista (lojas comerciais, distribuidoras e correlatas). Outra indagação inserida na pesquisa foi em relação ao foco de atuação da empresa em termos de sustentabilidade, cuja incidência de respostas é evidenciada na Tabela 1. 60 Revista Gestão Industrial Tabela 1- Ações socioambientais aferidas na pesquisa DISCRIMINAÇÃO SERVIÇOS INDUSTRIAL COMERCIAL Educação 36,4% 44,7% 29,5% Meio ambiente 11,9% 75,8% 43,1% Saúde 37,8% 22,3% 35,4% Ações Comunitárias 44,3% 31,1% 49,8% Fonte: Pesquisa de campo (2008) Fonte: Pesquisa de campo (2008) Pelas respostas, evidenciou-se uma preponderância de ações de proteção ambiental nas empresas industriais (75,8%). Nas demais empresas, serviços (11,9%) e comerciais (43,1%), notou- se menor ênfase com relação ao meio ambiente. Outras ações sociais e comunitárias (voluntariado, cultura, segurança, inclusão social, portadores de necessidades especiais, criança e adolescente, pessoas da terceira idade e proteção de animais) distribuíram-se, com ligeiro destaque aos setores comerciais e de serviços. As características socioambientais aferidas na pesquisa evidenciaram impactos de sustentabilidade diferenciados para cada tipo de empresa (vide Quadro 1). Quadro 1: Características socioambientais aferidas na pesquisa Quadro 1: Características socioambientais aferidas na pesquisa FATORES PESQUISADOS SERVIÇOS INDÚSTRIA COMÉRCIO a) cadeia produtiva sustentável Baixo Alto Médio b) impacto da produção no meio ambiente Nulo Alto Baixo c) impacto do produto no meio ambiente Nulo Alto Baixo d) fornecedores observam requisitos socioambientais Baixo Alto Médio e) barreiras institucionais/legais Baixo Alto Médio f) exigência recursos financeiros Baixo Alto Médio Fonte: dados da pesquisa Quadro 1: Características socioambientais aferidas na pesquisa Os resultados da análise destes fatores de influência pesquisados permitiram a identificação de características socioambientais intrínsecas a cada tipo de organização. Revista Gestão Industrial 61 61 Revista Gestão Industrial Tais tipos de organizações do universo empresarial brasileiro são inicialmente classificadas de forma simples, de acordo com a afinidade dos fatores pesquisados. Esses fatores (passivo socioambiental), posteriormente, foram ampliados, viabilizando dessa maneira, atingir o agrupamento de 10 tipos de organizações (vide Figura 2), conforme proposto neste trabalho. Na Figura 1, ilustrada a seguir, pode-se verificar os efeitos socioambientais diferenciados nas empresas dos três setores da economia. Figura 1: Efeitos socioambientais e os setores econômicos Figura 1: Efeitos socioambientais e os setores econômicos Fonte: Pesquisa de campo (2008) Fonte: Pesquisa de campo (2008) De acordo com a Figura 1, nota-se que a linha horizontal representa o tipo de empresa e na linha vertical o grau de efeito socioambiental, potencialmente causado pela empresa. Analisando os diferentes tipos de organizações, tem-se que as empresas prestadoras de serviços apresentam efeitos socioambientais quase que nulos, resumindo suas estratégias socioambientais às práticas de marketing institucional em termos de divulgação de balanços sociais e projetos sociais implementados nas áreas de: educação; cultura; voluntariado; e ações correlatas. No outro extremo, têm-se as empresas industriais causadoras, em potencial, de maiores impactos socioambientais, tais como aquelas vinculadas à siderurgia, cimento, papel e celulose, energia e similares. Fonte: Pesquisa de campo (2008) Fazendo uma análise, agora detalhada, dos fatores socioambientais pesquisados nas empresas da amostra (vide Quadro 1), puderam ser identificadas características de sustentabilidade diferenciadas, em função do tipo de organização, conforme pode ser observado na ilustração da Figura 2. 62 Revista Gestão Industrial Figura 2: Diferenciação das organizações em face de suas características socioambientais Figura 2: Diferenciação das organizações em face de suas características socioambientais Fonte: dados da pesquisa Fonte: dados da pesquisa Fonte: dados da pesquisa Nesta Figura 2, são apresentadas as características e grau de atuação socioambiental percebidos nos setores econômicos com relação aos fatores pesquisados. Tem-se um eixo de coordenadas onde a linha horizontal representa o tipo de empresa e na coluna tem-se o grau de impacto na comunidade e, portanto, exigências diferenciadas em termos de responsabilidade socioambiental, normalmente, praticadas pela empresa. Analisando os diferentes tipos de empresas, conforme modelo proposto deduz se que os bancos apresentam impactos ambientais quase que nulos, resumindo suas estratégias ambientais e sociais, praticamente, à divulgação de balanços sociais e projetos sociais comunitários. Ao lado dos bancos têm-se outras organizações como as instituições de ensino, empresas de serviços especializados que, além de enfatizarem essencialmente as estratégias sociais adotam, ainda, estratégias de tecnologias da informação que demandam efeitos favoráveis ao processo de gestão socioambiental, mesmo nestas organizações de baixíssimo impacto ambiental. No outro extremo, têm-se as empresas da indústria altamente concentrada, provocadoras de fortíssimos impactos ambientais, tais como: siderúrgicas, cimento, papel e celulose, hidrelétricas e afins. Entre estes dois extremos têm-se os outros tipos de empresas (empresas comerciais, empresas produtoras de bens de consumo duráveis, e outros tipos) que, normalmente, podem adotar estratégias socioambientais compatíveis com o grau de impactos ambientais causados pelos seus processos e estratégias sociais coerentes em função com o grau de expectativa da comunidade na qual está inserida. Os efeitos socioambientais nas empresas puderam ser graduados em 10 tipos de acordo com suas atividades econômicas e, portanto, em função do setor econômico a que pertencem. Desde Revista Gestão Industrial 63 63 Revista Gestão Industrial Revista Gestão Industrial empresas de baixo impacto socioambiental, como decorrência natural de suas atividades econômicas, que assumem comportamento ético também compatível com esta ínfima exigência em termos de responsabilidade social, até empresas do setor altamente concentrado, que adotam processo de avaliação de impactos dos produtos, processos e instalações, que precisa ser sistematizado, buscando antecipar às demandas e questões públicas. Fonte: Pesquisa de campo (2008) Esta última categoria de empresas, normalmente, necessita de certificação internacional do tipo selo verde e/ou equivalentes instituídos pela SA8000, AA1000, ISO14000 e congêneres. Os 10 tipos de organizações, classificadas segundo seus efeitos socioambientais (Passivo) pertinentes, são propostos e descritos a seguir. Organizações sociais (tipo A): a organização assume responsabilidades perante a sociedade e toma ações em relação ao exercício da cidadania coerente às suas atividades econômicas. A promoção do comportamento ético também é compatível com seu ramo de negócios de baixo impacto ambiental e ínfima exigência em termos de responsabilidade social. É o caso de cooperativas e associações; organizações sociais; e atividades correlatas. Empresas de serviços (tipo B): A organização reconhece os impactos causados por seus produtos, processos e instalações, apresentando algumas ações isoladas no sentido de minimizá-los. Enfatiza a promoção do comportamento ético. Posicionamento, normalmente, exigido para empresas de prestação de serviços especializados; firmas de engenharia; e organizações afins. Empresas comerciais (tipo C): A organização adota práticas socioambientais para atenuar os potenciais impactos dos produtos comercializados, processos e instalações. Tende a exercer certa liderança em questões de interesse da comunidade. Existe envolvimento das pessoas em esforços de desenvolvimento social. Médias e grandes organizações do setor de comércio varejistas e atacadistas; e afins. Instituições financeiras (tipo D): A organização adota práticas socioambientais para atenuar os impactos de seus serviços, processos e instalações. A organização promove o comportamento ético. Empresas prestadoras de serviços financeiros; bancos; seguradoras; e empresas de serviços em geral. Hospitais e Hotelaria (tipo E): A organização adota práticas socioambientais para atenuar os impactos de seus serviços, processos e instalações. A organização lidera questões de interesse da comunidade e do setor. O estímulo à participação das pessoas em esforços de desenvolvimento social é sistemático. Existem formas implementadas de avaliação e melhoria da atuação da organização no exercício da cidadania e no tratamento de suas responsabilidades públicas. Posicionamento, normalmente, exigido para hotéis, hospitais e organizações prestadoras de serviços de lazer e entretenimento. 64 Revista Gestão Industrial Empresas de médio efeito socioambiental (tipo F): A organização adota práticas socioambientais para atenuar os médios impactos de seus produtos, processos e instalações. Busca antecipar as questões públicas. A empresa publica balanços sociais e cumpre padrões anteriormente estruturados nos estágios anteriores. Posicionamento, normalmente, exigido para empresas de materiais de construção; do setor automotivo; confecções e têxteis; e higiene e cosméticos. Fonte: Pesquisa de campo (2008) Indústria de bens de consumo não-duráveis (tipo G): O processo de avaliação dos impactos dos produtos, processos e instalações precisa ser sistematizado, buscando antecipar as questões públicas. A empresa necessita, normalmente, de certificação internacional do tipo selo verde e/ou equivalentes instituídos pela SA8000, AA1000 e equivalentes. É o caso das empresas pertencentes a setores econômicos como: alimentos; agronegócios; e atividades correlatas de alto impacto socioambiental. Indústria de bens de consumo duráveis (tipo H): O processo de avaliação dos impactos dos produtos, processos e instalações precisa ser sistematizado, buscando antecipar as questões públicas. Adota, normalmente, certificação internacional do tipo selo verde e/ou equivalentes instituídos pela SA8000, AA1000 e equivalentes. É o caso das empresas pertencentes a setores econômicos como: construção pesada; plásticos e borracha; eletroeletrônicos; metalurgia e atividades correlatas de significativo impacto socioambiental. Indústrias de alto efeito socioambiental (tipo I): O processo de avaliação dos impactos dos produtos, processos e instalações precisa ser sistematizado, buscando antecipar as questões públicas. A empresa necessita de certificação internacional do tipo selo verde e/ou equivalentes instituídos pela SA8000, AA1000 e equivalentes. Deve adotar princípios de governança corporativa e cumpre padrões cumulativamente estruturados nos tipos anteriores. Neste nível alcançado pelas organizações deste tipo, deve ser considerada como uma exigência a ser “cobrada” das grandes organizações, cujas características socioambientais exigem tal posicionamento. É o caso das empresas pertencentes a setores econômicos como: papel e celulose; tabaco; farmacêutico; bebidas; química leve; e atividades correlatas de alto impacto socioambiental. São empresas de capital altamente concentrado e aplica-se, àquelas de grande porte com ações em bolsa de valores. Indústrias de altíssimo efeito socioambiental (tipo J): O processo de avaliação dos impactos dos produtos, processos e instalações precisa ser sistematizado, buscando antecipar as questões públicas. A empresa necessita de certificação internacional do tipo selo verde e/ou equivalentes instituídos pela SA8000, AA1000 e congêneres. Deve adotar princípios de governança corporativa e cumpre padrões cumulativamente estruturados nos tipos de organização anteriormente descritos. É o caso das empresas pertencentes a setores econômicos como: petroquímica; química pesada; mineração; hidrelétricas; termoelétricas e usinas nucleares; cimento; fabricantes de munições; armamento militar; fabricantes de agrotóxicos; produtoras de sementes transgênicas e Revista Gestão Industrial 65 65 Revista Gestão Industrial atividades correlatas de altíssimo impacto socioambiental. São empresas de capital altamente concentrado e aplica-se, àquelas de grande porte com ações em bolsa. Fonte: Pesquisa de campo (2008) A caracterização de cada tipo pode ser útil para sinalizar suas respectivas ações gerenciais correspondentes na forma de estratégias de sustentabilidade econômica. Esta tipologia permitiria, ainda, estruturar uma base de dados de forma a agrupar, em um mesmo lócus virtual, informações de referência acerca da sustentabilidade empresarial e, principalmente, de dados de custos socioambientais. 5.1. Mapeamento socioambiental Ficou evidente, pelos resultados da pesquisa, que uma empresa, qualquer que seja seu estilo de gestão, possui “efeitos” socioambientais, que são diferenciados em decorrência natural do setor econômico no qual está inserida. Estes efeitos, de acordo com o diagnóstico de sustentabilidade proposto neste trabalho (Quadro 2), podem ser representados na forma de passivo socioambiental. Para fazer frente a estes efeitos, a empresa necessita implementar ações correspondentes como contrapartida, na forma de deveres e obrigações (ativo socioambiental). Quadro 2. Ativo e Passivo Socioambiental Fonte: Autoria própria (2009) Fonte: Autoria própria (2009) Ou seja, o ativo (custos socioambientais) seria o quanto de ações socioambientais seria necessário adotar, por iniciativa da direção da empresa, para preservar os processos produtivos de forma sustentável. É o quanto de insumos produtivos e de providências gerenciais é necessário para continuar a produzir bens e serviços que consomem e absorvem recursos produtivos na forma de matérias-primas. A analogia, simplificada, que se pode fazer é com uma pessoa de classe média, que possui carro para trabalhar. Esta pessoa, com seu carro, consome gasolina que emite gás carbônico, Revista Gestão Industrial 66 66 Revista Gestão Industrial equivalente a uma árvore que teria que plantar, mensalmente, para compensar tal efeito ambiental nocivo. Neste raciocínio simplista, como há uma equivalência entre os efeitos socioambientais provocados pelo consumo mensal de gasolina (custos socioambientais pela metodologia ora proposta) com a “compensação” na forma da árvore plantada, o confronto entre “passivo socioambiental” e “ativo socioambiental” seria zero. Ou seja, não há saldo favorável nem desfavorável do desempenho individual desta pessoa no contexto de sua vida privada. Caso esta pessoa adotasse outras providências compensatórias como, além de plantar uma árvore por mês, por exemplo, instalasse um equipamento de gás veicular e adotasse transporte solidário, ela, certamente, teria um saldo socioambiental positivo a seu favor. O ativo (custos socioambientais), teoricamente, seria maior que o passivo (efeitos socioambientais) provocado pela emissão de gás carbônico, agora reduzido pelo consumo alternativo de gás veicular ao invés da gasolina. Fazendo uma analogia com o cenário empresarial, seria como se contentar com o levantamento de emissão e das projeções de neutralização de carbono, via plantação de mudas de árvores. Para calcular o impacto total do mundo dos negócios, devem ser somados os efeitos socioambientais de todo universo empresarial. Quanto maior for o mundo empresarial, menor será a quantidade de ativo socioambiental disponível por empresa. Como o consumo de insumos produtivos por parte das empresas estaria ocorrendo numa velocidade maior que a capacidade de reposição, significa que a continuidade das organizações está sob risco econômico de sobrevivência a longo prazo. Este diagnóstico socioambiental, coerentemente com os fatores de análise da sustentabilidade pesquisados (Quadro 1), pode ser representado na forma de um balanço de sustentabilidade. 5.2. Arquitetura da base de dados A base de dados de sustentabilidade empresarial está centrada na formulação de um indicador de desenvolvimento socioambiental – IDS que, de acordo com a proposta deste trabalho, refletiria o estágio em que se encontra a empresa em termos de sustentabilidade. A proposta de modelagem sistêmica desta arquitetura pode ser sintetizada na Figura 3, conforme evidenciada a seguir. 67 Revista Gestão Industrial Figura 3. Modelo conceitual de diagnóstico socioambiental Fonte: Autoria própria (2009) Fonte: Autoria própria (2009) Fonte: Autoria própria (2009) A modelagem lógica da base de dados (vide Figura 4), conceitualmente, foi estruturada em quatro níveis de análise para se chegar aos valores dos indicadores de desenvolvimento socioambiental – IDS e correspondente balanço de sustentabilidade. 68 Revista Gestão Industrial Figura 4. Modelagem da base de dados socioambientais Figura 4. Modelagem da base de dados socioambientais Fonte: dados da pesquisa Fonte: dados da pesquisa No primeiro nível é feito o diagnóstico socioambiental (critérios de diferenciação conforme enunciados na Figura 4) onde são analisadas as dimensões de sustentabilidade, transparência, governança corporativa, e capital humano. Em um segundo nível, é estruturada a base de dados de IDS (vide Figura 5) das empresas do universo empresarial brasileiro, classificadas por setor econômico, de forma apriorística (com padrões de IDS variando em intervalos pré-estabelecidos), a partir dos fatores analisados do passivo ambiental. A métrica, singular a cada empresa analisada, apurada a posteriori, a partir do cumprimento dos quesitos estabelecidos como ativo ambiental, pode variar nos intervalos do IDS de cada setor econômico. Empresas do tipo A, por exemplo, tiveram os valores de IDS variando de 0 a 1; as do tipo 2 com valores no intervalo entre 1,1 a 2,0 e assim sucessivamente até as empresas do tipo J, com valores variando de 9,1 a 10. 69 69 Revista Gestão Industrial 69 Revista Gestão Industrial Figura 5. Base de Dados com métricas de IDS Fonte: Autoria própria Como alternativa de custeio, poder-se-ia, simplesmente, efetuar o somatório em termos absolutos dos elementos que compõem o ativo socioambiental, apurando os custos de sustentabilidade. Porém nesta proposta, optou-se pela sinalização desses custos como referencial de atuação da gestão da controladoria da empresa, na forma de IDS. Ou seja, o IDS na forma proposta neste modelo, é um indicador derivado do desempenho socioambiental esperado na média das empresas que compõem cada um dos segmentos econômicos analisados. Isto permitiria o estabelecimento de uma escala para posicionar as empresas em face de seus diferentes estágios de sustentabilidade. No terceiro nível, são concebidos os recursos computacionais para geração do balanço socioambiental e, principalmente, de parâmetros de recuperação das informações de sustentabilidade. 70 Revista Gestão Industrial Figura 6. Representação da estrutura de programas do software SIMASE g p ç p g Fonte: Autoria própria Fonte: Autoria própria Fonte: Autoria própria E, no quarto nível, é criado o software de interface com o usuário, para permitir buscas, consultas e acesso on-line aos dados armazenados pelo sistema (balanço socioambiental, indicadores e demais informações de sustentabilidade). Em termos de programação e desenvolvimento de software, a modelagem da base de dados poderia ser explicitada conforme ilustração da Figura 6. Esses recursos podem ser sistematizados em um Sistema Informatizado de Monitoramento Ambiental e de Sustentabilidade Empresarial – SIMASE. A emissão desse balanço ocorre nesse quarto nível, observando enfoques diferenciados de sustentabilidade para diferentes organizações que, em razão de seu ramo de negócios, sofrem efeitos socioambientais distintos (Quadro 3). 71 Revista Gestão Industrial Quadro 3. Estrutura do Balanço Socioambiental Quadro 3. Estrutura do Balanço Socioambiental Fonte: Autoria própria (2009) Fonte: Autoria própria (2009) O balanço socioambiental, composto do ativo e passivo, pode ser estruturado na forma de planilha considerando um diagrama de dupla entrada (modelo em “T”). Evidencia de um lado, os efeitos socioambientais gerados pela organização (fatores de análise do Quadro 1 são quantificados, a priori, como passivo), e de outro, as decisões de sustentabilidade da sua Administração, com os correspondentes ônus econômico (custos socioambientais apurados, a posteriori, como ativo)) para fazer frente às exigências socioambientais decorrentes das características de sua cadeia produtiva. Dessa maneira poder-se-ia apurar os custos socioambientais de uma determinada organização, bem como o comportamento de organizações pertencentes a um mesmo setor econômico. Nesse quarto nível seria gerada analiticamente, ainda, tela de consulta, por empresa, conforme exemplificada graficamente na figura 8, para uma empresa hipotética “Alpha S/A”. Nela, são evidenciadas informações que caracterizam a empresa (nome da empresa, setor econômico e IDS) e aquelas inerentes ao cumprimento dos quesitos inerente aos custos socioambientais (Ativo). Estes mesmos quesitos de custos de sustentabilidade, de forma alternativa, poderiam ser apurados em termos absolutos, como elementos de custeio do ativo socioambiental. A depender do setor econômico a que pertence a empresa, pode-se alterar os quesitos de custos, uma vez que eles variam em função das características da cadeia produtiva. Uma empresa de serviços especializados, por exemplo, não precisaria de sistema de normatização do tipo ABNT/ISO14000, ou mesmo de boas práticas de governança corporativa, podendo dar lugar a outros quesitos pertinentes a esse tipo de empresa de prestação de serviços (por exemplo: implementação de projetos de cidadania corporativa, programas de capacitação de fornecedores, entre outros). Ou seja, sistêmicamente, as informações de criação do balanço socioambiental observaria a estrutura de dados ilustrada na Figura 7 explicitada a seguir. E que é, meramente, uma visão sistêmica interna, baseada na dimensão do balanço explicitada no Quadro 3. 72 Revista Gestão Industrial Figura 7. Visão sistêmica do programa de criação do balanço socioambiental g p g ç ç Fonte: Autoria própria (2009) Fonte: Autoria própria (2009) Nesta visão do balanço socioambiental evidenciada na Figura 7, é exemplificada a situação de uma empresa do tipo J (vide Quadro 1), que é uma organização de altíssimo efeito socioambiental. O Passivo deve ser o espelho da tipologia estabelecida no Quadro 1, enquanto o Ativo representa o quanto de ações de sustentabilidade a empresa adota. Quadro 3. Estrutura do Balanço Socioambiental Neste caso existe uma defasagem entre o que seria exigido, normalmente, para uma empresa de altíssimo efeito socioambiental e o que é adotado de práticas compensatórias desses impactos provenientes das peculiaridades dessa empresa. Ou seja, a empresa, sob análise, para ter efeito nulo (Ativo igual ao Passivo), teria que compensar cada ônus causado pela empresa, que é o Passivo, por equivalente prática do lado do Ativo. Os elementos que compõem o Passivo e correspondente contrapartida do lado do Ativo podem ser desdobrados em subitens de análise, a depender do tipo de empresa (como foi o caso do tópico balanço social e cargos e salários, que no exemplo da empresa do tipo J, se desmembraram em 2 subitens). Por exemplo, no caso de uma instituição financeira (bancos, e afins), não deve haver ênfase em termos de proteção ambiental e, portanto, não haveria exigência em termos de ISO14000. No entanto, elemento como a ética empresarial mereceria destaque neste tipo de organização podendo, seu código de ética ser desmembrado em itens como: ética em relação aos clientes; ética em relação aos fornecedores; ética em relação ao seu público interno; código de ética disponibilizado na Internet e Intranet. 73 Revista Gestão Industrial Figura 8. Representação da Tela de Consultas Customizada por Empresa Fonte: Autoria própria (2009) Fonte: Autoria própria (2009) Fonte: Autoria própria (2009) Fazendo uma análise das informações de sustentabilidade (tela de consultas da Figura 8), e comparando-a com outras organizações concorrentes do mesmo setor econômico (benchmarking), podem ser estabelecidas ênfases de estratégias ambientais e sociais específicas para a empresa focalizada. Isto permitiria criar condições para o aprimoramento das práticas empresariais corporativas na medida em que as estratégias de negócios passariam a se apoiar cada vez mais em metas de sustentabilidade. Ou seja, dada a convergência com os objetivos corporativos, as informações de sustentabilidade de uma base de dados, possibilitariam que as empresas definam qual a melhor estratégia para a geração de valor, explorando o potencial de crescimento econômico, de forma integrada com suas ações socioambientais. 6. Conclusões O objetivo deste trabalho foi conceber uma arquitetura de base de dados para subsidiar o monitoramento e direcionamento da gestão de custos e de controladoria nas empresas. Ela sinalizaria os requisitos necessários para o processo de tomada de decisões de sustentabilidade, de forma convergente com os objetivos estratégicos da organização. Tal modelo sugere que sejam adotados enfoques distintos de gestão de custos inerentes à sustentabilidade para diferentes tipos de organizações que, em razão de seu ramo de negócios, sofrem efeitos socioambientais diferenciados. Além de refletir o estágio de sustentabilidade em que se encontra a empresa analisada, a arquitetura permitiria o mapeamento socioambiental dos diferentes segmentos econômicos do universo empresarial brasileiro. No contexto específico de uma determinada organização, Revista Gestão Industrial 74 74 Abstract It presents an architecture of data for diagnosis of social and environmental of organizations in terms of economic sustainability. It proposed a model of the database of indicators of social differentiation of costs, based on empirical research, developed the method grounded theory. The emphasis of grounded theory is that learning from the data (interactive and inductive), and not from a theoretical vision existing (deductive). Such indicators, and reflect the stage of sustainability where the company tested, sinalyzing the mapping of different socio economic segments of the Brazilian business universe. The architecture, structured with the support of information technology resources would allow the planning decisions inherent in the management of social costs of their production chain. Key-words: sustainability; architecture of data sustainability; accountability indicator. Revista Gestão Industrial Revista Gestão Industrial possibilitaria que fossem planejadas decisões empresariais, indutoras de custos socioambientais, em sua cadeia produtiva e de ações voltadas à evidenciação (disclosure) contábil. O modelo proposto partiu do pressuposto de que os resultados corporativos passam a depender cada vez mais de decisões de controladoria que levem em conta que: a) não há conflito entre lucratividade e a questão socioambiental; b) clientes e comunidade passam a valorizar cada vez mais a proteção do meio ambiente; c) a demanda e, portanto, o faturamento das empresas sofre crescentes pressões e depende diretamente do comportamento de consumidores que enfatizarão suas preferências para produtos e organizações eticamente corretas. Outros resultados específicos, decorrentes da implementação dessa arquitetura, poderiam ser obtidos quanto à evidenciação externa, com oportunidade e clareza, possibilitando a perfeita compreensão, por parte dos stakeholders, da verdadeira situação que se encontra a organização. Ou seja, dados decorrentes do balanço de sustentabilidade e mesmo do IDS apurado, poderiam fazer parte do Relatório da Administração, que acompanha as demonstrações financeiras – DFs, e do próprio balanço social, normalmente, divulgado pela empresa. Referências bibliográficas BESSERMAN, Sérgio. A Lacuna das informações ambientais. In: TRIGUEIRO, André (Coord.). Meio Ambiente no século 21. Rio de Janeiro: Sextante, 2003. século 21. Rio de Janeiro: Sextante, 2003. BOLSA DE VALORES DE SÃO PAULO. BOVESPA. São Paulo, 2008. Disponível no site www.bovespa.com.br, Acesso em: 30 abr. 2008. Acesso em: 30 abr. 2008. BRONN P. S.; VRIONI, A. B. Corporate social responsibility and cause-related marketing: an overview. International Journal of Advertising, n.20, n.2, 2001. BRONN P. S.; VRIONI, A. B. Corporate social responsibility and cause-related marketing: an overview. International Journal of Advertising, n.20, n.2, 2001. BUYTENDIJK, F.; WOOD, B.; GEISHECKER, L. Mapping Road to Corporate Performance Management. Gartner Group, January, 2004. DRAPER, S. W.; DUNLOP, M.D. New IR-New Evaluation: the impact of interaction and multimedia on information retrieval and its evaluation, 2002. Disponível em: http://www.cs.strath.ac.uk/~mdd/research/publications/nrhm/new_IR_new_eval.pdf. Acesso em: 30 abr. 2008. 75 Revista Gestão Industrial EVANS, P.; WÜRSTER, T. Blown to Bits: How the Economics of Information Transforms Strategy, Boston. Harvard Business School Press, 2000. GLASER, B.; STRAUSS, A. The Discovery of Grounded Theory, Chicago: Aldine, 1967. GLASER, B. Doing grounded theory: issues and discussions. Mill Valley, Ca.: Sociology Press, 1998 GLASER, B. ; HOLTON, J. Remodeling Grounded Theory. The Grounded Theory Review. V.4, n.1, November 2004. HENDERSON, H. Transcendendo a economia. Tradução de Merle Scoss. 10ª ed. São Paulo: Editora Cultrix, 2001. INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA – IBGE. Indicadores de desenvolvimento sustentável – Brasil. Brasília: IBGE, 2008. INSTITUTO BRASILEIRO DE ANÁLISES SOCIAIS E ECONÔMICAS. Guia de Balanço Social – IBASE. Disponível em www.ibase.org.br. Acesso em: 01 mai. 2008. INSTITUTO ETHOS DE RESPONSABILIDADE SOCIAL. Matriz de Evidências de Sustentabilidade, Banco de Práticas e Ferramentas de gestão: Indicadores Ethos, Guia de Elaboração de Balanço Social. Disponível em www.ethos.org.br. Acesso em: 01 mai. 2008. Práticas e Ferramentas de gestão: Indicadores Ethos, Guia de Elaboração de Balanço Social. Disponível em www.ethos.org.br. Acesso em: 01 mai. 2008. LAUDON, K.C.; LAUDON, J.P. Management information systems: organization and technology in the networked enterprise. New Jersey: Prentice-Hall, 2000. ONU – Organização das nações Unidas. Contabilidad financiera y presentación de informes ambientales por las empresas. http://www.unetad.org/sp/sphome.htm. Acesso em 15 nov. 2007. ORGANIZATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT - OECD. Core set of Indicators for Environmental Performance Reviews. A synthesis report by the Group on the State of the Environment. Environment Monographs N° 83. Paris: OECD, 1993. Disponível em: http://lead.virtualcentre.org/en/dec/toolbox/Refer/gd93179.pdf. Acesso em 30 abr. 2008. Referências bibliográficas PROGRAMA DAS NAÇÕES UNIDAS PARA O DESENVOLVIMENTO. Relatório de Desenvolvimento Humano 2007/2008: Combater as alterações climáticas: Solidariedade humana num mundo dividido. PNUD, UN Plaza, New York, 2007. Disponível em: http://www.pnud.org.br/arquivos/rdh/rdh20072008/hdr_20072008_pt_complete.pdf. Acesso em: 01 fev 2008. RAYNARD, P.; FORSTARTER, M. Corporate Social Responsibility: Implications for Small and Medium Enterprises in Developing Countries. United Nations Industrial Development Organization, Viena, 2002. Disponível em: http://www.unido.org/doc/5162. Acesso em 01 fev. 2008. REVISTA EXAME. Melhores e Maiores e As Melhores Empresas em Cidadania Corporativa, São Paulo. Editora Abril, 2008. REVISTA EXAME. Melhores e Maiores e As Melhores Empresas em Cidadania Corporativa, São Paulo. Editora Abril, 2008. Dados dos autores: Revista Gestão Industrial Dados dos autores: Nome completo: Takeshy Tachizawa Filiação institucional: Faculdade Campo Limpo Paulista - FACCAMP Departamento: Programa de Mestrado Em Administração Função ou cargo ocupado: Professor/Pesquisador Endereço completo para correspondência: Avenida Francisco Jose Longo 555, São José dos Campos, São Paulo, CEP 04105-003. Telefones para contato: (12) 3922-8744 e-mail: usptakes@uol.com.br Nome completo: Jose Henrique Souza Filiação institucional: Pontifícia Universidade Católica de Campinas – PUCCAMP Departamento: Programa de Mestrado Em Administração Função ou cargo ocupado: Professor/Pesquisador Endereço completo para correspondência: R. Cel. Quirino, 910, ap. 102. Cep: 13025-001 - Campinas/SP Telefones para contato: (12) 3922-8744 e-mail: josehenriquesouza@yahoo.com.br REVISTA EXAME. Melhores e Maiores e As Melhores Empresas em Cidadania Corporativa, São Paulo. Editora Abril, 2008. 76 Revista Gestão Industrial STRAUSS, A.; CORBIN, J. Grounded Theory Methodology - An Overview. Handbook of Qualitative Research, N.K. Denzin and Y.S. Lincoln (eds.), Sage Publications, Thousand Oaks, CA, 1994. STRAUSS, A.; CORBIN, J. Grounded Theory in Practice. Sage Publications, London, 1997. TACHIZAWA, T. Gestão Ambiental e Responsabilidade Social Corporativa. 6a. edição revista e ampliada. São Paulo: Editora Atlas, 2009. TACHIZAWA, T. e ANDRADE, R. O. B. Gestão Socioambiental: estratégias na nova era da sustentabilidade. São Paulo: Campus Elsevier, 2008. Nome completo: Takeshy Tachizawa 77
https://openalex.org/W3120882664	https://zenodo.org/records/2183722/files/article.pdf	English	null	Radium in Medicine	Scientific American	1,903	public-domain	3,457	SCIENTIFIC AMERICAN HUPPLEMEN'l', No. 1455. But if, to make a purely hypothetical conjecture, some beneficent millionaire were to test the capacity of our old universities for undertaking this kind of work, and were to offer Rdequate meansfof the 1J1ITptrse, f feel pretty confi dent, from what I know of the spirit which dominates their governing bodies, that such an offer would be accepted both at Cambridge and here at Oxford, with few dissentients. If such a departure were placed within their power, I think that that great public which glories in the past achievements of these uni versities would rejoice in their new development. And I will further add that the creation of chairs of ap plied science would react upon and strengthen the teaching of al'l those sciences which are in any way connected with industrial productiveness. Radium Rays in TuberculOSis of the Lungs.-From the well known germicidal powers of radium, it is reasonable to suppose that it will prove a useful agent in tuberculosis of the lungs. Soddy, in the British Medical Journal, suggeȞts that radium be dissolved in water. He says that "instantly all its emanations are evolved in gas, and mixed with the air above the solu tion. Now let these emanations be removed by a cur rent of air passing through the solution. The all' with the emanations can now be stored in a gas holder in another room. Observations will show that one half of the emanations will disappear every four days, and in three weeks no emanations of radium will be left; but the solution from which it was obtained, has grown a fresh crop of emanations just ,as fast as the old ones disappear. That is, it takes about three weeks for the solution of radium to be as potent as it was in the beginning when the salts of radium were first dropped into it." i Inhalations of these emanations leave ·a thin film of radioactive substance on the lungs. This causes the phenomenon of induced radioactivity, which remains in the air cells of lungs, exercising a germicidal power over diseased tissue long after the emanations them selves have been exhaled. This "induced radioactivity" usually disappears in about three hours. For an in haler, an ordinary gas wash bottle provided with two taps could be used, so tliat there is no leakage of these precious and slowly formed emanations. CONCLUSIONS. 1. Thr discovȟry of radium may make it necessary to change our theories of the old hypothesis about matter and the conservation of energy. 1. Thr discovȟry of radium may make it necessary to change our theories of the old hypothesis about matter and the conservation of energy. 2. Radium may possibly open up the way for a cheaper and more wholesome lighting of houses by phosphorescence. f 2. Radium may possibly open up the way for a cheaper and more wholesome lighting of houses by phosphorescence. f p p 3. Radium is a practical agent to differentiate genu ine gems from artificial. gi 4. Radium is a useful agent to kill bacte-ria. gi 4. Radium is a useful agent to kill bacte-ria. 5 R di b id d l bl 5. Radium may be considered a valuable agent for the treatment of lupus, cancer, tuberculosis; and a possible agent to improve the eyesight and overcome partial blindness. No doubt, later discoveries will show it to be of service in other diseased conditions. Case I.-Dr. John McIntire, of Glasgow, reports a case of small lupus on the dorsal surface of the right hand, which was exposed to the radium rays daily for from twenty minutes to half an hour, the time uf exposure depending upon the skin reaction. The re sult was that within three weeks the lesion was entirely healed. The question of the recognition of app'lied science by our old universities must, as I said, be faced-the time is at hand for them to consider seriously whether it is desirable that they should cater for the train ing of those who are destined to be the founders and upholders of our national prosperity. The longer this question is shelved the smaller will grow the chances of their being able to participate in the work. At present we in this' country are not up to the German 'level so far as concerns the higher technical training of industrial leaders. Our universities, in other words, have not yet had to encounter the full force of com petition with newer institutions of the rank of the technical high schools. We have but very few, if any. SCIENTIFIC AMERICAN HUPPLEMEN'l', No. 1455. That is why I have taken advantage of the opportunity which has been placed in my hands for raising this note of alarm, because even if nothing practical results from this meeting, it may at any rate be useful to let it be known that many of us desire to see the most ancient and the most renowned of our educational foundations doing more for the edu{!ation of a nation the prosperity of which is so largely de pendent on productive industry. All these cases showed more or less improvement, and the two whOSE> history I now give, were cured. e > y Case I.-Male, aged thirty-seven years; operated on eight years ago for melanosarcoma of the left arm. He recently returned to the clinic because of a mUl tiple recurrence of the same. The numerous nodules were exposed to the radium rays for twenty-five min utes each day or two, depending upon the skin re action, and the cancerous nodule disappeared in one month. Case n.-Male, aged sixty-one yeȝrs; operated on for cancer of the mouth in 1888, 1891, and 1897, respective ly. He presented himself in the clinic again in April, 1903, with an inoperable cancer of the lip and palate. Radium was used as in the above case, and the can cerous .,.growth disappeared with complete ' healing in five -Weeks. p p y Whether as the outcome of the lectures delivered and the conferences held during this meeting the attitude of the universities toward applied science undergoes modification or not, the ventilation of opinions cannot but be of advantage in many ways. If, for example, it is made manifest that the current of national thought is moving slowly-alas! very slowly-toward the recognition' of science as the main factor of indus trial progress, it may help to emphasize the necessity for strengthening and developing the teaching of pure science. If the beneficent millionaires are not forth coming fo.r the purpose of endowing applMd science, there is, at any rate, ample scope for their beneficence in the endowment of pure science in our old universi ties. A school of active science workers would-to use a quasi-scientific expression found in the pages of many writers of fiction-"galvanize into life" the science of teaching of the schools. SCIENTIFIC AMERICAN HUPPLEMEN'l', No. 1455. If you can only help to mold the public mind into the belief that sci ence is a living reality veiling truths of inestimable value to humanity from every point of view, moral, social, and Ill11teria'l-truths that are to be wrested only by conscientious, laborious, and persistent re search-you will be assisting a great cause. If you will proclaim this dodrine from the housetops and assist ilJ. sweeping away that dustheap of formal text book knowledge which passes for science in our ex amination rooms you will be doing something toward raising the general level of opinion in this country. We need it badly! Think of all the creative inte'1lec tual power running to waste-the unrealized assets in the way of originality of thought which Great Britain might have at her disposal if the brain power of her teachers and students were only diverted into the right channels. The old universities, by virtue of their pres. tige, their traditions, and their past achievements, have still a powerful hold upon the public mind. They must open their doors still more widely to science if they wish to retain their hold. If their means are at present insufficient to enable them to meet the re quirements of the age, they can still forward the na tional cause by upholding the dignity of science, by insisting upon originality of thoughi as an essential qualification for its successful teaching, and by help ing to dispel the notion that it undergoes degradation by being applied to human welfare. It must be real ized, and it cannot be realized too soon, that the peace ful campaign of industria'l competition requires lead ers well trained in scientific method, and not crammed witnmere formal book learning-men as alert in mind amI resourceful in meeting difficulties, as upright in principle, as keen in enthusiasm, as far-seeing in imagination, and with as intimate a knowledge of hu man nature as the statesmen, warriors, divines, law yers, and schoolmasters which these old universities have given to the 'country. The victory of the future is with that nation which enables her children to approximate more closely toward Tennyson's ideal: iW Dr. Willy Meyer, and William J. Hammer, of New York, report a case of recurrent cancer in which the X-rays, and also Co-ley's flUid, had been used for about a year without avail. Last July, Dr. Meyer began ra dium treatment. SCIENTIFIC AMERICAN HUPPLEMEN'l', No. 1455. The radium was of 300,000 activity, and was applied every day for one minute. The large recurrent growth in the axillary line got smaller and less painful, and the smaller disseminated nodules broke 'more quickly. However, the case was so far advanced that, while improvement took place, a cure could not be accomplished. Dr., Andrew H. Smith, of New York, while abroad this summer, saw a case of rodent ulcer which had been treated by the X-rays without benefit. The ul cer was exposed to radium rays for five treatments, which resulted in perfect healing of the parts. Bacteriological Experiments with Radium.-The ob servations made with radium in bacteriology have been somewhat limited. However, Caspari and Ashkassi, of Italy, have exposed culture tubes of the Micrococcus prodi,giosus to' the rays of radium, and it has been found to have a fatal effect on this organism in three hours. . why they should not do so here. The form in which 'the question may be put is there fore whether, given the means, the older universities should develop their work in the direction of apB-li'd science. A large body-I cannot say how many-of outsiders who are well-wishers of these universities, is of opinion that they should, and there is an idea abroad that they are, suffering Rnrtncia'lly now from having neglected"'this -side of education in the past. There was, for example, a leading article in the Times of May 25 in the counie of which the writer suggests that they may have suffered through having a țalse reputation for being very wealthy bodies, and he adds: "Or is it, perchance, because the modern millionaire, being a man of his ag3, and an Englishman to boot, has no great belief in the economic value of knowl edge as such, and no great confidence in the capacity of our ancient universities to adapt themselves to the needs of the coming time?" Now, so far as the chem ical manufacturers of this country are concerned, I can say with some personal experiences of my own that they certainly have shown no great belief hitherto in the economic value of scientific knowledge, as they now know to their own cost. * Read at the .emi-annnal meeting of the Medical SOCiety of the St", of New York, held at the New York Academy of Medicine, Octobe" li- 1903. SCIENTIFIC AMERICAN HUPPLEMEN'l', No. 1455. SCIENTIFIC AMERICAN HUPPLEMEN'l', No. 1455. NOVEMBl<JR 21, 1903. 23315 university. But this is a matter of expediency, and not a real conflict between fundamental principles. I cannot find that the classical teaching of the American or German universities has been impaired by the splen did development of their scientific faculties; neither does it appear that the human products of their scien tific activities are in the least degree inferior as men to their classical scholars. Of course, I am a special pleader, and I am making the best use of my oppor tunities, and I repeat that I never could see where any antagonism existed between the older and the newer studies, excepting in the struggle for financial means. There are many educational authorities here and abroad who will tell you that th,e scientific devel opment of the German universities has reacted upon and improved the classical teaching by an infusion of scientific method into the latter. Moreover, it must be remembered that we who are advocates for the new education are not clamoring, as many people think, for the abolition of the old studies. I for one should deplore any falling off in the prestige of the old uni versities as seats of classical learning. Neither is it su'ggested that our future leaders of industry would never at any period of their studies derive benefit from that particular kind of culture which the ancient literature is capable of imparting. I firmly believe they would; but the question as to when and how would open up the whole field of education, elemen tary, secondary, and university, both pre- and post graduate, and I shou'ld find myself floundering among shoals and quicksands in no time. The ideal university is one that offers facilities for both the older and the, newer education; they are not mutually exclusive they can, and do, thrive side by side elsewhere, and there is no reason, at any rate no theoretical reason, why they should not do so here. educational standard. The effect cannot but be to cause the older universities to become of smaller im portance in the general scheme of national education as time goes on. CONCLUSIONS. schools of this status here now, but if I read the signs of the times correctly, the differentiation between the old and the new education-which has already become well marked- is bound with the progress of science to become more and more strongly pronounced. Our newer universities-especially those in large manu facturing centers-will be driven more and more into the teaching Of app'lied science, and our polytechnics and te(!hnical colleȜel$ will perforce have to raise their SCIENTIFIC AMERICAN HUPPLEMEN'l', No. 1455. From five to ten milligrammes of dry radium bromide should be introduced into the wash bottle and a few drops' of water drawn in to dissolve it, the taps being imme diately closed. For the first treatment, the first few bubblES of gas should be inhaled with a deep breath, gradually increasing the dose. Repeat this treatment every day. In this manner the emanations of radium and their radioactivity, which is inimical to germ life, would do their work at the seat of the disease. . . . the crowning race Of those that eye to eye shaH look On knowledge; under whose command Is Earth and Earth's; and in their hand Is Nature like an open book." On knowledge; under whose command p Of course, this is all hypothesis-the most nebulous of hypotheses. We all know, unfortunately, that the financial resources of the universities have been, and are, inadequate for the purpose of enabling them to meet the requirements of modern scientific education, either in the way of staff, accommodation, or equip ment. It can be said, and justly said, that so long as these univ"rsities are without the means of developing their schools of pure physical science to an extent worthy of their reputation, it is useless to talk a bout developing the teaching of app'lied science. So it may be. But I remind you that we are still in the region of hypothesis, and the captious critic might retort by saying they they have not done even as much as they might, and could, have done for the proper development of scientific teaching with the means al ready at their disposal-that they are still over weighted by ancient tradition, and that their internal scientific forces are still feeble as compared with the preponderating forces of the advocates of the older culture. There is no time, even if I knew enough about the inner mechanism of university administra tion, to discuss this aspect of the question, but if you want to know an American view of the case-a strong view!-I would invite attention to an address by Prof. Victor Alderson, Dean of the Armour Institute of Technology, de'livered before the Chicago Literary Club in October last year, an abstract of which was published in Nature of ' February 12. l Is Nature like an open book." RADIUM IN MEDICINE.* Perhaps the foregoing reports are somewhat rosy colored in their expectations; on the other hand, they may be in some respects not sufficiently extravagant time will tell. However, to the inexperienced it is well not to draw unwarrantable conclusions concern ing this wonderful substance, and to imagine that in radium we have a specific for all incurable diseases. No doubt, in time, scientific experimentation will show its value as a remedy, and perhaps reveal its mysteries, and the natural laws through which it works. By SAMUEL G. TRACY, B.S., M.D., New York. THERE is no doubt that the salts of radium by their radiations have a positive effeet on diseased tissues, and, even at this early stage of experimentation, it cer tainly looks as though their use were indicated in lu pus, rodent ulcer, superficial cancer, and some cases of deep cancer; in ce!'tain kinds of skin diseases, atrophy of the optic nerve, and partial blindness from other causes. In deep'seated cancers, the X-rays have, as a rule, not given satisfactory results. It would al most seem that, in the radium rays, which are more powerful and their emanations of radioactivity more penetrating, we have a new agent which is m?r€' likely to give better results in some of the cases which have heretofore been considered incurable. As yet, comparatively few physicia-ns have reported on the efficiency of radium rays in deep-seated cancer. Never theless, there is sufficient encouragement in the cases which I now collect and report, to make us feel that perhaps we are on the right road to find the specific cure for cancer and tuberculosis. NEW MORDANTING PROCESS. IT is well known that the ordinary method of mor danting wool with a bichromate and a reducing agent always makes the fiber more or less tender, and Amend proposed to substitute the use of a solution of chromic acid containing 1 to 2 per cent of the weight of the wool, at a temperature not exceeding 65 deg. C., and to treat it afterward with a solution of sodium bisul phite. Acco"rding to a recent French patent, better re sults are obtained with neutral or slightly basic, chro mium sulphocyali/'ide. This salt, if neutral or only very slightly basic, will mordant wool at 65 deg. C. The double sulphocyanide of chromiuip and ammo nium, got by dissolving chromic oxide In ammonium sulphocyanide, can also be used. Nevertbeless, in or der to precipitate chromium chromate on the fiber, it is advisable to have a soluble chromate and a nitrate present, as well as a soluble copper salt and a free Acid, One example of the process is as follows: Make the bath with 2 to 3 per cent of 'lmmonio-chromium Ca&e I I.-Female, , aged twenty-eight years, with lu pus of the nostril and nose about one inch in diameter. She had the same daily treatment as in the previous case, with the result that she was cured in four weeks. Case IlL-Rodent ulcer, under treatment only two' weeks at the time of the report. and even in this short time the discharge had dried up and the ulcer was in a fair way to heal. . Case IlL-Rodent ulcer, under treatment only two' weeks at the time of the report. and even in this short time the discharge had dried up and the ulcer was in a fair way to heal. . y . Dr. Oudin, of Paris, says that he has cured some cases of lupus with radium rays, but the report does 'not give the details. y Dr. Oudin, of Paris, says that he has cured some cases of lupus with radium rays, but the report does 'not give the details. Prof. Gassenbaurer reports to the Vienna Medical Society no less than twenty cases of cancer treated by radium during the six months ending July 1. Prof. Gassenbaurer reports to the Vienna Medical Society no less than twenty cases of cancer treated by radium during the six months ending July 1. © 1903 SCIENTIFIC AMERICAN, INC,
https://openalex.org/W2344913226	https://ojs.tdmu.edu.ua/index.php/here/article/download/1693/1641	Russian	null	АЛГОРИТМ КЛАСИФІКАЦІЇ ПОЛІТРАВМ МЕТОДОМ ІНДУКЦІЇ ДЕРЕВА РІШЕНЬ	Medična ìnformatika ta ìnženerìâ	2,013	cc-by	2,996	МЕДИЧНА ІНФОРМАТИКА ТА ІНЖЕНЕРІЯ УДК61:004.651 (075.8) Мета даної роботи - розробити і програмно реалі- © P. М. Борис, В. П. Марценюк АЛГОРИТМ КЛАССИФИКАЦИИ ПОЛИТРАВМ МЕТОДОМ ИНДУКЦИИ ДЕРЕВА РЕШЕНИЙ P. M. Борис, В. П. Марценюк1 ГП "Украинский научно-исследовательский институт медицины транспорта МЗ Украины" Тернопольский государственный медицинский университет имени И. Я. Горбачевского1 В работе разработан и программно реализован метод индукции дерева решений для задачи классификации политравм на основании ряда биохимических показателей. В работе разработан и программно реализован метод индукции дерева решений для задачи классификации политравм на основании ряда биохимических показателей. Алгоритм выбора атрибута использует значение прироста информации. Проект реализован в среде Netbeans на основе Java- классов. Алгоритм выбора атрибута использует значение прироста информации. Проект реализован в среде Netbeans на основе Java- классов. © P. М. Борис, В. П. Марценюк R. M. Borys, V. P. Martsenyuk1 SE "Ukrainian Scientific Research Institute of Transport Medicine of MPH of Ukraine Ternopil State Medical University by I. Ya. Horbachevsky1 R. M. Borys, V. P. Martsenyuk1 SE "Ukrainian Scientific Research Institute of Transport Medicine of MPH of Ukraine Ternopil State Medical University by I. Ya. Horbachevsky1 R. M. Borys, V. P. Martsenyuk1 SE "Ukrainian Scientific Research Institute of Transport Medicine of MPH of Ukraine Ternopil State Medical University by I. Ya. Horbachevsky1 The work program is developed and implemented induction decision tree method for classification of polytrauma based on a number of biochemical parameters. p algorithm uses the value of the attribute information gain. The project was implemented in the medium ased classes. The selection algorithm uses the value of the attribute information gain. The project was implemented Netbeans Java- based classes. АЛГОРИТМ КЛАСИФІКАЦІЇ ПОЛІТРАВМ МЕТОДОМ ІНДУКЦІЇ ДЕРЕВА РІШЕНЬ P. М. Борис, В. П. Марценюк1 ДП "Український науково-дослідний інститут медицини транспорту МОЗ України" Тернопільський державний медичний університет імені І. Я. Горбачевського1 P. М. Борис, В. П. Марценюк ДП "Український науково-дослідний інститут медицини транспорту МОЗ України" Тернопільський державний медичний університет імені І. Я. Горбачевського1 У роботі розроблено і програмно реалізовано метод індукції дерева рішень для задачі класифікації політравм на основі ряду біохімічних показників. У роботі розроблено і програмно реалізовано метод індукції дерева рішень для задачі класифікації політравм на основі ряду біохімічних показників. Алгоритм вибору атрибуту використовує значення приросту інформації. Проект реалізовано в середовищі Netbeans на основі Java-класів. Алгоритм вибору атрибуту використовує значення приросту інформації. Проект реалізовано в середовищі Netbeans на основі Java-класів. Ключові слова: політравма, прийняття рішень, дерево рішень, Java, SQL МЕДИЧНА ІНФОРМАТИКА ТА ІНЖЕНЕРІЯ нень, а також для зменшення переліку можливих діагнозів. нень, а також для зменшення переліку можливих діагнозів. сельних атрибутів грунтується, як правило, на мірах на основі ентропії, або індексі Джині [Han, 2001]. Про- цес розбиття рекурсивно повторюється до тих пір, поки не спостерігатиметься покращення точності прогнозування. Останній крок включає вилучення вузлів для уникнення оверфітінгу моделі. У резуль- таті ми повинні отримати множину правил, що йдуть від кореня до кожного термінального вузла, містять нерівності для чисельних атрибутів та умови вклю- чення для категоріальних атрибутів. - на дві під множини: . Розбиття чи- на терміналь- вання атрибуту класу, а саме них вузлах для довільного . Алгоритми індукції дерев рішень автоматично розбивають на вузлах значення чисельних атрибутів на два інтер- а категоріальних атрибутів вали: Сьогодні медичне діагностування може виконувати- ся автоматично з використанням комп'ютеризованих систем та алгоритмів. Такі системи переважно нази- ваються діагностичними системами підтримки прий- няття рішень або медичними діагностичними систе- мами. Вони належать до загальнішого класу клінічних систем підтримки прийняття рішень [Марценюк, 2004— 2012]. Метою таких систем є системний супровід ліка- ря в процесі диференційної діагностики. Багато з таких систем можуть надавати результати навіть коли не ви- стачає даних, тобто в умовах невизначеності, і що най- важливіше - вони не обмежені щодо кількості інфор- мації, яку можуть зберігати та обробляти. Одним з підходів, що відображає природний про- цес мислення при диференційній діагностиці, є метод індукції дерева рішень. У період кінця 1970 - почат- ку 1980 років J. R. Quinlan [Quinlan, 1986] розробив алгоритм побудови дерева рішень ID3 (ітеративний дихотомайзер). Пізніше J. R. Quinlan представив ал- горитм С4.5 (наступник ID3), який став еталоном, з яким часто порівнюються новітні алгоритми в галузі машинних знань. У 1984 році група статистиків (L. Breiman, J. Friedman, R. Olshen, С. Stone) опублі- кували роботу щодо Classification and Regression Trees (CART) [Breiman, 1984], в якій описали побудову бінарних дерев рішень. Алгоритми ID3 та CART, не зважаючи на те, що були розроблені незалежно і при- близно у той же час, реалізують подібний підхід до навчання дерев рішень на основі навчальних даних. При цьому дерева рішень будуються в результаті рекурсивної процедури типу «зверху-вниз». Більшість алгоритмів індукції дерев рішень також відповідають цьому загальному підходу. При цьому навчальна множина рекурсивно поділяється на менші підмно- жини по мірі того, як будується дерево. Метод індукції дерева рішень. За основу взято таку рекурсивну процедуру роботи [Han, 2001]. Метод індукції дерева рішень. За основу взято таку рекурсивну процедуру роботи [Han, 2001]. Key words: polytrauma, decision making, decision tree Java, SQL. Key words: polytrauma, decision making, decision tree Java, SQL. зувати алгоритм класифікації політравм з викорис- танням методу індукції дерева рішень. Вступ. Під політравмою мають на увазі складний патологічний процес, зумовлений пошкодженням кількох анатомічних ділянок або сегментів кінцівок. Проблему становить правильне та своєчасне діаг- ностування політравм, особливо в умовах надзвичай- них ситуацій або військових дій, коли пацієнт знахо- диться у стані без свідомості. Вступ. Під політравмою мають на увазі складний патологічний процес, зумовлений пошкодженням кількох анатомічних ділянок або сегментів кінцівок. Вирішувана проблема належить до широкого кла- су задач диференціальної діагностики. В медицині поняття «диференціальної діагностики» означає си- стемний підхід, який грунтується на доказовості, для визначення причини симптомів, що спостерігають- ся, у випадку, коли є кілька альтернативних пояс- Проблему становить правильне та своєчасне діаг- ностування політравм, особливо в умовах надзвичай- них ситуацій або військових дій, коли пацієнт знахо- диться у стані без свідомості. Мета даної роботи - розробити і програмно реалі- © P. М. Борис, В. П. Марценюк Мета даної роботи - розробити і програмно реалі- © P. М. Борис, В. П. Марценюк Медична інформатика та інженерія, № 2, 2013 12 Вихідні дані: дерево рішень Вихідні дані: дерево рішень Метод: МЕДИЧНА ІНФОРМАТИКА ТА ІНЖЕНЕРІЯ Генерація дерева рішень Вхідні дані: D - множина навчальних наборів даних Вихідні дані: дерево рішень Метод: 1. Створити вузол N. 1. Створити вузол N. 1. Створити вузол N. 2. Якщо усі набори в D належать до спільного кла- су С, тоді повернути вузол N як листок із назвою класу С. 3. Якщо список атрибутів (а отже і D) є порожнім, тоді повернути вузол N як листок із назвою найпоши- ренішого класу в D. 4. Застосувати Алгоритм відбору атрибуту із списку атрибутів і для множини D з метою відшу- кання «найкращого» атрибуту поділу. 5. Вилучити атрибут поділу із списку атрибутів. 6. Для кожної умови поділу j для атрибуту поділу розглянемо - множину наборів з D, що задоволь- няють умову поділу j. Задача індукції дерева рішень формулюється та- ким чином. Маємо множину D), що містить N на- борів навчальних даних. При цьому кожен і-й набір листок під заголовком найпоширенішого класу в інакше - приєднати до N вузол, що повертається ре- курсивним викликом методу Генерація дерева рішень з вхідними даними та список атрибутів. 7. Якщо - порожня, тоді приєднати до вузла N складається з вхідних даних складається з вхідних даних та вихідних даних - атрибуту - атрибутів класу С. Атрибути можуть приимати як чи- сельні, так і категоріальні значення. Атрибут класу С приймає одне з К дискретних значень: Метою є прогнозування деревом рішень значення ат- рибуту класу С на основі значень атрибутів При цьому слід максимізувати точність прогнозу- 8. Кінець циклу кроку 6. 9. Повернути вузол N В основу Алгоритму відбору атрибуту на j-му кроці рекурсії покладено такий інформаційний показ- ник (1) (1) Медична інформатика та інженерія, № 2, 2013 13 МЕДИЧНА ІНФОРМАТИКА Рис.1. Концептуальна модель інформаційної системи індукції дерева рішень. Тут (2) (2) - інформація, потрібна для класифікації набору - інформація, потрібна для класифікації набору (3) (3) - інформація, потрібна для класифікації після поділу в на підмно- жини відповідно до значень атрибуту У формулі (2) ймовірність того, що довільний набір з належить множині оцінюється як , де - множина наборів з для кількість еле- . Тут яких атрибут класу ментів в множині. , де - множина наборів з для кількість еле- . Тут яких атрибут класу ментів в множині. , де - множина наборів з для кількість еле- . Тут яких атрибут класу ментів в множині. У формулі (3) - оцінка ймовірності того, що атрибут множина наборів з довільний набір з належить множині для яких атрибут . Метод: Тут де У формулі (3) - оцінка ймовірності того, що атрибут множина наборів з довільний набір з належить множині для яких атрибут . Тут де ) Рис.1. Концептуальна модель інформаційної системи індукції дерева рішень. Рис.1. Концептуальна модель інформаційної системи індукції дерева рішень. МЕДИЧНА ІНФОРМАТИКА ТА ІНЖЕНЕРІЯ Клас DecisionTree є нащадком класу DefaultTreeModel пакету javax.swing.tree. Він має два елементи класу: m_dataManager - менеджер даних та m_htAttribute_list - хеш-таблиця із списком атри- бутів. Хеш-таблиця із списком атрибутів (у методах класу DecisionTree виступає під назвою htAttribute list) створюється для кожного вузла де- рева рішень. Вона має два призначення - поряд із списком включених для даного вузла атрибутів збе- рігати умови поділу (splitting conditions), які перейшли до даного вузла від вузлів-батьків. Кожен вузол де- рева рішень є об'єктом класу DefaultMutableTreeNode. В якості об'єкта кожен ву- зол зберігає об'єкт класу NodeObject, декларація якого наведена нижче: Метод Generate_decision_tree є безпосередньою реалізацією методу індукції дерева рішень. Заголо- вок методу має вигляд: private DefaultMutableTreeNode Generate_decision_ tree (Hashtable htAttribute list, DefaultMutable TreeNode dmtnSubroot, String splitting_criterion). private DefaultMutableTreeNode Generate_decision_ tree (Hashtable htAttribute list, DefaultMutable TreeNode dmtnSubroot, String splitting_criterion). В якості аргументів метод використовує корене- вий вузол дерева, список пов'язаних з ним атрибутів htAttribute list та умову поділу splitting criterion. В якості значення метод повертає дочірній вузол типу DefaultMutableTreeNode. Шляхом рекурсивного вик- лику методу Generate decision tree будується дере- во рішень. З метою візуалізації представлення дерева вико- ристано клас javax. swing JTree. При цьому дерево рішень створюється виводиться за допомогою опе- раторів: dtDecision tree = new DecisionTree(dmtnRoot, dataManager, htAttributelist); dtDecision tree = new DecisionTree(dmtnRoot, dataManager, htAttributelist); jTreel .setModel(dtDecision_tree); jTreel .setModel(dtDecision_tree); SQL-реалізація розрахунку інформаційних показників. Ключовим в реалізації методу Attribute selection method є розрахунок інформацій- Так, показник розра- них показників кроці рекурсії для атрибута ховується методом та на навчальних даних для кожного із вузлів і має таку структуру: Тут attribute - атрибут, який повертається мето- дом Attribute selection method, splitting criterion - умова поділу, яка переходить від батьківського вуз- ла, sLabel - надпис на вузлі. Хеш-таблиця htAttribute list використовується для побудови наборів Зауважимо, що множина наборів ся хеш-таблицею htAttribute list, з якої отримуємо перелік включених атрибутів sAttribute list та умов поділу sConditions. тут описуєть- Тип ключа int Тип об'єкта Attribute for list Структура об'єкта Attribute attribute; Hashtable htSplittingoutcomes; String splitting criterion; boolean included; Мова структурованих запитів SQL має досить гнучкі засоби для реалізації алгоритмів в галузі ма- шинних знань. Так, використавши вкладені запити, псевдоніми та агрегативні функції можна розрахува- ти в результаті виконання такого SQL-запиту: Тут included - булева змінна-прапорець належності атрибуту attribute до списку атрибутів даного вузла. Можна показати, що коли included=true, то вузол з назвою attribute є для даного вузла дочірнім (на пев- ному нижчому рівні ієрархії). наборів навчальних даних. Структура таблиць намові SQL для класифікації політравм наведена нижче: Отже, оцінює зменшення інформації, не- обхідної для класифікації довільного набору даних в за рахунок відомого значення атрибуту Таким з найбільшим значенням такого вибору для завершення процесу класифікації . У результаті рішень для умови поділу слід відбирати атрибут чином, з наявних атрибутів на кожному вузлі дерева мації. набору даних в вимагатиметься найменше інфор- Програмна реалізація. Метод реалізовано в се- редовищі розробки Netbeans на мові програмування Java. Базу навчальних даних розгорнуто на сервері MySQL. На рисунку 1 представлено концептуальну модель інформаційної системи. У класі DecisionTree безпосередньо реалізовано метод індукції дерева рішень. У клас DataManager надходять виклики від DecisionTree на виконання запитів до бази даних mysql щодо отримання навчальних даних. Програмні класи проекту включено до пакета decision_tree.model. Сюди входять beans-класи Attribute, Attribute_for_list та CategorisedData для ро- боти з даними відповідних таблиць. SQL-запити щодо отримання відповідних даних, включаючи розрахун- ки інформаційних показників, реалізовано в класі AttributeListPeer. База даних mysql складається з двох таблиць - таблиці attribute, призначеної для зберігання інфор- мації про атрибути, та таблиці categorized_data - для Медична інформатика та інженерія, № 2, 2013 14 МЕДИЧНА ІНФОРМАТИКА ТА ІНЖЕНЕРІЯ У випадку, коли атри- бут attribute не входить до списку атрибутів для да- ного вузла (included=false), то вузол з назвою attribute є батьківським (на певному рівні ієрархії), а в змінній splitting criterion зберігається умова поділу, якій підля- гає даний вузол відносно батьківського вузла attribute. Хеш-таблиця htSplitting_outcomes містить усі мож- ливі наслідки (умови поділу) щодо атрибуту attribute. розраховується методом Показник розраховується методом Показник Медична інформатика та інженерія, № 2, 2013 15 МЕДИЧНА ІНФОРМАТИКА ТА ІНЖЕНЕРІЯ dataManager, int і, Hashtable htAttribute list) - черепно-мозкова та скелетна травми мали місце 12 годин тому; - черепно-мозкова та скелетна травми з кровоте- чею мали місце 12 годин тому; обчислюється в резуль- таті виконання такого SQL-запиту: Остаточно обчислюється в резуль- таті виконання такого SQL-запиту: Остаточно - черепно-мозкова та скелетна травми мали місце 24 години тому; Q у - черепно-мозкова та скелетна травми з кровоте- чею мали місце 24 години тому. Використано таку таблицю атрибутів: INSERT INTO mysql.attribute (id, attributename, attribute field name) VALUES (1, 'Mass", " A 1 '). (2, 'Zag.bil.', "A2'). (3, "AsAT. 'A3'),(4, "A1AT. "A4'). (5, 'GP', 'A5'), (6, 'GR', 'A6'), (7, VG', 'A7'), (8, 'SOD", 'A8'), (9, 'Catalaza', 'A9'), (10, MDA', 'A10'), (11, DK', 'All'), (12, 'TsP', 'A12'),(13, 'TsIK', 'A13'), (14, 'API', 'A14'), (15, 'IgA', 'A15'),(16, 'IgM', 'A16'), (17, 'Ig G', 'A17'), (18, '11-2', 'A18'), (19, '11-6', 'A19'), (20, '11-10', 'A20'), (21, 'TNF-a', 'A21'); INSERT INTO mysql.attribute (id, attributename, attribute field name) VALUES (1, 'Mass", " A 1 '). (2, 'Zag.bil.', "A2'). (3, "AsAT. 'A3'),(4, "A1AT. "A4'). (5, 'GP', 'A5'), (6, 'GR', 'A6'), (7, VG', 'A7'), (8, 'SOD", 'A8'), (9, 'Catalaza', 'A9'), (10, MDA', 'A10'), (11, DK', 'All'), (12, 'TsP', 'A12'),(13, 'TsIK', 'A13'), (14, 'API', 'A14'), (15, 'IgA', 'A15'),(16, 'IgM', 'A16'), (17, 'Ig G', 'A17'), (18, '11-2', 'A18'), (19, '11-6', 'A19'), (20, '11-10', 'A20'), (21, 'TNF-a', 'A21'); Набори включають лише категоріальні дані (попе- редньо оброблені), наприклад: INSERT INTO mysql.categorised_data (id, Al, A2, A3, A4, A5, A6, A7, A8, A9, A10, Al 1, A12, A13, A14, A15,A16,A17,A18,A19, A20, A21, class) VALUES Далі за допомогою розроблених програмних класів побудуємо дерево рішень щодо класифікації політравм на основі даних 6-ти класифікаційних груп, а саме: (1, 'low', 'low', 'high', 'high', 'normal', 'low', 'low', 'low', 'low', 'high', 'high', 'high', 'high', 'low', 'high', 'high', 'high', 'high', 'high', 'high', 'high', 'craniocerebral_injury+orthopedic_trauma_2_hours'); - черепно-мозкова та скелетна травми мали місце 2 години тому; На рисунку 2 представлене побудоване дерево рішень. Час, затрачений на індукування дерева - 3104 мілісекунди. - черепно-мозкова та скелетна травми з кровоте- чею мали місце 2 години тому; Рис. 2. Дерево рішень для класифікації політравм. Рис 2 Дерево рішень для класифікації політравм Рис. 2. Дерево рішень для класифікації політравм. Рис. 2. Дерево рішень для класифікації політравм. можливості, які дозволяють розрахувати інформаційні показники на основі таблиць баз даних. Висновки. У роботі розглянуто питання розроб- ки і програмної реалізації методу індукції дерева рішень на основі інформаційних показників для побу- дови класифікаційного алгоритму політравм. За рахунок використання Java-класі в дана реалізації методу індукції дерева рішень є веб-інтегрованою. На даному експериментальному прикладі проде- монстровано, що такий підхід дозволяє розробити систему підтримки клінічних рішень. Перспективи подальших досліджень - аналіз продуктивності програмного продукту залежно від кількості біохімічних показників та обсягу наборів навчальних даних. Показано, що мова SQL має достатні синтаксичні Медична інформатика та інженерія, № 2, 2013 16 вец // Клиническая информатика и телемедицина - 2004. - №1.-С. 47-53. 1. Соколов В. А. Множественные и сочетанные травмы / В. А. Соколов. -М.: "ГЭОТАР", 2006 г. 2. J.Han and M.Kamber, Data Mining: Concepts and Techniques, Morgan Kaufmann, San Francisco, 1st edition 2001. 10. Математичні моделі в системі підтримки прийняття рішень страхового забезпечення лікування онкологічних захворювань: підхід на основі динаміки Гомперца / B. П. Марценюк, І. Є. Андрущак, І. С. Гвоздецька, Н. Я. Климук // Доповіді Національної академії наук Украї- ни. -2012. -№10. -С. 34-39. 3. T.Hastie, R.Tibshirani and J.H.Friedman The Elements of Statistical Learning, Springer, New York, 1st edition 2001. B. П. Марценюк, І. Є. Андрущак, І. С. Гвоздецька, Н. Я. Климук // Доповіді Національної академії наук Украї- ни. -2012. -№10. -С. 34-39. 4. C.Ordonez, Comparing association rules and decision trees for disease prediction. In Proc. ACM HIKM Workshop, 2006, pp. 17-24. 11. Марценюк В. П. Підхід на основі актуальних математич- них моделей до задач страхової медицини / В. П. Марце- нюк, І. Є. Андрущак, Н. Я. Климук //Медична інформати- ка та інженерія.-2010.-№4.-С. 85-87. 11. Марценюк В. П. Підхід на основі актуальних математич- них моделей до задач страхової медицини / В. П. Марце- нюк, І. Є. Андрущак, Н. Я. Климук //Медична інформати- ка та інженерія.-2010.-№4.-С. 85-87. 12. Марценюк В. П. О модели онкологического заболева- ния со временем пребывания на стадии в соответствии с распределением Гомперца / В. П. Марценюк, Н. Я. Кли- мук // Проблемы управления и информатики. Междуна- родный научно-технический журнал. - 2012. - № 6. - C. 137—143" 5. C.Ordonez, Integrating K-means clustering with a relational 5. C.Ordonez, Integrating K-means clustering with a relational DBMS using SQL, IEEE Transactions onKnowledge andData Engineering (IKDE) 18(2) (2006), 188-201. 6. J.R.Quinlan. Induction of decision trees. Machine Learning, 1:81-106,1986. DBMS using SQL, IEEE Transactions onKnowledge andData Engineering (IKDE) 18(2) (2006), 188-201. 12. Марценюк В. П. О модели онкологического заболева- ния со временем пребывания на стадии в соответствии с распределением Гомперца / В. П. Марценюк, Н. Я. Кли- мук // Проблемы управления и информатики. Междуна- родный научно-технический журнал. - 2012. - № 6. - C. 137—143" 6. J.R.Quinlan. Induction of decision trees. Machine Learning, 1:81-106,1986. 7. J.R.Quinlan. C4.5: Programs for Machine Learning. Morgan Kaufmann 1993. 8. LBreiman, J.Friedman, R.Olshen and C.Stone. Classification and Regression Trees. Wadsworth International Group, 1984. 13. Марценюк В. П. Медична інформатика. Інструмен- тальні та експертні системи/В. П. Марценюк, А. В. Семе- нець. - Тернопіль: Укрмедкнига. 2004. - 222 с. 9. МарценюкВ. П. Література вец // Клиническая информатика и телемедицина - 2004. - №1.-С. 47-53. вец // Клиническая информатика и телемедицина - 2004. - №1.-С. 47-53. вец // Клиническая информатика и телемедицина - 2004. - №1.-С. 47-53. О программной среде проектирования интеллектуальных баз данных/ В. П. Марценюк, Н. О. Кра-
https://openalex.org/W2919983752	https://discovery.dundee.ac.uk/files/32694856/1_s2.0_S1097276519300607_main_1_.pdf	English	null	Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes	Molecular cell	2,019	cc-by	21,481	Citation for published version (APA): Deegan, T., Baxter, J., Ortiz Bazan, M., Yeeles, J. T. P., & Labib, K. (2019). Pif1-family helicases support fork convergence during DNA replication termination in eukaryotes. Molecular Cell, 74(2), 231-244.e9. https://doi.org/10.1016/j.molcel.2019.01.040 University of Dundee Pif1-family helicases support fork convergence during DNA replication termination in eukaryotes Deegan, Thomas; Baxter, Jonathan; Ortiz Bazan, Maria; Yeeles, Joseph T. P.; Labib, Karim Published in: University of Dundee Pif1-family helicases support fork convergence during DNA replication termination in eukaryotes Deegan, Thomas; Baxter, Jonathan; Ortiz Bazan, Maria; Yeeles, Joseph T. P.; Labib, Karim Published in: Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes Graphical Abstract Graphical Abstract Authors Tom D. Deegan, Jonathan Baxter, Marı´a A´ ngeles Ortiz Baza´ n, Joseph T.P. Yeeles, Karim P.M. Labib Correspondence tdeegan@dundee.ac.uk (T.D.D.), kpmlabib@dundee.ac.uk (K.P.M.L.) In Brief To study the mechanism of DNA replication termination, Deegan et al. reconstituted the convergence of two replisomes using puriﬁed budding yeast proteins. Surprisingly, replisome convergence is inherently inefﬁcient but stimulated by the Pif1 and Rrm3 DNA helicases, which are also important during termination in vivo. Highlights University of Dundee Pif1-family helicases support fork convergence during DNA replication termination in eukaryotes Deegan, Thomas; Baxter, Jonathan; Ortiz Bazan, Maria; Yeeles, Joseph T. P.; Labib, Karim Published in: University of Dundee Pif1-family helicases support fork convergence during DNA replication termination in eukaryotes Deegan Thomas; Baxter Jonathan; Ortiz Bazan Maria; Yeeles Joseph T P ; Labib Karim Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): Deegan, T., Baxter, J., Ortiz Bazan, M., Yeeles, J. T. P., & Labib, K. (2019). Pif1-family helicases support fork convergence during DNA replication termination in eukaryotes. Molecular Cell, 74(2), 231-244.e9. https://doi.org/10.1016/j.molcel.2019.01.040 Citation for published version (APA): Deegan, T., Baxter, J., Ortiz Bazan, M., Yeeles, J. T. P., & Labib, K. (2019). Pif1-family helicases support fork convergence during DNA replication termination in eukaryotes. Molecular Cell, 74(2), 231-244.e9. https://doi.org/10.1016/j.molcel.2019.01.040 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Article SUMMARY otic species about the mechanisms that drive fork convergence and replisome encounter. The convergence of two DNA replication forks cre- ates unique problems during DNA replication termi- nation. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown. We studied the conver- gence of reconstituted yeast replication forks that include all core replisome components and both type I and type II topoisomerases. We found that most converging forks stall at a very late stage, indi- cating a role for additional factors. We showed that the Pif1 and Rrm3 DNA helicases promote efﬁcient fork convergence and completion of DNA synthesis, even in the absence of type II topoisomerase. Furthermore, Rrm3 and Pif1 are also important for termination of plasmid DNA replication in vivo. These ﬁndings identify a eukaryotic pathway for DNA repli- cation termination that is distinct from previously characterized prokaryotic mechanisms. During elongation, DNA unwinding by the replicative helicase causes torsional strain in the DNA template, which produces positive supercoils ahead of each replisome (Keszthelyi et al., 2016). The removal of these supercoils by type I or II topoiso- merases is essential for continued fork progression in both pro- karyotes and eukaryotes (Bermejo et al., 2007; Vos et al., 2011; Yeeles et al., 2015). When two forks converge, however, the remaining stretch of parental DNA becomes so short that super- coils cannot form (Vafabakhsh and Ha, 2012), and topoiso- merases are sterically excluded (Keszthelyi et al., 2016). Therefore, the ﬁnal stages of DNA unwinding and DNA synthesis are thought to require clockwise rotation of the two converging replication forks to transfer the topological stress behind the two replisomes in the form of intertwines or precatenanes be- tween the replicated sister chromatids (Keszthelyi et al., 2016; Schalbetter et al., 2015). ) During DNA replication termination in the bacterium E. coli and the virus SV40, the removal of precatenanes by type II topoiso- merases is essential for the converging replisomes to continue unwinding and complete DNA synthesis (Hiasa and Marians, 1996; Ishimi et al., 1992; Richter et al., 1987; Snapka et al., 1988). SUMMARY Moreover, the resolution of converged forks during termi- nation of SV40 replication in human cells appears to be a slow process that leads to the accumulation of a ‘‘late replication in- termediate’’ (Seidman and Salzman, 1979; Sundin and Varshav- sky, 1980). In contrast, however, type II topoisomerase activity is dispensable for the convergence of eukaryotic replisomes in budding yeast cells (Baxter and Difﬂey, 2008) and in Xenopus egg extracts (Dewar et al., 2015; Lucas et al., 2001). In addition, observations of DNA replication termination in Xenopus egg extracts indicated that two replisomes converge without detectable slowing or stalling (Dewar et al., 2015), in contrast to SV40 viral replication, despite the latter being dependent upon eukaryotic replication factors, apart from the viral DNA heli- case T-antigen. Until now, the pathways supporting fork conver- gence in eukaryotes have remained enigmatic. Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes Tom D. Deegan,1,* Jonathan Baxter,2 Marı´a A´ ngeles Ortiz Baza´ n,1 Joseph T.P. Yeeles,3 and Karim P.M. Labib1,4,* 1The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK 2Genome Damage and Stability Centre, Department of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK 3The MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK 4Lead Contact Correspondence: tdeegan@dundee.ac.uk (T.D.D.), kpmlabib@dundee.ac.uk (K.P.M.L.) https://doi.org/10.1016/j.molcel.2019.01.040 Correspondence: tdeegan@dundee.ac.uk (T.D.D.), kpmlabib@dundee.ac.uk (K.P.M.L.) https://doi.org/10.1016/j.molcel.2019.01.040 Molecular Cell 74, 231–244, April 18, 2019 ª 2019 The Authors. Published by Elsevier Inc. 231 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Highlights Highlights d In vitro reconstitution of the convergence of two eukaryotic replisomes d Converging replisomes stall at a late stage of DNA replication termination d The budding yeast DNA helicases Pif1 and Rrm3 stimulate fork convergence in vitro d Pif1 and Rrm3 promote fork convergence during DNA replication termination in vivo Molecular Cell Article INTRODUCTION In contrast to the initiation and elongation stages of eukaryotic DNA replication, which have been studied intensively over recent decades (Bell and Labib, 2016; Burgers and Kunkel, 2017; Dee- gan and Difﬂey, 2016), very little is known about the process of DNA replication termination, which occurs whenever two repli- somes from neighboring replication origins meet each other (Dewar and Walter, 2017). The ﬁnal stages of DNA synthesis involve at least ﬁve processes that are particular to termination (Dewar and Walter, 2017): the convergence and encounter of the two replisomes, gap ﬁlling between the leading strand at one fork and the lagging strand of the other (Dewar et al., 2015), regulated disassembly of the replisome (Dewar et al., 2017; Maric et al., 2014, 2017; Moreno et al., 2014; Sonneville et al., 2017), and decatenation of the sister chromatids that are the products of replication. The majority of these steps are un- derstood very poorly, and almost nothing is known in any eukary- Here we analyze eukaryotic DNA replication termination in vitro using a reconstituted system based on puriﬁed budding yeast proteins that has been shown previously to support the initiation and elongation stages of chromosome duplication (Devbhandari et al., 2017; Yeeles et al., 2015, 2017). Our data identify a eukaryotic pathway for fork convergence that is igure 1. Converging Replisomes Stall in the Absence of Accessory DNA Helicases A) A 3,189-bp plasmid template (pBS/ARS1WTA) and the products of complete DNA replication (left) or a defect in DNA replication termination (righ B) Puriﬁed S. cerevisiae Cdc9 (ligase) and Fen1 were visualized by SDS-PAGE and Coomassie staining. C) A 3,189-bp plasmid template (pBS/ARS1WTA) was replicated according to the schematic in Figure S1B, and Fen1 and Cdc9 were included as i ubsequently, the replication products were resolved in a denaturing agarose gel, and the radiolabeled nascent strands were detected by autoradio D) The products of replicating the 3,189-bp plasmid in the presence of Fen1 and ligase were analyzed in a denaturing agarose gel alongside the markers. Figure 1. Converging Replisomes Stall in the Absence of Accessory DNA Helicases (A) A 3,189-bp plasmid template (pBS/ARS1WTA) and the products of complete DNA replication (left) or a defect in DNA replication termination (rig (B) Puriﬁed S. cerevisiae Cdc9 (ligase) and Fen1 were visualized by SDS-PAGE and Coomassie staining. INTRODUCTION (C) A 3,189-bp plasmid template (pBS/ARS1WTA) was replicated according to the schematic in Figure S1B, and Fen1 and Cdc9 were included as Subsequently, the replication products were resolved in a denaturing agarose gel, and the radiolabeled nascent strands were detected by autoradi (D) The products of replicating the 3,189-bp plasmid in the presence of Fen1 and ligase were analyzed in a denaturing agarose gel alongside the markers. (legend continued on 232 Molecular Cell 74, 231–244, April 18, 2019 Figure 1. Converging Replisomes Stall in the Absence of Accessory DNA Helicases (A) A 3,189-bp plasmid template (pBS/ARS1WTA) and the products of complete DNA replication (left) or a defect in DNA replication termination (right). (B) Puriﬁed S. cerevisiae Cdc9 (ligase) and Fen1 were visualized by SDS-PAGE and Coomassie staining. (C) A 3,189-bp plasmid template (pBS/ARS1WTA) was replicated according to the schematic in Figure S1B, and Fen1 and Cdc9 were included as indicated. Subsequently, the replication products were resolved in a denaturing agarose gel, and the radiolabeled nascent strands were detected by autoradiography. (D) The products of replicating the 3,189-bp plasmid in the presence of Fen1 and ligase were analyzed in a denaturing agarose gel alongside the indicated markers. (l d ti d t ) Figure 1. Converging Replisomes Stall in the Absence of Accessory DNA Helicases (A) A 3,189-bp plasmid template (pBS/ARS1WTA) and the products of complete DNA replication (left) or a defect in DNA replication termination (right). (B) Puriﬁed S. cerevisiae Cdc9 (ligase) and Fen1 were visualized by SDS-PAGE and Coomassie staining. (C) A 3,189-bp plasmid template (pBS/ARS1WTA) was replicated according to the schematic in Figure S1B, and Fen1 and Cdc9 were included as indicated. Subsequently, the replication products were resolved in a denaturing agarose gel, and the radiolabeled nascent strands were detected by autoradiography. (D) The products of replicating the 3,189-bp plasmid in the presence of Fen1 and ligase were analyzed in a denaturing agarose gel alongside the indicated markers. 232 Molecular Cell 74, 231–244, April 18, 2019 Subsequently, however, a more careful analysis indicated that the ligated nascent strands were in fact between 3,000 and 3,100 nt in size (Figure 1D) and were thus about 90–190 nt shorter than the full-length plasmid. These data suggested that the majority of converging replisomes stall during the ﬁnal stages of DNA repli- cation in the reconstituted system. To explore this further, we resolved the products of similar reactions in native agarose gels. Converging Replisomes Stall in the Absence of Accessory DNA Helicases Previous work (Yeeles et al., 2015) established the minimal set of budding yeast proteins that is required in vitro to establish bi- directional forks from an origin of DNA replication on a circular plasmid template. In this system, the Mcm2-7 proteins (MCM [minichromosome maintenance]) that represent the catalytic core of the replicative helicase are ﬁrst loaded as double hexam- ers onto double stranded DNA (dsDNA) at origins of replication and then activated in a separate step to form two CMG (Cdc45-MCM-GINS) helicases. A ‘‘minimal replisome’’ then as- sembles around CMG at each nascent DNA replication fork, with DNA polymerase a making primers for lagging-strand syn- thesis, whereas DNA polymerase ε extends the leading strand, and the type II topoisomerase Top2 removes supercoils to allow fork progression. Pulse-chase experiments demonstrated that the appearance of these large products was coincident with the generation of leading strands of approximately half unit length (Figure 1F; note that ligase and Fen1 were omitted in this case so that lead- ing-strand synthesis could be monitored in denaturing gels). Overall, therefore, these data indicated that the reaction prod- ucts represented late replication intermediates (LRIs), which result from stalling of converging replisomes before the ﬁnal stretch of parental duplex DNA has been unwound. LRI formation was independent of vector size or sequence (Figures S1A and S1D) and was observed over a range of salt concentrations (Figure S1E). In addition, the LRIs persisted in longer time course experiments (Figure S1F), indicating that they are a terminal product of the replication reaction under these conditions. Because the replisome contains multiple fac- tors that modulate fork progression, such as Mrc1-Tof1-Csm3 (Calzada et al., 2005; Szyjka et al., 2005; Tourrie` re et al., 2005; Yeeles et al., 2017), we also performed reactions with a ‘‘mini- mal’’ replisome that lacks these factors, at the same time omit- ting Pol d and other proteins that are required for complete lagging-strand synthesis (Yeeles et al., 2015). Again, however, the large majority of converging forks arrested, producing LRIs (Figure S1G). These data indicated that LRI formation is an inherent consequence of fork convergence in the reconstituted replication system. Converging Replisomes Stall in the Absence of Accessory DNA Helicases Further development of this reconstituted replication system (Yeeles et al., 2017) added components of the replisome pro- gression complex that assembles around the yeast CMG heli- case (Gambus et al., 2006), including the type I topoisomerase Top1, and also added DNA polymerase d (Pol d) and other fac- tors that are required for lagging-strand synthesis. Under these conditions, the two replisomes move away from the origin at similar rates as those seen in vivo (Yeeles et al., 2017). Analogous to the situation at cellular replication forks, DNA polymerase a initiates every new DNA molecule, DNA polymerase ε (Pol ε) ex- tends the leading strands, and Pol d synthesizes each Okazaki fragment during lagging-strand synthesis (Yeeles et al., 2017). As a ﬁrst step toward addressing whether the reconstituted re- plisomes are able to support the completion of plasmid replica- tion when two forks converge, we monitored nascent strand formation in reactions containing the ﬂap endonuclease Fen1 and the DNA ligase Cdc9, which are required for Okazaki frag- ment processing and nascent strand ligation. Using a 3.2-kb plasmid template (Figures 1A and S1A; pBS/ARS1WTA), we observed the generation of approximately full-length nascent DNA in denaturing agarose gels, dependent on the presence of both Fen1 and ligase (Figures 1B and 1C; the replication reac- tions contained all of the factors indicated in Figures S1B and S1C), indicating that the reconstituted replisomes traverse the majority of the plasmid template. INTRODUCTION Digestion of complete replication products would produce 3.2-kb linear dsDNA (Figure 1A; Complete Replication). How- ever, the vast majority of the digested reaction products migrated in a native gel as replication intermediates of approxi- mately twice the plasmid size (Figure 1E; +SbfI), indicating that the replicated plasmids were still linked by a short stretch of parental dsDNA (Figure 1A, Termination defect). mediated by Pif1-family DNA helicases and is independent of type II topoisomerase activity. Moreover, these ﬁndings lay the foundations for future studies of the mechanisms and regulation that govern DNA replication termination in eukaryotes. mediated by Pif1-family DNA helicases and is independent of type II topoisomerase activity. Moreover, these ﬁndings lay the foundations for future studies of the mechanisms and regulation that govern DNA replication termination in eukaryotes. Figure 2. LRI Formation on Plasmids with a Single Origin of Repli- cation (A) After replication of naked (lanes 1 and 3) or chromatinized (lanes 2 and 4) versions of a 3.2-kb plasmid, the products were digested with SpeI and analyzed in a native agarose gel. Chromatin templates were generated in the presence of ISWI1, Nap1, and histones, and the histone chaperone FACT was included in the replication step as indicated. RIs = replication intermediates. (B) A 5.5-kb plasmid template was generated that contained the origin ARS306 but in which other putative binding sites for ORC were mutated so that repli- cation could only initiate from a single site (1). The plasmid was linearized with the indicated enzymes (2 and 3) so that the origin was located at either end of the linearized dsDNA, as indicated. (B) A 5.5-kb plasmid template was generated that contained the origin ARS306 but in which other putative binding sites for ORC were mutated so that repli- cation could only initiate from a single site (1). The plasmid was linearized with the indicated enzymes (2 and 3) so that the origin was located at either end of the linearized dsDNA, as indicated. (C) The DNA templates depicted in (B) were replicated and then analyzed by native agarose gel electrophoresis (sample 1 was digested with SmaI after purifying the replicated DNA to remove catenanes). Pol d, ligase, and Fen1 were omitted from these reactions so that un-ligated leading strands could be analyzed in denaturing agarose gels (Figure S2B) to monitor origin speciﬁcity. (D) The 3.2-kb plasmid template was replicated in the presence of the indi- cated concentrations of Cdt1-Mcm2-7 before DNA puriﬁcation, digestion with SmaI, and analysis by native gel electrophoresis. 234 Molecular Cell 74, 231–244, April 18, 2019 See also Figure S2. (or ORC) (Coster and Difﬂey, 2017), which are normally sup- pressed by chromatin (Azmi et al., 2017; Devbhandari et al., 2017; Kurat et al., 2017). This raised the possibility that the two replisomes from one origin might encounter inactive Mcm2-7 double hexamers elsewhere on the plasmid during the course of elongation in our reconstituted replication reactions. Because the inactive Mcm2-7 complexes can slide along dsDNA (Evrin et al., 2009; Gros et al., 2015; Remus et al., 2009), it was conceiv- able that the double hexamers might be pushed ahead of the two replisomes and form a barrier to fork convergence during DNA replication termination, producing the LRIs. Converging Hexamers Although yeast plasmids are replicated from a single replication origin in vivo, Mcm2-7 double hexamers can also be loaded at additional sites during reconstituted reactions with naked DNA templates (Remus et al., 2009) because of the presence of addi- tional ‘‘cryptic’’ binding sites for the origin recognition complex (E) Similar reactions as those in (C) were performed in the absence of Top2 (to avoid catenation of replication products under these conditions), digested with SbfI as indicated, and then analyzed by native agarose gel electrophoresis (top). Shown at the bottom is the same gel stained with ethidium bromide to illustrate the efﬁciency of digestion by SbfI. The percentage of full-length products was determined by autoradiography, as described in STAR Methods. LRI, late replication (F) A 9.7-kb plasmid template (pZN3) was replicated for 2 h and 50 min in the presence of 32P-dCTP (deoxycytidine triphosphate) before addition of a chase of cold dNTPs (see STAR Methods for details). Ligase and Fen1 were omitted from the reactions so that unligated leading strands could be monitored in denaturing agarose gels (bottom; we also omitted Pol d from these reactions). In parallel, aliquots of the same samples were digested with SmaI and monitored in a native gel (top), revealing the accumulation of LRIs at the end of the reaction. See also Figure S1. Molecular Cell 74, 231–244, April 18, 2019 233 Figure 2. LRI Formation on Plasmids with a Single Origin of Repli- cation 234 Molecular Cell 74, 231–244, April 18, 2019 (lane 7) before extraction of the replicated DNA and digestion with SmaI. The samples were then resolved in a native agarose gel and analyzed by autoradiography. Figure 2. LRI Formation on Plasmids with a Single Origin of Repli- cation At present, the mechanism by which active forks lead to the displacement of inactive Mcm2-7 double hexamers is not understood. As a ﬁrst way of addressing whether an excess of loaded Mcm2-7 complexes contributes to LRI formation, we generated a chromatinized version of the 3.2-kb plasmid template used above (Figure S2A). After loading the Mcm2-7 proteins onto DNA, replication reactions were performed in the presence or absence of the histone chaperone FACT (facilitates chromatin transactions), which is part of the replisome that assembles around the CMG helicase at replication forks (Foltman et al., 2013; Gambus et al., 2006). As seen previously (Kurat et al., 2017), the replication of naked plasmid DNA was independent of FACT (Figure 2A, lanes 1 and 3), but fork progression through chromatin was impeded in the absence of FACT, producing a broad smear of replication intermediates (Figure 2A, lane 2). Importantly, the products of chromatin replication in the pres- ence of FACT were indistinguishable from those seen upon replication of naked DNA, with the majority comprising LRIs (Fig- ure 2A, lane 4). These ﬁndings indicated that the defect in fork convergence is still observed when Mcm2-7 loading is restricted to ARS1 by chromatin assembly. In a second approach, we used plasmid templates in which the cryptic ORC binding sites were mutated (pZN3 and pVA18; Figure S1A) so that replication initiates speciﬁcally from the origin ARS306, even in the absence of chromatin (Taylor and Yeeles, 2018). As above, LRIs still formed during DNA replication (pZN3 in Figure 1F; pVA18 in Figure 2C, lane 1). Moreover, LRI 234 Molecular Cell 74, 231–244, April 18, 2019 234 Molecular Cell 74, 231–244, April 18, 2019 Figure 3. LRI Formation on Linearized Templates (A) A 9.7-kb plasmid (pZN3) with a single site of Mcm2-7 loading was re cated for 3 min and 50 s in the presence of 32P-dCTP to allow establishmen bi-directional replication forks. A chase of unlabeled dNTPs was then add followed immediately by addition of SmaI to linearize the vector at the i cated site adjacent to the origin. Nascent DNA strands are shown in Note that Pol d, ligase, and Fen1 were omitted in this experiment. (B) Control experiment illustrating that SmaI digestion of the 9.7-kb plasm 30C in replication reaction buffer leads to linearization within 10 s. Figure 2. LRI Formation on Plasmids with a Single Origin of Repli- cation DNA formation did not reﬂect an elongation defect under such condi- tions because the majority of replication products were full- length when the template was linearized close to the origin so that a single replication fork traversed almost the entire length of the plasmid (Figures 2B, 2C, and S2B, samples 2 and 3). Finally, we investigated the effect of modulating the Mcm2-7 loading conditions for the naked 3.2-kb plasmid template that contained cryptic ORC binding sites in addition to the origin ARS1. We varied the concentration of Cdt1 and the Mcm2-7 pro- teins in the MCM loading reaction because this has been shown to inﬂuence the number of Mcm2-7 double hexamers that are loaded onto the DNA template (Douglas et al., 2018). At the highest concentration used, we found that the proportion of full-length reaction products was reduced severalfold (Figure 2D, compare 200 nM and 40 nM Mcm2-7) so that almost all of the replicated molecules comprised LRIs. This suggests that an excess of loaded Mcm2-7 complexes can indeed interfere with DNA replication termination. However, LRIs still represented over 80% of the replication products when the concentration of Mcm2-7 was lowered dramatically, to levels that reduced the efﬁciency of replication and should only support loading of a single Mcm2-7 double hexamer (Figure 2D, 4 nM Mcm2-7). Taken together, these ﬁndings indicate that LRIs do not result from the encounter of converging replication forks with Mcm2-7 double hexamers nor from a defect in elongation (because an in- dividual fork can replicate the entire length of a linearized tem- plate DNA). Instead, LRIs are produced by an inherent problem that arises when two reconstituted replisomes converge during DNA replication termination. Figure 3. LRI Formation on Linearized Templates g p (A) A 9.7-kb plasmid (pZN3) with a single site of Mcm2-7 loading was repli- cated for 3 min and 50 s in the presence of 32P-dCTP to allow establishment of bi-directional replication forks. A chase of unlabeled dNTPs was then added, followed immediately by addition of SmaI to linearize the vector at the indi- cated site adjacent to the origin. Nascent DNA strands are shown in red. Note that Pol d, ligase, and Fen1 were omitted in this experiment. (B) Control experiment illustrating that SmaI digestion of the 9.7-kb plasmid at 30C in replication reaction buffer leads to linearization within 10 s. DNA was visualized with Sybr Safe stain. (C) The 9.7-kb plasmid template was linearized during replication as in (A), and g (A) A 9.7-kb plasmid (pZN3) with a single site of Mcm2-7 loading was repli- cated for 3 min and 50 s in the presence of 32P-dCTP to allow establishment of bi-directional replication forks. A chase of unlabeled dNTPs was then added, followed immediately by addition of SmaI to linearize the vector at the indi- cated site adjacent to the origin. Nascent DNA strands are shown in red. Note that Pol d, ligase, and Fen1 were omitted in this experiment. LRI Formation on Linear DNA We next examined whether the LRI was speciﬁcally produced by DNA replication termination on a circular plasmid or would also result from replisome convergence on a linearized template. Using the 9.7-kb origin-speciﬁc plasmid (pZN3; Figure S1A), we pulse-labeled the two nascent replication forks for 4 min (Fig- ure 3A). At that point, we added a cold ‘‘chase’’ of deoxyribonu- cleotide triphosphates (dNTPs) together with the SmaI restriction enzyme, which cleaves the template DNA extremely rapidly un- der these conditions (Figure 3B), proximal to the origin ARS306 and, thus, behind the two active replication forks. The progres- sion and convergence of the two forks were then monitored in native agarose gels. Although linearization of the template routinely led to an 10% increase in the abundance of full-length products (Figure 3C, compare lanes 6 and 7), the LRIs still repre- sented around 80% of the products synthesized under these conditions (Figure 3C, lane 6). Therefore, even when two repli- somes approach each other on linearized DNA, the majority still stall upon convergence. Figure 3. LRI Formation on Linearized Templates Convergence cerevisiae DNA helicases were puriﬁed and then visualized by SDS-PAGE and Coomassie staining. The asterisk denotes a con AR Methods for details). The reaction products were puriﬁed and then digested with SbfI as indicated before analysis in a nativ ndicated S. cerevisiae DNA helicases were puriﬁed and then visualized by SDS-PAGE and Coomassie staining. The asterisk denot see STAR Methods for details). The reaction products were puriﬁed and then digested with SbfI as indicated before analysis i B) The indicated S. cerevisiae DNA helicases were puriﬁed and then visualized by SDS-PAGE and Coomassie staining. The aster Dna2 sample. Dna2 sample. (C) The indicated DNA helicases (see STAR Methods for concentrations) were included in replication reactions with the 3.2-kb plasmid template. The reactions were stopped and quenched after 20 min, and then the replicated products were puriﬁed and linearized with SpeI before resolution in a native agarose gel. Pol d, Fen1, and ligase were omitted from the reactions to prevent strand displacement synthesis by Pol d, which has been shown previously to be enhanced by Pif1- Rrm3 (Osmundson et al., 2017; Rossi et al., 2008). Sgs1-T-R, Sgs1-Top3-Rmi1. (D) The ability of the indicated enzymes to unwind a 25-bp DNA duplex formed by annealing a 35-bp oligonucleotide to an M13 ssDNA template was monitored (C) The indicated DNA helicases (see STAR Methods for concentrations) were included in replication reactions with the 3.2-kb plasmid template. The reactions were stopped and quenched after 20 min, and then the replicated products were puriﬁed and linearized with SpeI before resolution in a native agarose gel. Pol d, Fen1, and ligase were omitted from the reactions to prevent strand displacement synthesis by Pol d, which has been shown previously to be enhanced by Pif1- Rrm3 (Osmundson et al., 2017; Rossi et al., 2008). Sgs1-T-R, Sgs1-Top3-Rmi1. (D) The ability of the indicated enzymes to unwind a 25-bp DNA duplex, formed by annealing a 35-bp oligonucleotide to an M13 ssDNA template, was monitored as described in STAR Methods. Fen1, and ligase were omitted from the reactions to prevent strand displacement synthesis by Pol d, which has been shown previously to be enhanced by Pif1- Rrm3 (Osmundson et al., 2017; Rossi et al., 2008). Sgs1-T-R, Sgs1-Top3-Rmi1. (D) The ability of the indicated enzymes to unwind a 25-bp DNA duplex, formed by annealing a 35-bp oligonucleotide to an M13 ssDNA template, was monitored as described in STAR Methods. Convergence Although the convergence of two reconstituted replisomes pro- duces a late replication intermediate, termination is extremely (B) Control experiment illustrating that SmaI digestion of the 9.7-kb plasmid at 30C in replication reaction buffer leads to linearization within 10 s. DNA was visualized with Sybr Safe stain. (C) The 9.7-kb plasmid template was linearized during replication as in (A), and samples were removed and quenched at the indicated times (lanes 1–6). As a control, undigested plasmid was replicated for 24 min in a parallel reaction (C) The 9.7-kb plasmid template was linearized during replication as in (A), and samples were removed and quenched at the indicated times (lanes 1–6). As a control, undigested plasmid was replicated for 24 min in a parallel reaction Molecular Cell 74, 231–244, April 18, 2019 235 Figure 4. The Budding Yeast Pif1 and Rrm3 DNA Helicases Stimulate Fork Convergence In Vitro (A) Mcm2-7 double hexamers were loaded and phosphorylated on the 3.2-kb plasmid template, which was then replicated in extracts of early S phase yeast cells ( STAR M th d f d t il ) Th ti d t iﬁd d th di t d ith SbfI i di t d b f l i i ti l Figure 4. The Budding Yeast Pif1 and Rrm3 DNA Helicases Stimulate Fork Convergence In Vitro g e loaded and phosphorylated on the 3.2-kb plasmid template, which was then replicated in extracts of early S phase yeast cells he reaction products were puriﬁed and then digested with SbfI as indicated before analysis in a native agarose gel. A helicases were puriﬁed and then visualized by SDS-PAGE and Coomassie staining. The asterisk denotes a contaminant in the (A) Mcm2-7 double hexamers were loaded and phosphorylated on the 3.2-kb plasmid template, which was then replicated in extracts of early S phase yeast cells (see STAR Methods for details). The reaction products were puriﬁed and then digested with SbfI as indicated before analysis in a native agarose gel. (B) The indicated S. cerevisiae DNA helicases were puriﬁed and then visualized by SDS-PAGE and Coomassie staining. The asterisk denotes a contaminant in the ble hexamers were loaded and phosphorylated on the 3.2-kb plasmid template, which was then replicated in extracts of early S ph ods for details). The reaction products were puriﬁed and then digested with SbfI as indicated before analysis in a native agaros S. Convergence (legend continued on next page) 236 Molecular Cell 74, 231–244, April 18, 2019 efﬁcient when plasmid replication is initiated in yeast cell ex- tracts (On et al., 2014), and around 90% of the linearized reaction products comprise full-length dsDNA (Figure 4A). This suggests that efﬁcient fork convergence relies on additional factors that are present in cells but absent from the puriﬁed replication system. Because the formation of LRIs indicates that the two converging replisomes cannot unwind the ﬁnal stretch of parental dsDNA, we considered whether additional ‘‘accessory’’ DNA helicases might be important to stimulate DNA replication termination. Therefore, we expressed and puriﬁed a range of budding yeast DNA helicases that have diverse roles in chromo- some duplication (Figure 4B) and incorporated them into the reconstituted replication reactions. press LRI formation on chromatinized or linearized DNA tem- plates (Figures S4A and S4B). Similarly, the Rrm3 helicase also stimulated the formation of full-length molecules in analogous re- actions (Figure 4C; Figure S3H). However, a bacterial ortholog of Pif1-Rrm3 was much less effective at supporting fork conver- gence in the reconstituted yeast replication system (Figure 4E), even when added at a 10-fold higher concentration than yeast Pif1, despite being highly active as a DNA helicase (Figure 4F; note that Bac Pif1 is better able to unwind a 40-bp duplex than S.c. Pif1). These ﬁndings indicate that the budding yeast Pif1 and Rrm3 helicases have a speciﬁc ability to promote the convergence of yeast replisomes. The addition of Pif1 and Rrm3 to the reconstituted replication reactions did not affect the rate of elongation (Figure S5A), and replication was still dependent on the CMG helicase (Figures S5B and S5C). To test directly whether the Pif1 and Rrm3 heli- cases can promote fork convergence, we examined the ability of wild-type or helicase-dead mutants of Pif1 and Rrm3 (Figures 5A and 5B) to resolve pre-formed LRIs. We generated LRIs as above in the absence of Pif1-Rrm3 and then added wild-type or mutant versions of Pif1 or Rrm3 together with a cold chase of unlabeled dNTPs. As shown in Figures 5C and 5D, both Pif1 and Rrm3 were able to resolve pre-formed LRIs and stimulate the production of full-length dsDNA, dependent on their helicase activity. (E) Bacteroides Pif1 was expressed and puriﬁed as described in STAR Methods and then visualized by SDS-PAGE and Coomassie staining (left). The 3.2-kb plasmid template was then replicated in the presence of 5 nM yeast Pif1 (S.c. [Saccharomyces cerevisiae]) or the indicated concentrations of Bacteroides Pif1 (BacPif1). The reaction products were puriﬁed, digested, and analyzed in native agarose gels. (F) The ability of the indicated concentrations of Bac Pif1 or S.c. Pif1 to unwind a 40-bp DNA duplex, formed by annealing a 55-bp oligonucleotide to an M13 ssDNA template, was monitored as above. See also Figures S3 and S4. Convergence Moreover, Pif1 could still unwind pre-formed LRIs in the presence of the DNA polymerase inhibitor aphidicolin (Figure 5E; Rrm3 was not tested in this assay) at doses that potently in- hibited DNA synthesis in the reconstituted replication system (Figure 5F). In summary, these ﬁndings indicate that the Pif1 and Rrm3 helicases are able to support fork convergence during DNA replication termination by helping to unwind the ﬁnal stretch of parental DNA. Furthermore, these data demonstrate that the LRIs are bona ﬁde precursors for full-length replication products. p Previous studies of DNA replication termination in E. coli showed that the RecQ helicase can unwind a late replication intermediate in the presence of Topoisomerase III and single- stranded DNA (ssDNA)-binding-protein, producing two daughter molecules that each have a short stretch of ssDNA (Suski and Marians, 2008). The yeast RecQ ortholog Sgs1 associates with Top3 and another factor known as Rmi1. This complex plays an important role in resolving recombination intermediates, including Holliday junctions (Bizard and Hickson, 2014; Larsen and Hickson, 2013), and can also catalyze the catenation and decatenation of dsDNA (Cejka et al., 2012; Figure S3A). However, addition of Sgs1-Top3-Rmi1 to the replication reactions had no effect on LRI formation, indicating that the yeast RecQ helicase was unable to promote fork convergence under these conditions (Figure 4C; Figures S3B and S3C; note that high concentrations of Sgs1-Top3-Rmi1 partially inhibited DNA replication in the re- constituted system). Similarly, the stalling of converging forks was unaffected by the presence of the anti-recombinase Srs2 (Ira et al., 2003), the Dna2 nuclease-helicase that contributes to DNA repair and Okazaki fragment processing (Stodola and Bur- gers, 2017), or the Chl1 helicase (Mayer et al., 2004; Samora et al., 2016; Skibbens, 2004) that plays an important but poorly deﬁned role in the establishment of sister chromatid cohesion during chromosome duplication (Figure 4C; Figures S3D–S3F). Importantly, the puriﬁed versions of Sgs1 (30 to 50), Srs2 (30 to 50), and Chl1 (50 to 30) were all active as DNA helicases in vitro (Fig- ure 4D; puriﬁed Dna2 was active as a nuclease, which compli- cated analysis of its 50 to 30 helicase activity). Figure 5. Pif1-Rrm3 Helicase Activity Supports LRI Resolution Figure 5. Pif1-Rrm3 Helicase Activity Supports LRI Resolution (A) Wild-type or ATP-binding mutants of Pif1 and Rrm3 were puriﬁed and then visualized by SDS-PAGE and Coomassie staining. (B) The ability of wild-type or helicase-dead versions of Rrm3 and Pif1 to unwind a 25-bp DNA duplex was monitored as in Figure 4. (C) LRIs were generated as above before addition of the indicated concentrations of wild-type PIf1 or a helicase-dead variant (Pif1-K264A) together with a cold chase of unlabeled dNTPs. The products were digested with SbfI before analysis in a native agarose gel. (D) Analogous reactions with wild-type Rrm3 or a helicase-dead equivalent (Rrm3-K260A). Products were digested with SpeI before native gel analysis. (E) The ability of Pif1 to unwind pre-formed LRIs was monitored in pulse-chase experiments as in (C) and (D) in the presence of DMSO or aphidicolin (10, 40, and 100 mg/mL) as indicated. (F) The effect of DMSO or aphidicolin (4, 10, and 40 mg/ mL) on DNA synthesis of a 5.5-kb template was monitored by denaturing agarose gel electrophoresis in reactions that lacked Pol d, ligase, and Fen1. chase of unlabeled dNTPs. The products were digested with SbfI before analysis in a native agarose gel. (D) Analogous reactions with wild-type Rrm3 or a helicase-dead equivalent (Rrm3-K260A). Products were digested with SpeI before native gel analysis. (E) The ability of Pif1 to unwind pre-formed LRIs was monitored in pulse-chase experiments as in (C) and (D) in the presence of DMSO or aphidicolin (10, 40, and 100 mg/mL) as indicated. (F) The effect of DMSO or aphidicolin (4, 10, and 40 mg/ mL) on DNA synthesis of a 5.5-kb template was monitored by denaturing agarose gel electrophoresis in reactions that lacked Pol d, ligase, and Fen1. (E) The ability of Pif1 to unwind pre-formed LRIs was monitored in pulse-chase experiments as in (C) and (D) in the presence of DMSO or aphidicolin (10, 40, and 100 mg/mL) as indicated. (F) The effect of DMSO or aphidicolin (4, 10, and 40 mg/ mL) on DNA synthesis of a 5.5-kb template was monitored by denaturing agarose gel electrophoresis in reactions that lacked Pol d, ligase, and Fen1. mg ) (F) The effect of DMSO or aphidicolin (4, 10, and 40 mg/ mL) on DNA synthesis of a 5.5-kb template was monitored by denaturing agarose gel electrophoresis in reactions that lacked Pol d, ligase, and Fen1. Pif1 and Rrm3 Support a Top2-Independent Pathway for DNA Replication Termination In E.coli, the type II topoisomerase TopoIV stimulates DNA replication termination by removing precatenanes behind the converging DNA replication forks, reducing torsional strain and allowing the ﬁnal stages of DNA unwinding to proceed (Hiasa and Marians, 1996). To investigate whether type II topoiso- merases contribute to fork convergence in the reconstituted yeast replication system, we compared the ability of yeast Top1 or Top2 and E. coli TopoIV to promote DNA replication termination. In the absence of any topoisomerase, forks stalled during elongation (Figure S6A, lane 1) because of the accumula- tion of positive supercoils in front of each replication fork (Yeeles et al., 2015). Although all three of the tested topoisomerases were able to support fork progression during elongation, the ma- jority of reaction products were LRIs in all cases (Figure S6A, lanes 2–5). However, slightly more full-length molecules were The budding yeast Pif1 and Rrm3 proteins are paralogs with 50 to 30 DNA helicase activity and partially overlapping functions during elongation. Pif1 and Rrm3 jointly help the replisome to bypass barriers at many sites around the genome, such as tRNA promoters, where non-nucleosomal proteins bind tightly to DNA (Ivessa et al., 2002, 2003; Osmundson et al., 2017; Tran et al., 2017). Strikingly, addition of Pif1 to the reconstituted replication reactions greatly stimulated the formation of full- length dsDNA, with a corresponding reduction in the appearance of LRIs (Figure 4C; Figure S3G). Moreover, Pif1 could also sup- Molecular Cell 74, 231–244, April 18, 2019 237 gure 5. Pif1-Rrm3 Helicase Activity Supports LRI Resolution Wild-type or ATP-binding mutants of Pif1 and Rrm3 were puriﬁed and then visualized by SDS-PAGE and Coomassie staining. The ability of wild-type or helicase-dead versions of Rrm3 and Pif1 to unwind a 25-bp DNA duplex was monitored as in Figure 4. LRIs were generated as above before addition of the indicated concentrations of wild-type PIf1 or a helicase-dead variant (Pif1-K264A) together with a cold ase of unlabeled dNTPs. The products were digested with SbfI before analysis in a native agarose gel. Analogous reactions with wild-type Rrm3 or a helicase-dead equivalent (Rrm3-K260A). Products were digested with SpeI before native gel analysis. The ability of Pif1 to unwind pre-formed LRIs was monitored in pulse-chase experiments as in (C) and (D) in the presence of DMSO or aphidicolin (10, 40, and 0 mg/mL) as indicated. Pif1 and Rrm3 Support a Top2-Independent Pathway for DNA Replication Termination The effect of DMSO or aphidicolin (4, 10, and 40 mg/ mL) on DNA synthesis of a 5.5-kb template was monitored by denaturing agarose gel electrophoresis in ctions that lacked Pol d ligase and Fen1 See also Figure S5. Rrm3 and Pif1 Are Required for Efﬁcient Termination of Plasmid Replication In Vivo To investigate whether Pif1-family helicases promote DNA replication termination independent of type II topoisomerase ac- tivity, we compared the ability of Pif1 to drive fork convergence in the presence of Top1, Top2, or both enzymes (Figure 6A). The stimulation of fork convergence by Pif1 was comparable in the presence of either Top1 or Top2 (Figure 6A; Figure S6C), indi- cating that the action of a type II topoisomerase is not required for Pif1 to promote fork convergence. Nevertheless, the produc- tion of full-length products was reproducibly enhanced in reac- tions containing Pif1 together with both Top1 and Top2 (Figures 6A and S6B). To explore the in vivo signiﬁcance of the Pif1 helicase family for DNA replication termination, we used native agarose gels and Southern blotting to screen for the appearance of LRIs during plasmid replication in synchronized yeast cells. Previous work showed that LRIs accumulated transiently when fork conver- gence was impeded in budding yeast cells by expression of a catalytically dead form of Top2 (Baxter and Difﬂey, 2008). Although LRIs were not observed during plasmid replication in control cells, they accumulated strikingly in rrm3D pif1-m2 dou- ble mutant cells before being resolved at later time points (Fig- ures 7A and 7B; note that rrm3D pif1D cells were too sick to examine in similar experiments, and so we used the pif1-m2 allele, which expresses the mitochondrial isoform of the protein but lacks nuclear Pif1). In contrast, LRI accumulation was less severe in rrm3D and absent in pif1D single mutant cells (Fig- ure S7A), indicating that the Rrm3 and Pif1 DNA helicases play a partially redundant role in promoting fork convergence during DNA replication termination in budding yeast cells. Finally, we tested whether Pif1 and Rrm3 can support the completion of DNA replication termination and the production of completely ligated products in the absence of type II topo- isomerase activity (reactions containing Top1 but not Top2). If plasmid replication were complete under such conditions, then the products should represent catenated dimers, which could then be decatenated with E. coli TopoIV after puriﬁcation of the replicated DNA (Figure 6B). This would produce relaxed monomers, into which negative supercoils could then be intro- duced by incubation with the DNA intercalator ethidium bromide. Figure 5. Pif1-Rrm3 Helicase Activity Supports LRI Resolution S l Fi S5 238 Molecular Cell 74, 231–244, April 18, 2019 produced in the presence of the type II topoisomerases Top2 or TopoIV (Figures S6A, lanes 3–5, and S6B) compared with a reac- tion containing the type I enzyme Top1 (Figures S6A, lane 2, and S6B). These ﬁndings indicate that type II topoisomerase activity has a modest ability, under these conditions, to promote fork convergence in the absence of Pif1-Rrm3. To investigate whether Pif1-family helicases promote DNA replication termination independent of type II topoisomerase ac- tivity, we compared the ability of Pif1 to drive fork convergence in the presence of Top1, Top2, or both enzymes (Figure 6A). The stimulation of fork convergence by Pif1 was comparable in the presence of either Top1 or Top2 (Figure 6A; Figure S6C), indi- cating that the action of a type II topoisomerase is not required for Pif1 to promote fork convergence. Nevertheless, the produc- tion of full-length products was reproducibly enhanced in reac- tions containing Pif1 together with both Top1 and Top2 (Figures 6A and S6B). Finally, we tested whether Pif1 and Rrm3 can support the completion of DNA replication termination and the production of completely ligated products in the absence of type II topo- isomerase activity (reactions containing Top1 but not Top2). If plasmid replication were complete under such conditions, then the products should represent catenated dimers, which could then be decatenated with E. coli TopoIV after puriﬁcation of the replicated DNA (Figure 6B). This would produce relaxed monomers, into which negative supercoils could then be intro- duced by incubation with the DNA intercalator ethidium bromide. Figure 6. Pif1-Family Helicases Support a Top2-Independent Pathway for DNA Replica- tion Termination (A) The 3.2-kb plasmid template was replicated in the presence of the indicated topoisomerases with or without Pif1. The products were digested with SmaI before analysis in a native agarose gel. (B) Reaction schematic for experiments to test whether Pif1 and Rrm3 can support complete plasmid duplication in the absence of Top2. Re- actions were performed in the presence of Pol d, Fen1, ligase, and Top1 with or without Pif1-Rrm3. Complete plasmid replication would produce cate- nated dimers, which could then be puriﬁed and decatenated by E. coli TopoIV. (C) Reactions performed as in (B) were analyzed in native agarose gels in the absence of the DNA in- tercalator ethidium bromide. (D) The same reaction products were resolved in a native agarose gel in the presence of 0.5 mg/mL ethidium bromide. See also Figure S6. containing Pif1 or Rrm3 did indeed pro- duce relaxed monomers (note that the reaction products were not linearized in these experiments, in contrast to those above). Moreover, a proportion of the reac- tion products were converted into super- coiled monomers in the presence of ethidium bromide, indicating that the prod- ucts comprised covalently closed circles (Figure 6D). Therefore, both Pif1 and Rrm3 are able to drive fork convergence and the completion of DNA synthesis in the complete absence of topoisomerase II activity. containing Pif1 or Rrm3 did indeed pro- duce relaxed monomers (note that the reaction products were not linearized in these experiments, in contrast to those above). Moreover, a proportion of the reac- tion products were converted into super- coiled monomers in the presence of ethidium bromide, indicating that the prod- ucts comprised covalently closed circles (Figure 6D). Therefore, both Pif1 and Rrm3 are able to drive fork convergence and the completion of DNA synthesis in the complete absence of topoisomerase II activity. produced in the presence of the type II topoisomerases Top2 or TopoIV (Figures S6A, lanes 3–5, and S6B) compared with a reac- tion containing the type I enzyme Top1 (Figures S6A, lane 2, and S6B). These ﬁndings indicate that type II topoisomerase activity has a modest ability, under these conditions, to promote fork convergence in the absence of Pif1-Rrm3. Figure 5. Pif1-Rrm3 Helicase Activity Supports LRI Resolution As shown in Figure 6C, decatenation of the products of reactions ucts comprised cova both Pif1 and Rrm3 the completion of D topoisomerase II act Rrm3 and Pif1 Are Plasmid Replicatio To explore the in vivo DNA replication term Southern blotting to plasmid replication i showed that LRIs ac gence was impeded catalytically dead fo Although LRIs were control cells, they ac ble mutant cells befo ures 7A and 7B; not examine in similar e allele, which express but lacks nuclear Pif severe in rrm3D and ure S7A), indicating t a partially redundant DNA replication term To conﬁrm that the and Rrm3 were caus during DNA replicatio produced in the presence of the type II topoisomerases Top2 or TopoIV (Figures S6A, lanes 3–5, and S6B) compared with a reac- ti t i i th t I T 1 (Fi S6A l 2 d ucts comprised cov both Pif1 and Rrm th l ti f Figure 6. Pif1-Family Helicases Support a Top2-Independent Pathway for DNA Replica- tion Termination Rrm3 and Pif1 Are Required for Efﬁcient Termination of Plasmid Replication In Vivo As shown in Figure 6C, decatenation of the products of reactions To conﬁrm that the LRIs observed in yeast cells lacking Pif1 and Rrm3 were caused by a speciﬁc defect in fork convergence during DNA replication termination rather than being produced Molecular Cell 74, 231–244, April 18, 2019 239 by the pausing of forks at replication fork barriers, we explored the nature of the LRI by native-native two-dimensional gel elec- trophoresis. DNA isolated from the 40-min time point in the experiment in Figure 7B was digested with a restriction enzyme that cuts within the origin (Figure S7B), and the samples were then analyzed by native-native two-dimensional gel electropho- resis. If cells lacking Pif1 and Rrm3 have a speciﬁc defect in fork convergence during termination, then the digested LRI should comprise a large ‘‘double Y’’ structure that approximates an X-shaped molecule with arms of roughly equal length (LRI in Fig- ure 7C, left, and Figure S7B). However, if the LRI is formed by one fork stalling at a barrier during elongation until the arrival of the second fork, then the digestion of the plasmid at the origin would produce an X-shaped LRI with uneven arm length, which would migrate further down the ‘‘X arc’’ in the two-dimensional gel (Fig- ure 7C, left, black dotted line). In contrast to plasmid DNA from control cells (Figure 7C, center), the digested DNA sample from pif1-m2 rrm3D cells contained a single LRI species that migrated as a very large double Y near the top of the X arc (Fig- ure 7C, right). This demonstrates that the LRI was produced by a defect in fork convergence on the opposite side of the plasmid to the origin. Figure 7. Pif1-Rrm3 Are Required for Efﬁcient Termination of Plasmid DNA Replication In Vivo (A) Control (W303-1a) and pif1-m2 rrm3D (yMO291) yeast strains containing the plasmid pRS425 were arrested in G1 phase with mating pheromone and then released into S phase for the indicated times. DNA content was monitored by ﬂow cytometry. (B) Samples from each time point were used to prepare total DNA, which was then analyzed by native agarose gel electrophoresis and Southern blotting (C) The DNA samples from the 40-min time point in (B) were digested at the origin of replication with SnaBI before resolution in a two-dimensional neutral-neutral agarose gel, as described in STAR Methods. Left: the positions of linear molecules (black dot, bottom right) and replication intermediates (double Y structures of increasing size, located along the line from the linear dot to the LRI). DISCUSSION We reconstituted the ﬁnal stages of eukaryotic DNA replication and found that the majority of converging replisomes stall when CMG is the only DNA helicase in the reaction, producing late replication intermediates in which the ligated nascent strands are 90–190 bp shorter than the full-length plasmid (Fig- ure 1D). The deﬁcit in nascent strand size reﬂects the 30-bp footprint of the CMG helicase at each fork (R€aschle et al., 2008), together with any parental dsDNA between the converged replisomes, and also the gap between the 50 end of the lagging strand and the 30 end of the leading strand at each fork (Fig- ure 1A). The observed difference between the size of ligated nascent strands and the full-length plasmid is comparable with the situation when two replisomes converge at an inter-strand DNA cross-link in Xenopus egg extracts (R€aschle et al., 2008). Therefore, the reconstituted yeast replisomes must be very close to each other in the LRI, and the remaining amount of parental dsDNA must be less than the minimal value of about 150 bp that is needed to form a loop or supercoil (Vafabakhsh and Ha, 2012). This implies that the converging replisomes stall after the point at which fork progression becomes dependent on repli- some rotation and precatenane formation. The defect in fork convergence is seen in the presence of type I or II topoisomerases, regardless of whether the reactions involve Figure 7. Pif1-Rrm3 Are Required for Efﬁcient Termination of Plasmid DNA Replication In Vivo (A) Control (W303-1a) and pif1-m2 rrm3D (yMO291) yeast strains containing the plasmid pRS425 were arrested in G1 phase with mating pheromone and then released into S phase for the indicated times. DNA content was monitored by ﬂow cytometry. (B) Samples from each time point were used to prepare total DNA, which was then analyzed by native agarose gel electrophoresis and Southern blotting Figure 7. Pif1-Rrm3 Are Required for Efﬁcient Termination of Plasmid DNA Replication In Vivo (A) Control (W303-1a) and pif1-m2 rrm3D (yMO291) yeast strains containing the plasmid pRS425 were arrested in G1 phase with mating pheromone and then released into S phase for the indicated times. DNA content was monitored by ﬂow cytometry. (Brewer and Fangman, 1987). The positions of various plasmid isoforms are indicated, and the red arrow indicates the accumulation of LRIs in pif1-m2 rrm3D cells. (Brewer and Fangman, 1987). The positions of various plasmid isoforms are indicated, and the red arrow indicates the accumulation of LRIs in pif1-m2 rrm3D cells. (C) The DNA samples from the 40-min time point in (B) were digested at the origin of replication with SnaBI before resolution in a two-dimensional neutral-neutral agarose gel, as described in STAR Methods. Left: the positions of linear molecules (black dot, bottom right) and replication intermediates (double Y structures of increasing size, located along the line from the linear dot to the LRI). See also Figure S7 DISCUSSION coli and SV40, the removal of precatenanes by type II top- oisomerases relieves torsional strain at converging forks and allows continued replisome rotation and completion of DNA unwinding. We note that Top2 does make a minor contribution to the efﬁciency of fork convergence in the reconstituted yeast replication system (Figure S6), but depletion of type II topoisom- erase does not block fork convergence in yeast cells (Baxter and Difﬂey, 2008) or Xenopus egg extracts (Dewar et al., 2015). This indicates that eukaryotic cells have other pathways that can drive fork convergence during DNA replication termination, inde- pendent of type II topoisomerase activity. Our data identify one such pathway that is driven by the Pif1 helicase family and is distinct from the previously described prokaryotic mechanism of fork convergence. We note that our data are consistent with past studies that monitored fork convergence at protein-DNA barriers in vivo and that observed termination defects in budding yeast cells lacking Rrm3 (Ivessa et al., 2000; Mohanty et al., 2006) or in ﬁssion yeast cells lacking the Pfh1 ortholog of Pif1-Rrm3 (Sabo- uri et al., 2012; Steinacher et al., 2012). However, our ﬁndings establish that Pif1 and Rrm3 have a direct role in fork conver- gence per se rather than simply being required to remove a pro- tein-DNA barrier that otherwise would block fork convergence. The reconstitution of Pif1-dependent DNA replication termina- tion sets the scene for future mechanistic studies of this enig- matic and fascinating area of chromosome duplication. It will be particularly interesting to determine how the termination of DNA synthesis provides a signal for disassembly of the eukary- otic replisome, which is initiated by ubiquitylation of the CMG helicase and represents the ﬁnal regulated step in eukaryotic chromosome replication (Dewar et al., 2015, 2017; Maric et al., 2014; Moreno et al., 2014; Sonneville et al., 2017). The CMG helicase is essential for replisome progression and can unwind the DNA template over many kilobases, aided by stable entrapment of the leading-strand DNA template within the hexameric Mcm2-7 ring. DISCUSSION (B) Samples from each time point were used to prepare total DNA, which was then analyzed by native agarose gel electrophoresis and Southern blotting (B) Samples from each time point were used to prepare total DNA, which was then analyzed by native agarose gel electrophoresis and Southern blotting See also Figure S7. 240 Molecular Cell 74, 231–244, April 18, 2019 240 Molecular Cell 74, 231–244, April 18, 2019 ‘‘minimal’’ (Yeeles et al., 2015) or ‘‘complete’’ (Yeeles et al., 2017) versions of the replisome or whether fork convergence occurs on circular or linearized DNA templates (Figure 3). Several potential explanations for LRI formation could be envisaged. First, the two replisomes might clash when they encounter each other. How- ever, previous work indicated that the CMG helicase encircles the template of the leading strand and can bypass protein bar- riers on the lagging strand (Fu et al., 2011; Langston et al., 2017). Furthermore, recent evidence indicates that the activation of an Mcm2-7 double hexamer during initiation produces two ‘‘converged’’ CMG helicases that must bypass each other before leaving the origin and establishing bi-directional replica- tion forks (Douglas et al., 2018). This suggests that two converging replisomes should also be able to pass each other unimpeded during DNA replication termination. ‘‘minimal’’ (Yeeles et al., 2015) or ‘‘complete’’ (Yeeles et al., 2017) versions of the replisome or whether fork convergence occurs on circular or linearized DNA templates (Figure 3). Several potential explanations for LRI formation could be envisaged. First, the two replisomes might clash when they encounter each other. How- ever, previous work indicated that the CMG helicase encircles the template of the leading strand and can bypass protein bar- riers on the lagging strand (Fu et al., 2011; Langston et al., 2017). Furthermore, recent evidence indicates that the activation of an Mcm2-7 double hexamer during initiation produces two ‘‘converged’’ CMG helicases that must bypass each other before leaving the origin and establishing bi-directional replica- tion forks (Douglas et al., 2018). This suggests that two converging replisomes should also be able to pass each other unimpeded during DNA replication termination. replication forks. Both Pif1 and Rrm3 unwind DNA 50 to 30 and, thus, interact with the template of the lagging strand, comple- menting the 30 to 50 action of the CMG helicase along the lead- ing-strand template. DISCUSSION Notably, a bacterial Pif1 helicase was very inefﬁcient at LRI resolution despite being a highly active DNA helicase in vitro (Figures 4E and 4F). Moreover, the Chl1 50 to 30 helicase was also unable to promote fork convergence in the reconstituted system (Figures 4C and 4D). These ﬁndings indicate that the budding yeast Pif1 helicases have a speciﬁc ability to support DNA replication termination, perhaps aided by a direct interaction with the budding yeast replisome. Consis- tent with this view, Rrm3 was found to interact with the catalytic subunit of Pol ε (Azvolinsky et al., 2006). In agreement with our in vitro data, Pif1 and Rrm3 share a partially overlapping role during the termination of plasmid repli- cation in budding yeast cells, leading to the accumulation of LRIs in rrm3D pif1-m2 cells (Figure 7; Figure S7). Fork convergence is delayed but not abolished under such conditions, indicating that additional pathways contribute to termination in vivo, possibly including the removal of precatenanes by type II topoisomerases (Fachinetti et al., 2010). The existence of multiple pathways that promote fork convergence likely explains why budding yeast cells lacking both Pif1 and Rrm3 are very sick but able to form colonies. Functional redundancy with other fork convergence pathways might also explain the viability of mice that lack the sin- gle mammalian ortholog of Pif1 (Snow et al., 2007). However, we cannot exclude that a penetrant defect in fork convergence in the absence of Pif1 helicases is counterbalanced by other pathways that can resolve X-shaped DNA molecules before cell division; for example, through the action of nucleases and recombination factors. A second potential explanation for LRI formation was that inactive Mcm2-7 double hexamers might block the convergence of two replisomes in the absence of factors (potentially including Pif1-Rrm3) that help forks to displace such ‘‘pre-replicative’’ complexes. However, LRIs are still seen under conditions where only a single double hexamer is loaded per plasmid, and our data indicate that the major defect in fork convergence in the recon- stituted replication systems arises for another reason, although our ﬁndings do not exclude that inactive Mcm2-7 double hexam- ers have the potential to block progression of the CMG helicase. A further possibility is that converging CMG helicases become arrested at a stage when continued replisome rotation is impaired by torsional strain. During DNA replication termination in E. DISCUSSION In contrast, Pif1 and Rrm3 are monomeric DNA helicases that have low processivity (Byrd and Raney, 2017; Singleton et al., 2007) and play important roles at speciﬁc points during elongation, unwinding highly stable structures, such as G4 quadruplexes, that might otherwise pre- sent a barrier to CMG (Paeschke et al., 2013; Paeschke et al., 2011), and assisting with the replication of centromeres (Chen et al., 2018). It is possible that the robust helicase activity of Pif1-Rrm3 helps to overcome torsional strain at converging forks, driving replisome rotation and helping to unwind the ﬁnal stretch of parental dsDNA. AUTHOR CONTRIBUTIONS Deegan, T.D., and Difﬂey, J.F. (2016). MCM: one ring to rule them all. Curr. Opin. Struct. Biol. 37, 145–151. J.B. analyzed plasmid replication in budding yeast cells (Figures 7 and S7), M.A.O.B. constructed and characterized the pif1-m2 rrm3D strains, and T.D.D. performed all other experiments. J.T.P.Y. supplied the origin-speciﬁc plasmids pZN3 and pVA18, reagents for chromatin replication, and invaluable advice and support. T.D.D. and K.P.M.L. designed the project together with J.B. The manuscript was written by T.D.D. and K.P.M.L. with invaluable input from J.B. and J.T.P.Y. Devbhandari, S., Jiang, J., Kumar, C., Whitehouse, I., and Remus, D. (2017). Chromatin Constrains the Initiation and Elongation of DNA Replication. Mol. Cell 65, 131–141. Dewar, J.M., and Walter, J.C. (2017). Mechanisms of DNA replication termina- tion. Nat. Rev. Mol. Cell Biol. 18, 507–516. Dewar, J.M., Budzowska, M., and Walter, J.C. (2015). The mechanism of DNA replication termination in vertebrates. Nature 525, 345–350. d METHOD DETAILS Azvolinsky, A., Dunaway, S., Torres, J.Z., Bessler, J.B., and Zakian, V.A. (2006). The S. cerevisiae Rrm3p DNA helicase moves with the replication fork and affects replication of all yeast chromosomes. Genes Dev. 20, 3104–3116. B Yeast methods Baxter, J., and Difﬂey, J.F.X. (2008). Topoisomerase II inactivation prevents the completion of DNA replication in budding yeast. Mol. Cell 30, 790–802. Bell, S.P., and Labib, K. (2016). Chromosome Duplication in Saccharomyces cerevisiae. Genetics 203, 1027–1067. Bermejo, R., Doksani, Y., Capra, T., Katou, Y.M., Tanaka, H., Shirahige, K., and Foiani, M. (2007). Top1- and Top2-mediated topological transitions at replica- tion forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev. 21, 1921–1936. Bermejo, R., Doksani, Y., Capra, T., Katou, Y.M., Tanaka, H., Shirahige, K., and Foiani, M. (2007). Top1- and Top2-mediated topological transitions at replica- tion forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev. 21, 1921–1936. Biswas, D., Yu, Y., Prall, M., Formosa, T., and Stillman, D.J. (2005). The yeast FACT complex has a role in transcriptional initiation. Mol. Cell. Biol. 25, 5812–5822. B Two-dimensional native-native agarose gel electro- phoresis Bizard, A.H., and Hickson, I.D. (2014). The dissolution of double Holliday junc- tions. Cold Spring Harb. Perspect. Biol. 6, a016477. B Gel imaging and presentation Brewer, B.J., and Fangman, W.L. (1987). The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51, 463–471. d QUANTIFICATION AND STATISTICAL ANALYSIS d QUANTIFICATION AND STATISTICAL ANALYSIS Burgers, P.M.J., and Kunkel, T.A. (2017). Eukaryotic DNA Replication Fork. Annu. Rev. Biochem. 86, 417–438. SUPPLEMENTAL INFORMATION Byrd, A.K., and Raney, K.D. (2017). Structure and function of Pif1 helicase. Biochem. Soc. Trans. 45, 1159–1171. Supplemental Information can be found with this article online at https://doi. org/10.1016/j.molcel.2019.01.040. Calzada, A., Hodgson, B., Kanemaki, M., Bueno, A., and Labib, K. (2005). Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev. 19, 1905–1919. ACKNOWLEDGMENTS Cejka, P., Plank, J.L., Dombrowski, C.C., and Kowalczykowski, S.C. (2012). Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes. Mol. Cell 47, 886–896. We gratefully acknowledge support from the Wellcome Trust (reference 204678/Z/16/Z for a Sir Henry Wellcome postdoctoral fellowship to T.D.D. and 102943/Z/13/Z for an investigator award to K.P.M.L.), the Medical Research Council (core grants MC_UU_12016/13 to K.P.M.L. and MC_UP_1201/12 to J.T.P.Y.), BBSRC (project grant BB/N007344/1 to J.B.), and the Royal Society (a research fellowship to J.B.). We thank Valentina Aria for plasmid pVA18, John Difﬂey for strains and for sharing unpublished data, Steve Kowalczykowski for puriﬁed Sgs1 and Top3-Rmi1, Lumir Krejci for plasmids and protocols for expression of Srs2, Martin Singleton for providing baculoviruses that express Chl1, Fabrizio Villa for help with insect cell growth and infection, and MRC PPU Reagents and Services (https:// mrcppureagents.dundee.ac.uk) for support. Chen, C.F., Pohl, T.J., Pott, S., and Zakian, V.A. (2018). Two Pif1-family DNA Helicases Cooperate in Centromere Replication and Segregation in Saccharomyces cerevisiae. Genetics 211, 1105–1119. Christianson, T.W., Sikorski, R.S., Dante, M., Shero, J.H., and Hieter, P. (1992). Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119–122. Coster, G., and Difﬂey, J.F.X. (2017). Bidirectional eukaryotic DNA replication is established by quasi-symmetrical helicase loading. Science 357, 314–318. Coster, G., Frigola, J., Beuron, F., Morris, E.P., and Difﬂey, J.F. (2014). Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase. Mol. Cell 55, 666–677. The authors declare no competing interests. Douglas, M.E., Ali, F.A., Costa, A., and Difﬂey, J.F.X. (2018). The mechanism of eukaryotic CMG helicase activation. Nature 555, 265–268. Received: August 9, 2018 Revised: November 26, 2018 Accepted: January 29, 2019 Published: March 5, 2019 Received: August 9, 2018 Revised: November 26, 2018 Accepted: January 29, 2019 Published: March 5, 2019 Evrin, C., Clarke, P., Zech, J., Lurz, R., Sun, J., Uhle, S., Li, H., Stillman, B., and Speck, C. (2009). A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. Proc. Natl. Acad. Sci. USA 106, 20240–20245. DECLARATION OF INTERESTS Dewar, J.M., Low, E., Mann, M., R€aschle, M., and Walter, J.C. (2017). CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replica- tion termination. Genes Dev. 31, 275–290. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS d KEY RESOURCES TABLE d CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS The polarity of Pif1 helicases is likely to be an important deter- minant of their ability to assist with DNA unwinding at converging Molecular Cell 74, 231–244, April 18, 2019 241 d METHOD DETAILS B Yeast methods B Protein puriﬁcation buffers B Protein puriﬁcation B DNA templates B Molecular weight markers B In vitro replication assays B Preparation of substrates for helicase assays B Helicase assays B Catenation assay with Sgs1-Top3-Rmi1 B DNA preparation for agarose gel electrophoresis B Decatenation of replicated plasmids with E. coli TopoIV B One-dimensional agarose gel electrophoresis and Southern blotting B Two-dimensional native-native agarose gel electro- phoresis B Gel imaging and presentation d QUANTIFICATION AND STATISTICAL ANALYSIS REFERENCES Post-licensing Speciﬁcation of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA. Mol. Cell 60, 797–807. On, K.F., Beuron, F., Frith, D., Snijders, A.P., Morris, E.P., and Difﬂey, J.F. (2014). Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication. EMBO J. 33, 605–620. Hiasa, H., and Marians, K.J. (1996). Two distinct modes of strand unlinking during theta-type DNA replication. J. Biol. Chem. 271, 21529–21535. Osmundson, J.S., Kumar, J., Yeung, R., and Smith, D.J. (2017). Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes. Nat. Struct. Mol. Biol. 24, 162–170. Ira, G., Malkova, A., Liberi, G., Foiani, M., and Haber, J.E. (2003). Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115, 401–411. Paeschke, K., Capra, J.A., and Zakian, V.A. (2011). DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase. Cell 145, 678–691. Ishimi, Y., Sugasawa, K., Hanaoka, F., Eki, T., and Hurwitz, J. (1992). Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro. J. Biol. Chem. 267, 462–466. Paeschke, K., Bochman, M.L., Garcia, P.D., Cejka, P., Friedman, K.L., Kowalczykowski, S.C., and Zakian, V.A. (2013). Pif1 family helicases suppress genome instability at G-quadruplex motifs. Nature 497, 458–462. Ivessa, A.S., Zhou, J.Q., and Zakian, V.A. (2000). The Saccharomyces Pif1p DNA helicase and the highly related Rrm3p have opposite effects on replica- tion fork progression in ribosomal DNA. Cell 100, 479–489. R€aschle, M., Knipscheer, P., Enoiu, M., Angelov, T., Sun, J., Grifﬁth, J.D., Ellenberger, T.E., Sch€arer, O.D., and Walter, J.C. (2008). Mechanism of repli- cation-coupled DNA interstrand crosslink repair. Cell 134, 969–980. Ivessa, A.S., Zhou, J.Q., Schulz, V.P., Monson, E.K., and Zakian, V.A. (2002). Saccharomyces Rrm3p, a 50 to 30 DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA. Genes Dev. 16, 1383–1396. Remus, D., Beuron, F., Tolun, G., Grifﬁth, J.D., Morris, E.P., and Difﬂey, J.F. (2009). Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell 139, 719–730. Ivessa, A.S., Lenzmeier, B.A., Bessler, J.B., Goudsouzian, L.K., Schnakenberg, S.L., and Zakian, V.A. (2003). The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA com- plexes. Mol. Cell 12, 1525–1536. Richter, A., Strausfeld, U., and Knippers, R. (1987). Effects of VM26 (teniposide), a speciﬁc inhibitor of type II DNA topoisomerase, on SV40 DNA replication in vivo. Nucleic Acids Res. 15, 3455–3468. REFERENCES Fachinetti, D., Bermejo, R., Cocito, A., Minardi, S., Katou, Y., Kanoh, Y., Shirahige, K., Azvolinsky, A., Zakian, V.A., and Foiani, M. (2010). Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements. Mol. Cell 39, 595–605. Azmi, I.F., Watanabe, S., Maloney, M.F., Kang, S., Belsky, J.A., MacAlpine, D.M., Peterson, C.L., and Bell, S.P. (2017). Nucleosomes inﬂuence multiple steps during replication initiation. eLife 6, e22512. 242 Molecular Cell 74, 231–244, April 18, 2019 Maric, M., Mukherjee, P., Tatham, M.H., Hay, R., and Labib, K. (2017). Ufd1- Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication. Cell Rep. 18, 3033–3042. Foltman, M., Evrin, C., De Piccoli, G., Jones, R.C., Edmondson, R.D., Katou, Y., Nakato, R., Shirahige, K., and Labib, K. (2013). Eukaryotic replisome com- ponents cooperate to process histones during chromosome replication. Cell Rep. 3, 892–904. Marini, V., and Krejci, L. (2012). Unwinding of synthetic replication and recom- bination substrates by Srs2. DNA Repair (Amst) 11, 789–798. Frigola, J., Remus, D., Mehanna, A., and Difﬂey, J.F. (2013). ATPase-depen- dent quality control of DNA replication origin licensing. Nature 495, 339–343. Mayer, M.L., Pot, I., Chang, M., Xu, H., Aneliunas, V., Kwok, T., Newitt, R., Aebersold, R., Boone, C., Brown, G.W., and Hieter, P. (2004). Identiﬁcation of protein complexes required for efﬁcient sister chromatid cohesion. Mol. Biol. Cell 15, 1736–1745. Fu, Y.V., Yardimci, H., Long, D.T., Ho, T.V., Guainazzi, A., Bermudez, V.P., Hurwitz, J., van Oijen, A., Sch€arer, O.D., and Walter, J.C. (2011). Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA heli- case. Cell 146, 931–941. of protein complexes required for efﬁcient sister chromatid cohesion. Mol. Biol. Cell 15, 1736–1745. Mohanty, B.K., Bairwa, N.K., and Bastia, D. (2006). The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 103, 897–902. Gambus, A., Jones, R.C., Sanchez-Diaz, A., Kanemaki, M., van Deursen, F., Edmondson, R.D., and Labib, K. (2006). GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat. Cell Biol. 8, 358–366. Moreno, S.P., Bailey, R., Campion, N., Herron, S., and Gambus, A. (2014). Polyubiquitylation drives replisome disassembly at the termination of DNA replication. Science 346, 477–481. Gros, J., Kumar, C., Lynch, G., Yadav, T., Whitehouse, I., and Remus, D. (2015). REFERENCES Rossi, M.L., Pike, J.E., Wang, W., Burgers, P.M., Campbell, J.L., and Bambara, R.A. (2008). Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal. J. Biol. Chem. 283, 27483–27493. Keszthelyi, A., Minchell, N.E., and Baxter, J. (2016). The Causes and Consequences of Topological Stress during DNA Replication. Genes (Basel) 7, 134. Kingston, I.J., Yung, J.S., and Singleton, M.R. (2011). Biophysical character- ization of the centromere-speciﬁc nucleosome from budding yeast. J. Biol. Chem. 286, 4021–4026. Ruone, S., Rhoades, A.R., and Formosa, T. (2003). Multiple Nhp6 molecules are required to recruit Spt16-Pob3 to form yFACT complexes and to reorga- nize nucleosomes. J. Biol. Chem. 278, 45288–45295. Kurat, C.F., Yeeles, J.T.P., Patel, H., Early, A., and Difﬂey, J.F.X. (2017). Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates. Mol. Cell 65, 117–130. Sabouri, N., McDonald, K.R., Webb, C.J., Cristea, I.M., and Zakian, V.A. (2012). DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase. Genes Dev. 26, 581–593. Labib, K., Difﬂey, J.F.X., and Kearsey, S.E. (1999). G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus. Nat. Cell Biol. 1, 415–422. Samora, C.P., Saksouk, J., Goswami, P., Wade, B.O., Singleton, M.R., Bates, P.A., Lengronne, A., Costa, A., and Uhlmann, F. (2016). Ctf4 Links DNA Replication with Sister Chromatid Cohesion Establishment by Recruiting the Chl1 Helicase to the Replisome. Mol. Cell 63, 371–384. Langston, L.D., Mayle, R., Schauer, G.D., Yurieva, O., Zhang, D., Yao, N.Y., Georgescu, R.E., and O’Donnell, M.E. (2017). Mcm10 promotes rapid isomer- ization of CMG-DNA for replisome bypass of lagging strand DNA blocks. eLife 6, e29118. Schalbetter, S.A., Mansoubi, S., Chambers, A.L., Downs, J.A., and Baxter, J. (2015). Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability. Proc. Natl. Acad. Sci. USA 112, E4565–E4570. Larsen, N.B., and Hickson, I.D. (2013). RecQ Helicases: Conserved Guardians of Genomic Integrity. Adv. Exp. Med. Biol. 767, 161–184. Lucas, I., Germe, T., Chevrier-Miller, M., and Hyrien, O. (2001). Topoisomerase II can unlink replicating DNA by precatenane removal. EMBO J. 20, 6509– 6519. Seidman, M.M., and Salzman, N.P. (1979). Late replicative intermediates are accumulated during simian virus 40 DNA replication in vivo and in vitro. J. Virol. 30, 600–609. Marahrens, Y., and Stillman, B. (1992). A yeast chromosomal origin of DNA replication deﬁned by multiple functional elements. Science 255, 817–823. REFERENCES Singleton, M.R., Dillingham, M.S., and Wigley, D.B. (2007). Structure and mechanism of helicases and nucleic acid translocases. Annu. Rev. Biochem. 76, 23–50. Maric, M., Maculins, T., De Piccoli, G., and Labib, K. (2014). Cdc48 and a ubiq- uitin ligase drive disassembly of the CMG helicase at the end of DNA replica- tion. Science 346, 1253596. Skibbens, R.V. (2004). Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion. Genetics 166, 33–42. Molecular Cell 74, 231–244, April 18, 2019 243 Taylor, M.R.G., and Yeeles, J.T.P. (2018). The Initial Response of a Eukaryotic Replisome to DNA Damage. Mol. Cell 70, 1067–1080.e12. Snapka, R.M., Powelson, M.A., and Strayer, J.M. (1988). Swiveling and deca- tenation of replicating simian virus 40 genomes in vivo. Mol. Cell. Biol. 8, 515–521. Tourrie` re, H., Versini, G., Cordo´ n-Preciado, V., Alabert, C., and Pasero, P. (2005). Mrc1 and Tof1 promote replication fork progression and recovery inde- pendently of Rad53. Mol. Cell 19, 699–706. Snow, B.E., Mateyak, M., Paderova, J., Wakeham, A., Iorio, C., Zakian, V., Squire, J., and Harrington, L. (2007). Murine Pif1 interacts with telomerase and is dispensable for telomere function in vivo. Mol. Cell. Biol. 27, 1017–1026. Tran, P.L.T., Pohl, T.J., Chen, C.F., Chan, A., Pott, S., and Zakian, V.A. (2017). PIF1 family DNA helicases suppress R-loop mediated genome instability at tRNA genes. Nat. Commun. 8, 15025. Sonneville, R., Moreno, S.P., Knebel, A., Johnson, C., Hastie, C.J., Gartner, A., Gambus, A., and Labib, K. (2017). CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis. Nat. Cell Biol. 19, 468–479. Vafabakhsh, R., and Ha, T. (2012). Extreme bendability of DNA less than 100 base pairs long revealed by single-molecule cyclization. Science 337, 1097–1101. Steinacher, R., Osman, F., Dalgaard, J.Z., Lorenz, A., and Whitby, M.C. (2012). The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability. Genes Dev. 26, 594–602. Vos, S.M., Tretter, E.M., Schmidt, B.H., and Berger, J.M. (2011). All tangled up: how cells direct, manage and exploit topoisomerase function. Nat. Rev. Mol. Cell Biol. 12, 827–841. Stodola, J.L., and Burgers, P.M. (2017). Mechanism of Lagging-Strand DNA Replication in Eukaryotes. Adv. Exp. Med. Biol. 1042, 117–133. Sundin, O., and Varshavsky, A. (1980). Terminal stages of SV40 DNA replica- tion proceed via multiply intertwined catenated dimers. Cell 21, 103–114. Yeeles, J.T., Deegan, T.D., Janska, A., Early, A., and Difﬂey, J.F. (2015). 244 Molecular Cell 74, 231–244, April 18, 2019 REFERENCES cerevisiae strains are detailed in Table S5 Recombinant DNA pAM3 (Cdc6 puriﬁcation) Frigola et al., 2013 N/A pJY19 (PCNA puriﬁcation) Yeeles et al., 2017 N/A pRJ1228-Nhp6 (Nhp6 puriﬁcation) Ruone et al., 2003 N/A pCDFduet.H2A-H2B (histones puriﬁcation) Kingston et al., 2011 N/A pCDFduet.H3-H4(histones puriﬁcation) Kingston et al., 2011 N/A pJFDJ5 (GINS puriﬁcation) Yeeles et al., 2015 N/A pET28a-Mcm10 (Mcm10 puriﬁcation) Yeeles et al., 2015 N/A pTF175 (FACT puriﬁcation) Biswas et al., 2005 N/A pJW22 (FACT puriﬁcation) Biswas et al., 2005 N/A pCFK1 (Nap1 puriﬁcation) Kurat et al., 2017 N/A pET11d-Srs2 (Srs2 puriﬁcation) Marini and Krejci, 2012 N/A pTDK10 (Pif1 puriﬁcation) This study N/A pTDK24 (Pif1 K264A puriﬁcation) This study N/A pTDK4 (generation of yTDK4 for Csm3-Tof1 puriﬁcation) This study N/A pTDK15 (generation of yTDK9 for Rrm3 puriﬁcation) This study N/A pTDK8 (generation of yTDK6 for Top1 puriﬁcation) This study N/A pTDK34 (generation of yTDK17 for Rrm3 K260A puriﬁcation) This study N/A pFV36 (generation of yFV43 for Dna2 puriﬁcation) This study N/A pTDK18 (generation of yTDK18 for Fen1 puriﬁcation) This study N/A pTDK19 (generation of yTDK19 for Cdc9 puriﬁcation) This study N/A pTDK13 (template for generation of molecular weight markers) This study N/A (Continued on next page) REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-ﬂuorescein-AP Fab fragments Roche 000000011426338910 Bacterial and Virus Strains Escherichia coli: Rosetta (DE3) pLysS cells: F ompT hsdSB(rB mB ) gal dcm (DE3) pLysSRARE (CamR) Novagen 70956 Chemicals, Peptides, and Recombinant Proteins Anti-FLAG M2 afﬁnity gel Sigma-Aldrich A2220 Glutathione Sepharose 4B GE Healthcare 17075601 Ni-NTA agarose QIAGEN 30210 Calmodulin Sepharose 4B GE Healthcare 17052901 Streptactin superﬂow resin IBA Life Sciences 2-1206-002 IgG Sepharose 6 Fast Flow GE Healthcare 17096901 3Flag peptide Sigma-Aldrich F4799 Roche Complete EDTA-free protease inhibitor cocktail Roche 000000011873580001 Sigma protease inhibitor cocktail Sigma-Aldrich P8215 Critical Commercial Assays CDP-Star GE Healthcare GERPN3682 Recombinant proteins are detailed in Table S4 Experimental Models: Cell Lines Sf21 insect cells ThermoFisher Scientiﬁc 11497013 Experimental Models: Organisms/Strains S. REFERENCES Regulated eukaryotic DNA replication origin ﬁring with puriﬁed proteins. Nature 519, 431–435. Suski, C., and Marians, K.J. (2008). Resolution of converging replication forks by RecQ and topoisomerase III. Mol. Cell 30, 779–789. Yeeles, J.T.P., Janska, A., Early, A., and Difﬂey, J.F.X. (2017). How the Eukaryotic Replisome Achieves Rapid and Efﬁcient DNA Replication. Mol. Cell 65, 105–116. Szyjka, S.J., Viggiani, C.J., and Aparicio, O.M. (2005). Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae. Mol. Cell 19, 691–697. 244 Molecular Cell 74, 231–244, April 18, 2019 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-ﬂuorescein-AP Fab fragments Roche 000000011426338910 Bacterial and Virus Strains Escherichia coli: Rosetta (DE3) pLysS cells: F ompT hsdSB(rB mB ) gal dcm (DE3) pLysSRARE (CamR) Novagen 70956 Chemicals, Peptides, and Recombinant Proteins Anti-FLAG M2 afﬁnity gel Sigma-Aldrich A2220 Glutathione Sepharose 4B GE Healthcare 17075601 Ni-NTA agarose QIAGEN 30210 Calmodulin Sepharose 4B GE Healthcare 17052901 Streptactin superﬂow resin IBA Life Sciences 2-1206-002 IgG Sepharose 6 Fast Flow GE Healthcare 17096901 3Flag peptide Sigma-Aldrich F4799 Roche Complete EDTA-free protease inhibitor cocktail Roche 000000011873580001 Sigma protease inhibitor cocktail Sigma-Aldrich P8215 Critical Commercial Assays CDP-Star GE Healthcare GERPN3682 Recombinant proteins are detailed in Table S4 Experimental Models: Cell Lines Sf21 insect cells ThermoFisher Scientiﬁc 11497013 Experimental Models: Organisms/Strains S. EXPERIMENTAL MODEL AND SUBJECT DETAILS The Saccharomyces cerevisiae strain yJF1 (MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 bar1D::hphNT pep4D:: kanMX) was transformed with linearized plasmids using standard genetic procedures to generate the protein expression strains, as detailed in the ‘Key Resources Table’, Table S1 and Table S5. The codon usage of the synthetic gene constructs that were used for the protein expression strains was optimized for high-level expression in Saccharomyces cerevisiae (Yeeles et al., 2015). For expression of proteins in E. coli, the corresponding plasmids (listed in the Key Resources Table and Table S1) were transformed into Rosetta (DE3) pLysS cells (Novagen) (F- ompT hsdSB(rB - mB -) gal dcm (DE3) pLysSRARE (CamR). The Saccharomyces cerevisiae strain yJF1 (MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 bar1D::hphNT pep4D:: kanMX) was transformed with linearized plasmids using standard genetic procedures to generate the protein expression strains, as detailed in the ‘Key Resources Table’, Table S1 and Table S5. The codon usage of the synthetic gene constructs that were used for the protein expression strains was optimized for high-level expression in Saccharomyces cerevisiae (Yeeles et al., 2015). For expression of proteins in E coli the corresponding plasmids (listed in the Key Resources Table and Table S1) were transformed For expression of proteins in E. coli, the corresponding plasmids (listed in the Key Resources Table and Table S1) were transformed into Rosetta (DE3) pLysS cells (Novagen) (F- ompT hsdSB(rB - mB -) gal dcm (DE3) pLysSRARE (CamR). REFERENCES cerevisiae strains are detailed in Table S5 Recombinant DNA pAM3 (Cdc6 puriﬁcation) Frigola et al., 2013 N/A pJY19 (PCNA puriﬁcation) Yeeles et al., 2017 N/A pRJ1228-Nhp6 (Nhp6 puriﬁcation) Ruone et al., 2003 N/A pCDFduet.H2A-H2B (histones puriﬁcation) Kingston et al., 2011 N/A pCDFduet.H3-H4(histones puriﬁcation) Kingston et al., 2011 N/A pJFDJ5 (GINS puriﬁcation) Yeeles et al., 2015 N/A pET28a-Mcm10 (Mcm10 puriﬁcation) Yeeles et al., 2015 N/A pTF175 (FACT puriﬁcation) Biswas et al., 2005 N/A pJW22 (FACT puriﬁcation) Biswas et al., 2005 N/A pCFK1 (Nap1 puriﬁcation) Kurat et al., 2017 N/A pET11d-Srs2 (Srs2 puriﬁcation) Marini and Krejci, 2012 N/A pTDK10 (Pif1 puriﬁcation) This study N/A pTDK24 (Pif1 K264A puriﬁcation) This study N/A pTDK4 (generation of yTDK4 for Csm3-Tof1 puriﬁcation) This study N/A pTDK15 (generation of yTDK9 for Rrm3 puriﬁcation) This study N/A pTDK8 (generation of yTDK6 for Top1 puriﬁcation) This study N/A pTDK34 (generation of yTDK17 for Rrm3 K260A puriﬁcation) This study N/A pFV36 (generation of yFV43 for Dna2 puriﬁcation) This study N/A pTDK18 (generation of yTDK18 for Fen1 puriﬁcation) This study N/A pTDK19 (generation of yTDK19 for Cdc9 puriﬁcation) This study N/A pTDK13 (template for generation of molecular weight markers) This study N/A (Continued on next page) Molecular Cell 74, 231–244.e1–e9, April 18, 2019 e1 Continued REAGENT or RESOURCE SOURCE IDENTIFIER pBS/ARS1WTA (3.2 kb replication template) Marahrens and Stillman, 1992 N/A pCFK1_WT (5.8 kb replication template) Yeeles et al., 2015 N/A pVA18 (5.5 kb replication template) Valentina Aria N/A pZN3 (9.7 kb replication template) Taylor and Yeeles, 2018 N/A pRS425 Baxter laboratory N/A l DNA-HindIII Digest: molecular weight markers New England Biolabs N3012S M13 ssDNA New England Biolabs N4040S Software and Algorithms ImageJ National Institute of Health https://imagej.nih.gov/ij/ CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to the Lead Contact, Karim Labib (kpmlabib@ dundee.ac.uk). Further information and requests for resources and reagents should be directed to the Lead Contact, Karim Labib (kpmlabib@ dundee.ac.uk). Yeast methods For expression of proteins, the corresponding strains (see Table S5) were grown at 30C in YP + 2% rafﬁnose to a density of 2–4 3 107 cells / ml. For expression of Mrc1, Csm3-Tof1, Rrm3, Top1, Fen1 and Cdc9, the cells were arrested in G1-phase by incubation for 3 h with 200 ng / ml alpha factor mating pheromone (Pepceuticals). Protein expression was then induced by addition of galactose to 2% for 3 h at 30C. RFC, Pol d and Dna2 expression was induced in the same way, but in asynchronous cultures. Following expression, cells were collected by centrifugation and washed once with lysis buffer (see puriﬁcation protocols for de- tails of buffers used) without protease inhibitors. The cell pellets were then resuspended in 0.3–0.4 volumes of lysis buffer + protease inhibitors (see puriﬁcation protocols for details) and the resulting suspensions were frozen drop-wise in liquid nitrogen. The frozen cells were crushed in a freezer mill (SPEX CertiPrep 6850 Freezer/Mill) with 4 cycles of 20 at a rate of 15. The resulting powders were stored at 80C until required. For the experiments in Figure 7 and Figure S7, cells containing the plasmid pRS425 were grown to early log phase at 30C, in min- imal media supplemented with 2% glucose but lacking leucine, before transfer to rich medium supplemented with 2% Glucose (YPD: 1% yeast extract, 2% peptone), supplemented with 40 mg / ml of adenine. The cells were then grown to mid-log phase and arrested in G1-phase by addition of 10 mg / ml alpha factor per hour, until 95% of cells were unbudded (typically 120’). Cells were then washed three times in YPD medium lacking alpha factor, before further incubation in YPD. ‘Time 0’ was designated as the time of the addition of the ﬁrst wash to the pelleted cells. Samples were taken at the indicated time points and used to prepare genomic DNA (2x108 cells were pelleted and frozen on dry ice) or to monitor DNA content by ﬂow cytometry (Labib et al., 1999). Protein puriﬁcation buffers p Buffer A: 25 mM HEPES KOH pH 7.6, 10% glycerol, 0.02% NP-40-S, 1 mM DTT Buffer B: 25 mM Tris-Cl pH 7.2, 10% glycerol, 1 mM DTT Buffer C: 25 mM Tris-Cl pH 8.5, 10% glycerol, 0.02% NP-40-S, 1mM DTT Buffer D: 25 mM Tris-Cl pH 7.2, 10% glycerol, 0.02% NP-40-S, 1 mM DTT Buffer E: 25 mM HEPES KOH pH 7.6, 10% glycerol, 5 mM MgOAc, 1 mM DTT Buffer F: 50 mM HEPES KOH pH 7.6, 10% glycerol, 1 mM DTT, 0.02% NP-40 e2 Molecular Cell 74, 231–244.e1–e9, April 18, 2019 Chl1 A 20 mL aliquot of P5 viral supernatant, expressing full-length Chl1 including an N-terminal 2HA-6His-2Strep tag (a kind gift from Martin Singleton) was added to 2.8 l SF21 insect cells at a cell density of 1 million cells / ml. The cells were grown at 27C with shaking at 110 rpm for 48 h. The cells were then harvested by centrifugation at 2500 rpm for 15 min at 4C in a JLA9.1000 rotor (Beckman). The cell pellets were washed once in PBS + 5 mM MgOAc, resuspended in 6 volumes 25 mM HEPES pH 7.6, 0.02% Tween-20, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 15 mM KCl, 2 mM MgOAc, 0.4 mM PMSF, 2 mM 2-mercaptoethanol, Roche protease inhibitor tablets, then snap frozen in aliquots. Subsequently, the frozen cell suspension was thawed at room temperature, left on ice for 10 min, then lysed in a dounce homog- enizer (20x compressions). KCl was added to 0.3 M and the sample centrifuged at 40,000 x g for 30 min at 4C in a JA30.5 rotor (Beck- man). The soluble fraction was recovered and mixed with 3 mL Streptactin superﬂow resin (IBA Life Sciences) for 90 min at 4C with rotation. The beads were collected in a disposable column and washed extensively in 50 mM Tris-HCl pH 8.5, 0.3 M NaCl, 10% Glyc- erol, 1 mM DTT, then Chl1 was eluted in 2 column-volumes of 50 mM Tris-HCl pH 8.5, 0.3 M NaCl, 10% Glycerol, 1 mM DTT, 2.5 mM desthiobiotin. The eluate fractions were pooled and the tag removed by overnight cleavage with 100 mg TEV protease at 4C. The sample was diluted 3-fold in 20 mM Tris-HCl pH 8.5, 10% Glycerol, 1 mM DTT and then loaded onto a 1 mL HiTrap Q column. Chl1 was eluted with a 20 column-volume gradient from 0.1 – 0.4 M NaCl in 20 mM Tris-HCl pH 8.5, 0.1 M NaCl, 10% Glycerol, 1 mM DTT. The peak frac- tions were pooled, diluted with 20 mM Tris-HCl pH 8.5, 10% Glycerol, 1 mM DTT to a conductivity equivalent of 0.1 M NaCl, and loaded onto a 0.24 mL MiniQ column. Chl1 was eluted with a 5 mL gradient from 0.1 – 0.4 M NaCl. The peak fractions containing Chl1 were pooled, aliquoted and snap frozen. Homemade protease inhibitor cocktail 1 mM PMSF, 5 mM benzamidine HCl, 1 mM AEBSF, 1 mg/ml pepstatin A, 1 mg/ml aprotinin 1 mM PMSF, 5 mM benzamidine HCl, 1 mM AEBSF, 1 mg/ml pepstatin A, 1 mg/ml aprotinin One protease inhibitor tablet (Roche, 000000011873580001) was used per 25 mL of lysis buffer where indicate 1 mL of Sigma protease inhibitor cocktail (Sigma Aldrich P8215) was used per 100 mL of lysis buffer where in One protease inhibitor tablet (Roche, 000000011873580001) was used per 25 mL of lysis buffer whe 1 mL of Sigma protease inhibitor cocktail (Sigma-Aldrich, P8215) was used per 100 mL of lysis buffe p ( , ) p y 1 mL of Sigma protease inhibitor cocktail (Sigma-Aldrich, P8215) was used per 100 mL of lysis buffer where indicated. Protein puriﬁcation p Yeast protein expression strains, and expression plasmids for protein puriﬁcation from Escherichia coli are detailed in the Key Resources Table, Table S1 and Table S5. Proteins puriﬁed in this study are listed in Table S4. During extract preparation, thawed yeast cell powder was resuspended in 2-3 volumes of the initial lysis buffer in all cases. After afﬁnity puriﬁcation from yeast or bac- terial cell lysates, the afﬁnity resin was washed with at least 40 column volumes of wash buffer in all instances. Cd 9 p Csm3-Tof1 Powder was thawed in buffer A / 0.2 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and the insoluble material removed by centrifugation (235,000 x g, 4C, 1 h). The soluble extract was supplemented with 2 mM CaCl2, 2 mL calmodulin afﬁnity resin was added, and the mixture incubated with rotation at 4C for 90 min. The resin was collected and washed extensively with buffer A / 0.2 M NaCl. The washed resin was resuspended in one column volume of buffer A / 0.2 M NaCl and incubated with 200 mg TEV protease for 4 h at 4C. The ﬂow-through was collected and HIS- tagged TEV protease was depleted with 1 mL Ni-NTA beads (QIAGEN, 30210). The Csm3-Tof1 sample was then separated on a 24 mL Superose 6 column in buffer A / 0.2 M NaCl. Csm3-Tof1 peak fractions were pooled, dialysed against buffer A / 0.3 M KOAc, snap frozen and stored at 80C. D 2 Cdc9 Cdc9 Powder was thawed in buffer C / 0.2 M NaCl / protease inhibitors (Roche tablets + Sigma protease inhibitors + homemade cocktail). Insoluble material was removed by centrifugation (235,000 x g, 4C, 1 h) and the supernatant was mixed with 2.5 mL anti-FLAG M2 afﬁnity gel (Sigma-Aldrich) at 4C for 90 min. y g g The resin was collected and washed with buffer C / 0.2 M NaCl. Cdc9 was eluted in 1 column volum 0.5 mg / ml 3FLAG peptide, then 1 column volume of buffer C / 0.2 M NaCl / 0.25 mg / ml 3FLAG pept The resin was collected and washed with buffer C / 0.2 M NaCl. Cdc9 was eluted in 1 column volume of buffer C / 0.2 M NaCl / 0.5 mg / ml 3FLAG peptide, then 1 column volume of buffer C / 0.2 M NaCl / 0.25 mg / ml 3FLAG peptide. The eluate fraction was diluted 2-fold in buffer C then loaded onto a 1 mL HiTrap Q column in buffer C / 0.1 M NaCl. Cdc9 was eluted with a 20 column-volume gradient from 0.1 – 0.7 M NaCl in buffer C. Cdc9 containing fractions were pooled, dialysed versus buffer A / 0.2 M KOAc, snap frozen and stored at 80. Mrc1 Mrc1 Powder was thawed in buffer A / 0.5 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and insoluble material was removed by centrifugation (235,000 x g, 4C, 1 h). The soluble extract was mixed with 4 mL anti-FLAG M2 afﬁnity gel (Sigma-Aldrich, A2220) and the mixture incubated with rotation at 4C for 90 min. The resin was collected and washed extensively with 120 mL buffer A / 0.5 M NaCl / protease inhibitors, then 20 mL buffer A / 0.5 M NaCl, then 40 mL buffer A / 0.5 M NaCl / 10 mM MgOAc / 1 mM ATP, then 20 mL buffer A / 0.2 M NaCl. Mrc1–5FLAG was eluted in 1 column volume of buffer A / 0.2 M NaCl / 0.5 mg / ml 3FLAG peptide, followed by 2 column volumes of buffer A / 0.2 M NaCl / 0.25 mg / ml 3FLAG peptide. The eluate was diluted to a conductivity equivalent to 0.1 M NaCl with buffer A. The resulting sample was loaded onto 1 mL HiTrap Q column equilibrated in buffer A / 0.1 M NaCl. Mrc1 was eluted with a 20 column-volume gradient from 0.1 – 1 M NaCl in buffer A. Mrc1-containing fractions were pooled and dialysed against buffer A / 40% glycerol / 0.3 M KOAc for 4 h at 4C. The dialysed sam- ple was recovered, aliquoted and snap frozen. Pol a / primase Buffer A / 0.4 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) was added to cell powder and the resuspension was centrifuged (235,000 x g, 4C, 1 h). The soluble extract was supplemented with 2 mM CaCl2 and Pol a / primase was puriﬁed by calmodulin afﬁnity chromatography using 2 mL calmodulin afﬁnity resin (GE Healthcare, 17052901). The eluate fractions were pooled and separated on a 24 mL Superdex 200 column in buffer B + 0.4 M fractions were pooled, concentrated, aliquoted and snap frozen. The eluate fractions were pooled and separated on a 24 mL Superdex 200 column in buffer B + 0.4 M KOAc. Pol a / primase peak fractions were pooled, concentrated, aliquoted and snap frozen. P l d Pif1 (S. cerevisiae) Protein was eluted with a 20 column-volume gradient from 0.15 – 1 M NaCl. Pif1 containing fractions were pooled and dil t d t d ti it i l t f 0 15 M N Cl ith b ff E l ki lt Th lt t f ti th l d d t 1 L The eluate was diluted 2-fold in buffer E lacking salt and then loaded onto a 1 mL HiTrap SP FF column pre-equilibrated in buffer E / 0.15 M NaCl. Protein was eluted with a 20 column-volume gradient from 0.15 – 1 M NaCl. Pif1 containing fractions were pooled and diluted to a conductivity equivalent of 0.15 M NaCl with buffer E lacking salt. The resultant fraction was then loaded onto a 1 mL HiTrap Heparin HP column pre-equilibrated in buffer E / 0.15 M NaCl. Protein was eluted with a 20 column-volume gradient from 0.15 - 1 M NaCl. Pif1 containing fractions were pooled and dialysed against 25 mM HEPES / 40% glycerol / 5 mM MgOAc / 0.3 M KOAc / 1 mM DTT for 4 h at 4C. The dialysed sample was recovered, aliquoted and snap frozen. BacPif1 BacPif1 Pif1 from Bacteroides sp 2 1 16 (BacPif1) was puriﬁed as for budding yeast Pif1, until the Ni2+ afﬁnity puriﬁcation step. Following elution from Ni-NTA resin, the eluate was diluted 2-fold in buffer E lacking salt and then loaded onto a 1 mL HiTrap Q HP column pre-equilibrated in buffer E / 0.15 M NaCl. Protein was eluted with a 20 column-volume gradient from 0.15 – 1 M NaCl. BacPif1 con- taining peak fractions were pooled and dialysed against 25 mM HEPES / 40% glycerol / 5 mM MgOAc / 0.3 M KOAc / 1 mM DTT at 4C for 4 h. The dialysed sample was recovered, aliquoted and snap frozen in liquid nitrogen. Pol a / primase p Dna2 Powder was thawed in buffer A / 0.2 M KCl / protease inhibitors (Roche tablets + homemade cocktail) and insoluble material removed by centrifugation (235,000 x g, 4C, 1 h). The soluble extract was supplemented with 2 mM CaCl2, 2 mL calmodulin afﬁnity resin was added, and the mixture incubated with rotation at 4C for 1 h. The resin was collected and washed extensively with buffer A / 0.2 M KCl. The washed resin was then resuspended in 1 column- volume of buffer A / 0.2 M KCl and incubated with 240 mg TEV protease at room temperature for 1 h. The ﬂow-through was collected and diluted to a conductivity equivalent of 0.1 M KCl with buffer A. The resulting sample was loaded onto 1 mL HiTrap Q column equilibrated in buffer A / 0.1 M KCl. Dna2 was then eluted with a 20 column-volume gradient from 0.1 – 0.7 M KCl in buffer A. Dna2 containing fractions were pooled, concentrated and separated on a 24 mL Superdex 200 column in buffer A / 0.3 M KOAc without NP-40. Dna2 containing fractions were then pooled, concentrated, aliquoted and snap frozen. Molecular Cell 74, 231–244.e1–e9, April 18, 2019 e3 Fen1 Powder was thawed in buffer C / 0.2 M NaCl / protease inhibitors (Roche tablets + Sigma protease inhibitors + homemade cocktail). Insoluble material was removed by centrifugation (235,000 g, 4C, 1 h) and the supernatant was mixed with 2.5 mL anti-FLAG M2 afﬁnity gel (Sigma-Aldrich) at 4C for 90 min. y g ( g ) The resin was collected and washed with buffer C / 0.2 M NaCl. Fen1 was eluted in 1 column volume of buffer C / 0.2 M NaCl / 0.5 mg / ml 3FLAG peptide, then 1 column volume of buffer C / 0.2 M NaCl / 0.25 mg / ml 3FLAG peptide. The eluate fraction was diluted 2-fold in buffer C then loaded onto a 1ml HiTrap heparin HP column in buffer D / 0.1 M NaCl. Fen1 was eluted with a 20 column-volume gradient from 0.1 – 1 M NaCl in buffer D. Fen1 containing fractions were pooled, dialysed versus buffer D / 0.3 M KOAc, snap frozen and stored at 80. Pif1 (S. cerevisiae) Rosetta E. coli cells (Novagen) were transformed with the relevant Pif1 expression vector (see Key Resources Table). The transform- ant colonies were inoculated into a 250 mL LB / ampicillin (50 mg/ml) / chloramphenicol (35 mg/ml) culture, which was grown overnight at 37C with shaking at 200 rpm. The following morning, the culture was diluted into 1-2 l of LB / ampicillin (50 mg/ml) / chloramphen- icol (35 mg/ml) to a ﬁnal OD600 of 0.15. The culture was left to grow at 37C until an OD600 of 0.6 was reached. Cells were cooled on ice for 30 min, 1 mM IPTG was added to induce expression, and then the cells were incubated overnight at 23C. The cells were har- vested by centrifugation at 5000 rpm for 10 min in a JLA-9.1000 rotor (Beckman). For lysis, cell pellets were resuspended in 30 mL of buffer E / 0.3 M NaCl / 30 mM imidazole / Roche protease inhibitor tablets. Lysozyme was added to a ﬁnal concentration of 500 mg/ml and the mixture then left for 20 min on ice. Subsequently, the sample was sonicated for 90 s (15 s on, 30 s off) at 40% on a Branson Digital Soniﬁer. Insoluble material was removed by centrifugation at 15000 rpm for 30 min in an SS-34 rotor (Sorvall). The supernatant was subjected to Ni2+ afﬁnity puriﬁcation by incubation with 1 mL packed bead volume of Ni-NTA resin (QIAGEN) for 90 min at 4C. The beads were recovered in a disposable gravity ﬂow column and washed extensively with buffer E / 0.3 M NaCl / 30 mM imidazole / Roche protease inhibitor tablets. Pif1 was eluted with 5 column volumes of buffer E / 0.3 M NaCl / 0.4 M imidazole. The supernatant was subjected to Ni2+ afﬁnity puriﬁcation by incubation with 1 mL packed bead volume of Ni-NTA resin (QIAGEN) for 90 min at 4C. The beads were recovered in a disposable gravity ﬂow column and washed extensively with buffer E / 0.3 M NaCl / 30 mM imidazole / Roche protease inhibitor tablets. Pif1 was eluted with 5 column volumes of buffer E / 0.3 M NaCl / 0.4 M imidazole. The eluate was diluted 2-fold in buffer E lacking salt and then loaded onto a 1 mL HiTrap SP FF column pre-equilibrated in buffer E / 0.15 M NaCl. Srs2 Rosetta E. coli cells were transformed with pET11d-Srs2 (see Key Resources Table). A 200 mL LB / ampicillin (50 mg/ml) / chloram- phenicol (35 mg/ml) culture was inoculated with transformant colonies and grown overnight at 37C with shaking at 200 rpm. The following morning, the culture was diluted 20-fold into 2 l of LB / ampicillin (50 mg/ml) / chloramphenicol (35 mg/ml) and then left to grow at 37C until an OD600 of 1 was reached. 0.1 mM IPTG was added and Srs2 expression induced overnight at 16C. Cells were harvested by centrifugation for 10 min in a JLA-9.1000 rotor (Beckman) at 5000 rpm. y g ( ) p For lysis, cell pellets were resuspended in 30 mL of buffer A / 0.6 M KCl / 30 mM imidazole / Roche protease inhibitor tablets. Son- ication was performed as described for Pif1. Insoluble material was removed by centrifugation at 15,000 rpm for 30 min in an SS-34 rotor (Sorvall). ( ) The supernatant was subjected to Ni2+ afﬁnity puriﬁcation by incubation with 1 mL packed bead volume of Ni-NTA resin (QIAGEN) for 90 min at 4C. The beads were recovered in a disposable gravity ﬂow column and washed extensively with buffer A / 0.6 M KCl / 30 mM imidazole / Roche protease inhibitor tablets. Srs2 was eluted with 10 column volumes of buffer A / 0.6 M KCl / 0.4 M imidazole. The eluate was diluted 4-fold in buffer A lacking salt and then loaded onto a 1 mL HiTrap SP FF column pre-equilibrated in buffer A / 0.15 M KCl. Protein was eluted with a 20 column-volume gradient from 0.15 – 1 M KCl. Srs2 containing fractions were then pooled and loaded onto a 120 mL Superdex 200 column in buffer A / 0.2 M KCl. Peak fractions were then re-loaded onto a 1 mL HiTrap SP FF and Srs2 was eluted with a 10 column-volume gradient from 0.2 – 1 M KCl. The Srs2 containing fractions were pooled, concentrated and snap frozen. RFC RFC Powder was thawed in buffer A / 0.15 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and then centrifuged (235,000 x g, 4C, 1 h). The soluble extract was recovered and supplemented with 2 mM CaCl2 and RFC puriﬁed by camodulin afﬁnity chromatography using 1 mL resin. Powder was thawed in buffer A / 0.15 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and then centrifuged (235,000 x g, 4C, 1 h). The soluble extract was recovered and supplemented with 2 mM CaCl2 and RFC puriﬁed by camodulin afﬁnity chromatography using 1 mL resin. Eluate fractions were pooled and loaded onto a 1 mL HiTrap SP FF column in buffer A / 0.15 M NaCl. RFC was eluted with a 20 col- umn-volume gradient from 0.15 – 1 M NaCl in buffer A. RFC containing fractions were pooled and dialysed against buffer A / 0.3 M KOAc for 4h at 4C. The dialysed sample was recovered, concentrated, aliquoted and snap frozen. Rrm3 Rrm3 The frozen cell powder was resuspended in 3 volumes of buffer F / 0.5 M KCl / protease inhibitors (Roche tablets + Sigma inhibitors + homemade cocktail) and centrifuged (235,000 x g, 4C, 1 h). Solid ammonium sulfate was added gradually to the soluble extract to 30% ﬁnal concentration with stirring (10 min, 4C). The insoluble material was then removed by centrifugation (27,000 x g, 4C, 20 min) and the supernatant was mixed with 4 mL anti-FLAG M2 afﬁnity gel (Sigma-Aldrich) at 4C for 30 min. The resin was collected and washed with 40 column volumes of buffer A / 0.5 M KCl / protease inhibitors, then 10 column volumes of buffer A / 0.5 M KCl / 5 mM MgOAc / 1 mM ATP, then 10 column volumes of buffer A / 0.5 M KCl. At this point, 3FLAG-Rrm3 was eluted in 1 column volume of buffer A / 0.5 M KCl / 0.5 mg / ml 3FLAG peptide, then 2 column volumes of buffer A / 0.5 M KCl / 0.25 mg / ml 3FLAG peptide. The eluate was dialysed against buffer A / 0.3 M KCl for 3 h at 4C, then loaded onto a 1 mL HiTrap heparin column equilibrated in buffer A / 0.3 M KCl / 10 mM MgOAc / 1 mM ATP. Rrm3 was eluted with a 15 column-volume gradient from 0.3 – 1 M KCl in buffer A / 10 mM MgOAc / 1 mM ATP. Peak fractions containing Rrm3 were pooled and dialysed against buffer A / 40% glycerol / 0.35 M KCl at 4C for 4 h. The dialysed sample was recovered, aliquoted and snap frozen. Pol d Buffer A / 0.2 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) was added to thawed powder and the sample was centrifuged (235,000 x g, 4C, 1 h). The soluble extract was recovered and supplemented with 2 mM CaCl2. At this point, 1.5 mL calmodulin afﬁnity resin was added and the mixture incubated at 4C for 90 min with rotation. e4 Molecular Cell 74, 231–244.e1–e9, April 18, 2019 The resin was collected and washed extensively with buffer A / 0.2 M NaCl / 2 mM CaCl2, and Pol d was then eluted with buffer A / 0.2 M NaCl / 2 mM EDTA / 2 mM EGTA. The eluate fractions were pooled, concentrated and loaded onto a 24 mL Superdex 200 column in buffer A / 0.3 M KOAc without NP-40. The peak fractions containing Pol d were pooled, concentrated, aliquoted and snap frozen. RFC Sgs1 and Top3-Rmi1 were a kind gift from Dr. Stephen Kowalczykowski. E. coli TopoIV was purchased from Inspiralis (T4001). ORC, Cdc6, Cdt1-Mcm2-7, DDK, S-CDK, Sld3/7, Cdc45, Dpb11, Pol ε, Sld2, GINS, Mcm10, Ctf4, Top2, PCNA, RPA, ISWI, Nap1, Nhp6, FACT and histones were puriﬁed by adapting previously established protocols (Coster et al., 2014; Frigola et al., 2013; Kurat et al., 2017; On et al., 2014; Yeeles et al., 2015). A brief puriﬁcation strategy for each of these proteins is listed in the Table S3. Top1 p Powder was thawed in buffer A / 0.3 M NaCl / protease inhibitors (Roche tablets + homemade cocktail) and insoluble material removed by centrifugation (235,000 x g, 4C, 1 h). At this point, 2 mM CaCl2 was added to the soluble extract, followed by 2 mL calmodulin afﬁnity resin, and the mixture incubated for 90 min at 4C. The resin was collected and washed extensively with buffer A / 0.3 M NaCl. Top1 was eluted in 6 column volumes buffer A / 0.3 M NaCl / 2 mM EDTA / 2 mM EGTA. The Top1-containing fractions were pooled, concentrated and separated on a 24 mL Superdex 200 column in buffer A / 0.4 M KOAc. The peak fractions were pooled, concentrated, aliquoted and snap frozen. Other proteins Other proteins Sgs1 and Top3-Rmi1 were a kind gift from Dr. Stephen Kowalczykowski. E. coli TopoIV was purchased from Inspiralis (T4001). ORC, Cdc6, Cdt1-Mcm2-7, DDK, S-CDK, Sld3/7, Cdc45, Dpb11, Pol ε, Sld2, GINS, Mcm10, Ctf4, Top2, PCNA, RPA, ISWI, Nap1, Nhp6, FACT and histones were puriﬁed by adapting previously established protocols (Coster et al., 2014; Frigola et al., 2013; Kurat et al., 2017; On et al., 2014; Yeeles et al., 2015). A brief puriﬁcation strategy for each of these proteins is listed in the Table S3. Molecular Cell 74, 231–244.e1–e9, April 18, 2019 e5 DNA templates The DNA templates pBS/ARS1WTA (3.2 kb), pCFK1_WT (5.8 kb), pZN3 (9.7 kb) and pRS425 have been described previously (Chris- tianson et al., 1992; Marahrens and Stillman, 1992; Taylor and Yeeles, 2018; Yeeles et al., 2015). pVA18 (5.5 kb) is a smaller derivative of the origin-speciﬁc plasmid pZN3. The DNA sequence of pVA18 (5589 bp) is provided below: DNA templates CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTT GGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTT CCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTT TAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTC TTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCC TTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAAT ACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCA AGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGA TTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCA TCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAG GACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTAC CTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTAT TCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATC GGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAA TGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCG ACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACAC CACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGTATACGGTACTACTGCACAACAAACTTTGG AAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAA AGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAA CTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGA AATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACA AAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGA ATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTC AACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTC AGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAAC CCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGGTACGCTGGGAAGTCAGCCTTTA GCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCG GCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGG GAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTGCGGC CGCTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAA TGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTGTCAGTTCCTTCAGTATGGTGTCCTTATATA CTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATT TTCACGTAAAACGCCACAGTCCGATTTTTCTAGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAG AGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGACGTCAGGAGAAGGAGCGGGTCTGGTGAATA GTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGG TGTACGTTGACGTTAGTAAGGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTG TTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTT TTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACT TCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTC TGGCAAGGGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATCGATGTTTCCTGTG TGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAG CTAACTCACATTAATTAAGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCA ACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCT GCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTC TCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAG CCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTA GAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAA ACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC e6 Molecular Cell 74, 231–244.e1–e9, April 18, 2019 TTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGG GAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGC TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGG TATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT CGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGT CATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTG CTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGG GGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTT TTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGT TGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC mids for in vitro replication reactions were puriﬁed using alkaline lysis followed by caesium chloride density Covalently closed plasmids for in vitro replication reactions were puriﬁed using alkaline lysis followed by caesium chloride density gradient centrifugation. For the preparation of linear DNA templates, 4 mg plasmid DNA was incubated with 2 mL restriction enzyme (New England Biolabs) in 1X CutSmart Buffer (New England Biolabs) in a 40 mL ﬁnal reaction volume. After incubating for 4h at 37C, the sample was treated with SDS / proteinase K and the DNA puriﬁed by phenol / chloroform extraction. The DNA was then precipitated from the aqueous phase with 0.2 M NaCl and 3 volumes 100% ethanol, then left at 20C overnight. The following morning, the DNA pellet was har- vested by centrifugation, washed with 70% ethanol, harvested again, air-dried, and then resuspended in 15-20 mL TE. Molecular weight markers Standard molecular weight markers were prepared by dephosphorylating 17 mg l DNA-HindIII Digest (New England Biolabs N3012S) with 10 U Antarctic Phosphatase (New England Biolabs M0289S) in total volume of 40 mL for 1 h at 37C. The phosphatase was then inactivated by incubation at 80C for 10 min. Subsequently, 6.8 mg of dephosphorylated DNA was labeled with g-[32P]-ATP using 40 units of T4 Polynucleotide Kinase (New England Biolabs M0201S) for 1 h at 37C, in a total reaction volume of 40 ml. Unincorpo- rated g-[32P]-ATP was removed using Illustra MicroSpin G-50 columns (GE Healthcare) and 5 mM EDTA was added to the recovered sample. For Figure 1D, end-labeled 3189 bp plasmid was prepared by digesting 4 mg plasmid DNA with 2 mL SmaI (Roche) in 1X CutSmart Buffer (New England Biolabs) in a 40 mL ﬁnal reaction volume for 2 h at 25C. The linearized plasmid was then column puriﬁed using the High Pure PCR Product Puriﬁcation Kit (Roche) and then dephosphorylated and end-labeled with g-[32P]-ATP, as described for standard molecular weight markers. Other labeled markers in Figure 1D were generated by PCR ampliﬁcation using oligonucleotides 7272 – 7275 (see Table S2) and pTDK13 plasmid template. In each case, 50 mL PCR reactions were assembled in the presence of 33 nM a-[32P]-dCTP and the PCR products were puriﬁed over Illustra MicroSpin G-50 columns (GE Healthcare). In vitro replication assays Mcm2-7 loading and DDK phosphorylation was performed as follows. First, 5 nM plasmid DNA template, 5-10 nM ORC, 20 nM Cdc6, 40 nM Cdt1/Mcm2-7 and 20 nM DDK were incubated in 25 mM HEPES-KOH (pH 7.6), 100 mM potassium acetate, 0.02% NP-40-S, 0.1 mg / ml BSA, 1 mM DTT, 10 mM Mg(OAc)2, 5 mM ATP at 30C for 10 min. Subsequently, separate buffer and replication protein mixtures were added sequentially to the Mcm2-7 loading mixture to give a ﬁnal replication reaction containing 25 mM HEPES-KOH (pH 7.6), 100 mM potassium acetate (unless otherwise indicated), 0.02% NP-40-S, 0.1 mg/ml BSA, 1 mM DTT, 10 mM Mg(OAc)2, 2.75 mM ATP, 30 mM dATP-dCTP-dGTP-dTTP, 33 nM a-[32P]-dCTP, 400 mM CTP-GTP-UTP, 20 nM S-CDK, 30 nM Dpb11, 8 nM GINS, 40 nM Cdc45, 30 nM Pol ε, 5 nM Mcm10, 5 nM RFC, 20 nM PCNA, 20 nM Top1, 20 nM Top2, 20 nM Pol aprimase, 25 nM Sld3-7, 10 nM Ctf4, 100 nM RPA, 20 nM Csm3-Tof1, 20 nM Mrc1, 50 nM Sld2, 5 nM Pol d, 10 nM Fen1 and 20 nM Cdc9 (unless otherwise indicated). Subsequently, separate buffer and replication protein mixtures were added sequentially to the Mcm2-7 loading mixture to give a ﬁnal replication reaction containing 25 mM HEPES-KOH (pH 7.6), 100 mM potassium acetate (unless otherwise indicated), 0.02% NP-40-S, 0.1 mg/ml BSA, 1 mM DTT, 10 mM Mg(OAc)2, 2.75 mM ATP, 30 mM dATP-dCTP-dGTP-dTTP, 33 nM a-[32P]-dCTP, 400 mM CTP-GTP-UTP, 20 nM S-CDK, 30 nM Dpb11, 8 nM GINS, 40 nM Cdc45, 30 nM Pol ε, 5 nM Mcm10, 5 nM RFC, 20 nM PCNA, 20 nM Top1, 20 nM Top2, 20 nM Pol aprimase, 25 nM Sld3-7, 10 nM Ctf4, 100 nM RPA, 20 nM Csm3-Tof1, 20 nM Mrc1, 50 nM Sld2, 5 nM Pol d, 10 nM Fen1 and 20 nM Cdc9 (unless otherwise indicated). Pol d, Fen1 and Cdc9 were generally omitted from replication reactions including accessory DNA helicases (except in Figures 6C and 6D and Figure S4A), in order to prevent strand displacement synthesis by Pol d, which has previously been shown to be enhanced by Pif1-Rrm3 (Osmundson et al., 2017; Rossi et al., 2008). The ﬁnal concentration of potassium acetate was adjusted to 250 mM in those reactions that included Pol d. In vitro replication assays Pol d, Fen1 and Cdc9 were generally omitted from replication reactions including accessory DNA helicases (except in Figures 6C and 6D and Figure S4A), in order to prevent strand displacement synthesis by Pol d, which has previously been shown to be enhanced by Pif1-Rrm3 (Osmundson et al., 2017; Rossi et al., 2008). The ﬁnal concentration of potassium acetate was adjusted to 250 mM in those reactions that included Pol d. 2-5 mL of the Mcm2-7 loading mixture was generally used per sample and this was typically diluted 4-fold in the ﬁnal reaction. The extra contribution from protein storage buffers to the ﬁnal reaction was approximately 22 mM chloride and 50-60 mM acetate, and the corresponding potassium counter-ions. Replication reactions were conducted at 30C for 20 min unless otherwise indicated. Pif1 and Rrm3 were included at 5 nm and 12.5 nM, respectively, unless otherwise indicated. For Figure 4C, Sgs1 and Top3-Rmi1 were included at 10 nM. Srs2, Chl1 and Dna2 were included at 20 nM. 2-5 mL of the Mcm2-7 loading mixture was generally used per sample and this was typically diluted 4-fold in the ﬁnal reaction. The extra contribution from protein storage buffers to the ﬁnal reaction was approximately 22 mM chloride and 50-60 mM acetate, and the corresponding potassium counter-ions. Replication reactions were conducted at 30C for 20 min unless otherwise indicated. 2-5 mL of the Mcm2-7 loading mixture was generally used per sample and this was typically diluted 4-fold in the ﬁnal reaction. The extra contribution from protein storage buffers to the ﬁnal reaction was approximately 22 mM chloride and 50-60 mM acetate, and the corresponding potassium counter-ions. Replication reactions were conducted at 30C for 20 min unless otherwise indicated. Pif1 and Rrm3 were included at 5 nm and 12.5 nM, respectively, unless otherwise indicated. For Figure 4C, Sgs1 and Top3-Rmi1 were included at 10 nM. Srs2, Chl1 and Dna2 were included at 20 nM. 2-5 mL of the Mcm2-7 loading mixture was generally used per sample and this was typically diluted 4-fold in the ﬁnal reaction. The extra contribution from protein storage buffers to the ﬁnal reaction was approximately 22 mM chloride and 50-60 mM acetate, and the corresponding potassium counter-ions. Replication reactions were conducted at 30C for 20 min unless otherwise indicated. RFC, PCNA, Top2, Ctf4, Csm3-Tof1, Mrc1, Pol d, Fen1 and Cdc9 were omitted from reactions with th Figure S1G. Helicase assays Helicase assays (10 mL volume) were carried out using 1 nM M13-based substrate in buffer containing 25 mM HEPES-KOH (pH 7.6), 0.1 mg/ml BSA, 2 mM Mg(OAc)2 and 2 mM ATP. Reactions were assembled on ice, equilibrated to room temperature and the respec- tive helicases added to 50 nM ﬁnal concentration. Reactions were incubated at 30C for 30 min. Reactions were stopped by the addition of EDTA (20 mM), SDS (0.4%) and proteinase K (1/25 volumes) and the incubation continued at 37C for 10 min. The samples were supplemented with Novex Hi-Density TBE Sample Buffer (ThermoFisher Scientiﬁc LC6678) and analyzed in 4%–20% Novex TBE Gels (ThermoFisher Scientiﬁc EC62252BOX) at 200 V for 30 min in 1X TBE. Gels were mounted onto chromatography paper (GE Healthcare, 3030-861) and exposed to Amersham Hyperﬁlm ECL (GE Healthcare) at 80C. Catenation assay with Sgs1-Top3-Rmi1 The catenation assay in Figure S3A was carried out using a modiﬁed version of an existing protocol (Cejka et al., 2012). The 3.2 kb plasmid (5 nM) was incubated with Sgs1 (60 nM), Top3-Rmi1 (400 nM) and RPA (100 nM) at 30C for 30 min in a 10 mL reaction con- taining 25 mM HEPES-KOH (pH 7.6), 0.1 mg/ml BSA, 1 mM Mg(OAc)2, 1 mM ATP, 100 mg / ml creatine phosphate kinase and 40 mM creatine phosphate. The reaction was stopped by addition of EDTA (50 mM), SDS (0.1%) and proteinase K (1 / 100 volume) and the incubation continued at 37C for 60 min. Reaction products were then separated in a 1% native agarose gel, which was subsequently stained with EtBr (0.5 mg / ml) for 20 min at room temperature. Preparation of substrates for helicase assays 28 pmol of PAGE-puriﬁed oligonucleotides purchased from Sigma (see Table S2 for oligonucleotide sequences) were ﬁrst labeled with g-[32P]-ATP using 20 U T4 Polynucleotide Kinase (New England Biolabs M0201S) for 1 h at 37C in a total reaction volume of 20 mL. Unincorporated g-[32P]-ATP was then removed using illustra MicroSpin G-50 columns (GE Healthcare). For annealing, a 3-fold molar excess of labeled oligonucleotide was incubated with 1.3 pmol of M13 ssDNA (New England Biolabs N4040S) in a total reaction volume of 20 mL with heating to 95C for 10 min in a metal heating block. The metal block was then placed at room temperature and the sample left to cool for 3 h. Free oligonucleotide was removed from the sample by applying a 20 mL annealing reaction to a 400 mL Sephacryl S-400 column (prepared in 0.8 mL Pierce Centrifuge Columns (Thermo Scientiﬁc 89868)) pre-equilibrated in TE. The ﬁnal concentration of the recovered substrate was estimated to be 38 nM. Substrates were stored at 4C. In vitro replication assays For the experiments in Figures 3C and S4B, SmaI (Roche) was added to a ﬁnal concentration of 0.5 U/ml concomitant with the chase. Chromatin replication experiments were carried out using a modiﬁed version of an existing protocol (Taylor and Yeeles, 2018). Brieﬂy, for chromatin assembly, ISW1 (30 nM), Nap1 (3 mM) and histone octamers (150 nM) were incubated in chromatin assembly buffer (25 mM HEPES-KOH (pH 7.6), 10 mM Mg(OAc)2, 100 mM KOAc, 0.01% NP-40-S, 5% glycerol, 0.1 mg/ml BSA) for 10 min on ice. Creatine phosphate (40 mM), creatine phosphate kinase (100 mg/ml) and ATP (3 mM) were then added and chromatin assembly initiated by the addition of plasmid template (3 nM). Chromatin assembly was performed at 30C for 1 h, with addition of ORC (10 nM) after 10 min incubation. Chromatin replication experiments were carried out using a modiﬁed version of an existing protocol (Taylor and Yeeles, 2018). Brieﬂy, for chromatin assembly, ISW1 (30 nM), Nap1 (3 mM) and histone octamers (150 nM) were incubated in chromatin assembly buffer (25 mM HEPES-KOH (pH 7.6), 10 mM Mg(OAc)2, 100 mM KOAc, 0.01% NP-40-S, 5% glycerol, 0.1 mg/ml BSA) for 10 min on ice. Creatine phosphate (40 mM), creatine phosphate kinase (100 mg/ml) and ATP (3 mM) were then added and chromatin assembly initiated by the addition of plasmid template (3 nM). Chromatin assembly was performed at 30C for 1 h, with addition of ORC (10 nM) after 10 min incubation. Chromatinised plasmids were puriﬁed by applying a 40 mL chromatin assembly reaction to a 400 mL Sephacryl S-400 column (pre- pared in a 0.8 mL Pierce Centrifuge Column (Thermo Scientiﬁc 89868)) pre-equilibrated in 25 mM HEPES-KOH (pH 7.6), 100 mM potassium acetate, 0.02% NP-40-S, 1 mM DTT, 10 mM Mg(OAc)2, and then centrifuging at 700 x g for 2 min. Chromatin replication was performed as for naked DNA templates with the addition of FACT (80 nM) and Nhp6 (400 nM) in the replication step. Pol d was included at 10 nM in chromatin replication experiments. were conducted as with the reconstituted system reactions, except that S-phase extract (8 mg / ml) (On ad of puriﬁed proteins during the replication step. For Figure 4A, reactions were conducted as with the reconstituted system reactions, except that S- et al., 2014) was used instead of puriﬁed proteins during the replication step. In vitro replication assays extra contribution from protein storage buffers to the ﬁnal reaction was approximately 22 mM chloride and 50 60 mM acetate, and the corresponding potassium counter-ions. Replication reactions were conducted at 30C for 20 min unless otherwise indicated. Pif1 and Rrm3 were included at 5 nm and 12.5 nM, respectively, unless otherwise indicated. For Figure 4C, Sgs1 and Top3-Rmi1 were included at 10 nM. Srs2, Chl1 and Dna2 were included at 20 nM. p g p p Pif1 and Rrm3 were included at 5 nm and 12.5 nM, respectively, unless otherwise indicated. For Figure 4C, Sgs1 and Top3-Rmi1 were included at 10 nM. Srs2, Chl1 and Dna2 were included at 20 nM. p g p p Pif1 and Rrm3 were included at 5 nm and 12.5 nM, respectively, unless otherwise indicated. For Figure 4C, Sgs1 and Top3-Rmi1 were included at 10 nM. Srs2, Chl1 and Dna2 were included at 20 nM. RFC, PCNA, Top2, Ctf4, Csm3-Tof1, Mrc1, Pol d, Fen1 and Cdc9 were omitted from reactions with the ‘minimal replisome’, as in Figure S1G. RFC, PCNA, Top2, Ctf4, Csm3-Tof1, Mrc1, Pol d, Fen1 and Cdc9 were omitted from reactions with the ‘minimal replisome’, as in Fi S1G RFC, PCNA, Top2, Ctf4, Csm3-Tof1, Mrc1, Pol d, Fen1 and Cdc9 were omitted from reactions with the ‘minimal replisome’, as in Figure S1G. Csm3-Tof1, Mrc1, Pol d, Fen1 and Cdc9 were omitted from reactions with the ‘minimal replisome’, as in Molecular Cell 74, 231–244.e1–e9, April 18, 2019 e7 Origin-speciﬁc replication reactions were conducted as above, except that Mcm2-7 loading was conducted at 24C for 10 min and 80 nM S-CDK was then added directly to this reaction at 24C for 5 min before the replication step. For pulse-chase experiments, dATP, dTTP, dGTP and dCTP were each added to 600 mM in the chase at the indicated times. For the experiments in Figures 1F, 3C, S4B, S5A, the dCTP concentration was adjusted to 2.5 mM in the pulse. For the experiments in Figures 3C and S4B, SmaI (Roche) was added to a ﬁnal concentration of 0.5 U/ml concomitant with the chase. For pulse-chase experiments, dATP, dTTP, dGTP and dCTP were each added to 600 mM in the chase at the indicated times. For the experiments in Figures 1F, 3C, S4B, S5A, the dCTP concentration was adjusted to 2.5 mM in the pulse. Gel imaging and presentation For in vitro replication experiments, native gels were dried directly onto chromatography paper (GE Healthcare, 3030-861). Dena- turing gels were ﬁxed by two incubations (20 min, 4C) in cold 5% trichloroacetic acid and then dried onto chromatography paper. The dried gels were typically exposed to both Amersham Hyperﬁlm ECL (GE Healthcare) and BAS-MS Imaging Plates (Fujiﬁlm), which were then developed on a Typhoon phosphorimager (GE Healthcare). Decatenation of replicated plasmids with E. coli TopoIV For post-reaction treatment with E. coli TopoIV (Figure 6), replication reactions were processed as above and then treated with TopoIV (Inspiralis, T4001) at 37C for 30 min as per the manufacturer’s instructions. The reactions were quenched and the samples prepared for gel analysis as above. DNA preparation for agarose gel electrophoresis For all in vitro replication experiments, reactions were quenched by addition of EDTA to 25 mM. SDS (0.1%) and proteinase K (1 / 100 volumes) were subsequently added and the incubation continued at 37C for 30 min. To this was added an equal volume of phenol:chloroform:isoamyl alcohol 25:24:1 (Sigma-Aldrich P2069) saturated with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and the DNA was then extracted. The aqueous phase was buffer exchanged to TE and unincorporated nucleotides were removed with Illustra MicroSpin G-50 columns (GE Healthcare). For the experiments in Figures 7B and S7, frozen cell pellets corresponding to 2 3 108 cells were re-suspended in 400 mL lysis buffer (50 mM Tris-HCl pH 8.0, 0.1 M NaCl, 10 mM EDTA, 1% SDS) and the cell wall removed by incubation with 80 units / ml Lyticase (Sigma) and 1% b-mercaptoethanol at 37C for 5 min. DNA was then extracted with phenol/chloroform/isoamylalcohol (25:24:1) and e8 Molecular Cell 74, 231–244.e1–e9, April 18, 2019 the aqueous layer collected using phase lock tubes (SLS, 2302800). The DNA was then precipitated with 2 volumes of 100% ethanol and washed with 70% ethanol before being re-solubilized in 120 mL 10 mM Tris pH 8.0. the aqueous layer collected using phase lock tubes (SLS, 2302800). The DNA was then precipitated with 2 volumes of 100% ethanol and washed with 70% ethanol before being re-solubilized in 120 mL 10 mM Tris pH 8.0. One-dimensional agarose gel electrophoresis and Southern blotting Samples for denaturing agarose gels were supplemented with 20 mM EDTA, and 1/10 volume alkaline loading dye (0.5 M NaOH, 10% sucrose, xylene cyanol) and then left to denature at room temperature for 10 min. For native gels, samples were mixed with 1/6 vol- ume native loading dye (30% glycerol, 0.25% bromophenol blue). For restriction digestion of the replicated products, 8-14 mL of sam- ple was incubated in 1x CutSmart buffer with 0.25 mL restriction enzyme at 37C for 30 min, except for SmaI that was incubated at 25C for 30 min. Samples were analyzed in 0.6 - 0.8% denaturing agarose gels at 21 V overnight in 30 mM NaOH, 2 agarose gels at 20 V overnight in 1X TAE. EtBr was included at 0.5 mg/ml for Figure 6D. For the experiments in Figure 7B and Figure S7, the samples were electrophoresed at 0.4 V / cm for 7 days in 0.8% agarose (Mega- sieve) in 0.5x TBE. Following neutral Southern blotting onto Hybond-N+ membrane (GE Healthcare), the plasmids were detected by hybridization with a LEU2 DNA probe that also detected the genomic leu2 locus. Labeling and detection were performed with the ‘random prime labeling module’ incorporating ﬂuorescein tagged dUTP (Roche, 11585622910). After hybridization and washing, ﬂuorescein tagged dUTP was detected with alkaline phosphatase tagged anti ﬂuorescein Fab fragments (Roche, 000000011426338910), and revealed with CDP-Star (GE Healthcare, GERPN3682). Images were acquired with the ImageQuant 4000 system (GE Healthcare). Two-dimensional native-native agarose gel electrophoresis The method was adapted from that described previously (Brewer and Fangman, 1987). In an analogous experiment to that in Fig- ure 7B, involving control and pif1-m2 rrm3D cells containing the plasmid pRS425, DNA equivalent to 4 3 107 cells was prepared from the 40 minute time point. The DNA was then digested with 110 units of SnaB1 enzyme (New England Biolabs R0130M) in 1x ‘CutSmart buffer’ in a total volume of 50 mL at 37C for 4 h. 20 mL of the digested volume was then loaded onto a 0.4% native agarose gel which was run 1 V / cm in 1x TBE for 15 h at room temperature. The lanes were then cut out and reset in a second dimension of 1% agarose containing 0.3 mg/ml EtBr and run in 1x TBE containing 0.3 mg/ml EtBr for 8 h at 5 V / cm in the cold room. The resultant gel was then prepared for Southern blotting and non- radioactive hybridization, as described above for one-dimensional gels. Molecular Cell 74, 231–244.e1–e9, April 18, 2019 e9 QUANTIFICATION AND STATISTICAL ANALYSIS For quantiﬁcation of replication products, gel images generated on a Typhoon phosphorimager were converted to 16-Bit Tiff ﬁles using the Linearize GelData plugin in ImageJ (National Institute of Health). Boxes were drawn around each lane and the peaks corresponding to LRIs and full-length products were selected manually from the resultant lane proﬁles. The ‘percentage full-length products’ was then calculated, relative to all the replication products in a given lane. The corresponding experiments were performed the following number of times: Figure 1C (3x), Figure 1D (3x), Figure 1F (2x), Fig- ure 2A (3x), Figure 2C (2x), Figure 2D (2x), Figure 3B (2x), Figure 3C (3x), Figure 4A (2x), Figure 4C (2x), Figure 4D (3x), Figure 4E (3x), Figure 4F (2x), Figure 5B (2x), Figure 5C (3x), Figure 5D (3x), Figure 5E (1x), Figure 5F (1x), Figure 6A (3x), Figures 6C and 6D (3x), Figure 7B (3x), Figure 7B (2x), Figure S1D (2x), Figure S1E (2x), Figure S1G (2x), Figure S2A (2x), Figure S2B (2x), Figure S3A (1x), Figures S3B and S3C (3x), Figure S3D (1x), Figure S3E (1x), Figure S3F (2x), Figure S4A (2x), Figure S4B (2x), Figure S5A (1x), Fig- ure S5B (1x), Figure S5C (2x), Figure S6A (3x), Figure S7A (3x). The analyses in Figures 1E, S1F, S3G and S3H formed part of a number of other experiments, and were thus repeated many times. Molecular Cell 74, 231–244.e1–e9, April 18, 2019 e9
W3009512890.txt	https://link.springer.com/content/pdf/10.1007/s10503-020-09512-4.pdf	en		Schemes, Critical Questions, and Complete Argument Evaluation	Argumentation	2,020	cc-by	12,114	Argumentation (2020) 34:469–498 https://doi.org/10.1007/s10503-020-09512-4 ORIGINAL RESEARCH Schemes, Critical Questions, and Complete Argument Evaluation Shiyang Yu1 · Frank Zenker2 Published online: 6 March 2020 © The Author(s) 2020 Abstract According to the argument scheme approach, to evaluate a given scheme-saturating instance completely does entail asking all critical questions (CQs) relevant to it. Although this is a central task for argumentation theorists, the field currently lacks a method for providing a complete argument evaluation. Approaching this task at the meta-level, we combine a logical with a substantive approach to the argument schemes by starting from Toulmin’s schema: ‘data, warrant, so claim’. For the yet more general schema: ‘premise(s); if premise(s), then conclusion; so conclusion’, we forward a meta-level CQ-list that is arguably both complete and applicable. This list should inform ongoing theoretical efforts at generating appropriate object-level CQs for specific argument schemes. Keywords Argument · Complete evaluation · Critical question · Scheme · Standardization 1 Introduction For the purpose of evaluating natural language arguments, argument(ation) schemes and their associated critical questions (CQs) have remained central to contemporary argumentation theory (e.g., Walton et al. 2014, 89). Probably the first scholar to use ‘critical question’ (CQ) in today’s technical sense was Hastings (1962). Yet earlier, * Frank Zenker f.zenker@ans.pw.edu.pl 1 College of Philosophy, Nankai University, Tianjin, People’s Republic of China 2 Center for Formal Ontology, Warsaw University of Technology, Pl. Politechniki 1, 00‑661 Warsaw, Poland 13 Vol.:(0123456789) 470 S. Yu, F. Zenker Perelman and Olbrechts-Tyteca (1958) had introduced ‘argumentative scheme’ (French: schème argumentatif) with reference to the schemes’ historical ancestors: the topoi (Greek: places; Latin: loci).1 While the topoi serve primarily in argument construction, whereas the schemes and CQs serve primarily in argument evaluation, both concepts, of course, are closely related. Even when restricted to argument evaluation, however, a unified theoretical understanding of argument schemes and CQs has so far remained absent. Indeed, already a unified taxonomy of argument schemes can hardly arise as long as “existing classifications of arguments are unsatisfying in a number of ways” (Wagemans 2016, 1).2 For instance, pragma-dialecticians treat an argument scheme as a kind of “characteriz[ation] [of] the way in which the reason given in support of a standpoint is supposed to bring about a transfer of acceptance to the standpoint in a particular type of argumentation” (van Eemeren 2018, 7). Further, informal logicians and scholars in AI & law tend to view schemes as “forms of argument (structures of inference) that represent structures of common types of arguments used in everyday discourse, as well as in special contexts like those of legal argumentation and scientific argumentation” (Walton et al. 2008, 1). Virtually all scholars, moreover, let ‘argument scheme’ denote a linguistic structure, while some few also view such structures as externalizing an (internal) reasoning scheme (Blair 2001; Hitchcock 2006, 218).3 We adopt the former use. Schemes and CQs clearly relate to other methods for evaluating arguments. For example, a normative application of fallacy theory assumes that “many [possibly most] of the fallacies are failed instances of good argument schemes or forms” (Tindale 2007, xiv), although such instances as “[a] ‘Straw Man’ argument would seem to be always incorrect and have no redeemable instances” (ibid., 12). Argument schemes also are indispensable in computational versions of acceptance-based approaches like Walton (2016), who—viewing schemes and CQs as basic (ibid., 339)—finds them compatible with probabilistic approaches like Verheij’s (2014). Hahn and Hornikx (2016, 1833) even argue “that the most fruitful approach to developing normative models of argument quality […] combines the argumentation scheme approach with [the Pascalian probability approach known as] Bayesian argumentation.” 1 For additional background, see Garssen’s (2001) historical overview, Rubinelli (2009) on Aristotle’s and Cicero’s topoi, Rapp and Wagner (2013, 22–24) on how the topoi relate to argument schemes, and Walton et al. (2008) for a collection of schemes and associated CQs. 2 Wagemans’ own periodic table of arguments is based on three distinctions (ibid., 3–7): subject argument versus predicate argument, a distinction referencing the constituents of arguments under a formallinguistic analysis; first-order versus second-order argument, a distinction seemingly running parallel to the classical one between internal and external topics (Hitchcock and Wagemans 2011, 197–199); and finally the distinction—originating in debate theory—between propositions that constitute policy-, value-, or fact-related argumentation (Schut and Wagemans 2014, 25–39). 3 Argument tends to differ from reasoning in its direction. Argument runs typically (but not necessarily) from a claim to its supporting reason(s), whereas reasoning runs from the reason(s) to a claim. Only the former direction assumes that a claim is yet in need of reasoned support. In fact, the argument-to-reasoning relation is yet more complex: “[a]rguments can also […] stimulate reflection on one’s own reasoning,” which is one of their “cognitive functions” (Hoffmann 2016, 366). 13 Schemes, Critical Questions, and Complete Argument Evaluation 471 Contemporary scholarship associates lists of CQs (or CQ-lists) for the most part to the schemes themselves, and only in rare cases to their applied instances. This association occurs at what we call the theoretical level, respectively the applied level. At both levels, however, agreed-upon stopping criteria for a complete CQ-list are unavailable, thus leaving it unclear whether analysts must ask yet further CQs. Our main objective in this paper, therefore, is to develop meta-level conditions under which a CQ-based evaluation is complete. Similar in motivation is Rigotti and Greco Morasso’s (2006, 2009, 2010) argumentum model of topics (AMT).4 Closest to our own approach probably comes the epistemological approach of Lumer (2011), who treats an argument scheme as the “general form of an ideal, elementary or molecular argument” (ibid., 8), and considers “the whole system of (ideal [i.e., fully explicit, valid]) elementary argument schemes [as] the argument tree” (ibid., 10; his italics). This tree’s three “branches [should] be defined […] according to the argument schemes’ epistemological underpinnings,” namely: “logic, probability theory and (prudential) desirability theory” (ibid., 10), which thus serve as meta-level evaluative principles. Our approach differs from Lumer’s in that we treat ARGUMENT SCHEME as a primarily logical concept.5 Moreover, Lumer seems to downplay the influence of the Aristotelian Topics (ibid., 2), whereas our own attempt seeks to incorporate them by enriching a logical with a substantive approach. The following thus offers a meta-level hybrid-account of argument schemes and CQs, that is, a logical account (Sect. 2) complemented by a substantive account (Sect. 3). We submit that the Toulmin model proves sufficient to develop this account (Sect. 4). Upon reviewing related work (Sect. 5), we propose a complete CQ-list, justify its applicability (Sect. 6), and then conclude (Sect. 7). 2 Why a Logical Account of Argument Schemes? 2.1 Levels One may associate CQs to an argument scheme or to its specific instance. The former level we call theoretical because it abstracts from the argument’s specific content; the latter level we call applied. Accordingly, one may separate theoretical from applied CQs, which specify the theoretical CQs by saturating their placeholders. 4 Devised for argument reconstruction, the AMT builds upon the pragma-dialectical distinction between procedural and material starting points. According to the AMT, an “argument scheme combines a procedural starting point, coinciding with the inferential connection (maxim) that is activated, with a material starting point guaranteeing […] the applicability of the maxim to the actual situation considered in the argument” (Rigotti and Greco Morasso 2010, 493). Regarding the procedural starting point, they distinguish three levels: (i) ontological relations, represented as locus; (ii) a series of inferential connections (‘maxims’) arising from each ontological relation; and (iii) a logical form activated by each maxim. Our own approach is more fundamental because our theoretical level can house all of their three levels. 5 We borrow the term ‘logical’ from Prakken (2010), who obtains a logical account by formalizing argument schemes such that ‘formalization’ denotes his method, while ‘logical account’ names the method’s outcome. As an anonymous reviewer has rightly pointed out, one may alternatively call our account ‘formal’ rather than ‘logical’. 13 472 S. Yu, F. Zenker This distinction also applies to argument schemes. As an example, consider the ‘from a position to know’-scheme at the theoretical level (1a–c),6 and compare three of its instances at the applied level (1a′–c′), where substantive rather than logical information saturates the placeholders a and A. (1a) Argument scheme ‘from a position to know’; theoretical level, instance 1 a is in a position to know whether A is true. a asserts that A is true. A is true. (1b) Argument scheme ‘from a position to know’; theoretical level, instance 2 a is in a position to know whether A is true. a asserts that A is true. If a is in a position to know whether A is true, and a asserts that A is true, then A is true. A is true. (1c) Argument scheme ‘from a position to know’; theoretical level, instance 3 a is in a position to know whether A is true. a asserts that A is true. a is an honest (trustworthy, reliable) source. A is true. (1a′) Argument scheme ‘from a position to know’; applied level, instance 1 Harry is in a position to know whether ‘Mary is the murderer’ is true. Harry asserts, “‘Mary is the murderer’ is true.”7 ‘Mary is the murderer’ is true. 6 See Prakken (2010) and our Sect. 2.2 for alternative scheme-versions, whose names variously carry ‘a’, ‘the’ or no article), and the wording of which is always a matter of choice. Walton et al. (2008, 309), for instance, list this scheme as “Major Premise: Source a is in position to know about things in a certain subject domain S containing proposition A. Minor Premise: a asserts that A is true (false). Conclusion: A is true (false).”. 7 At the applied level, one may find the alternative version: “Harry asserts ‘Mary is the murderer’.” Following a deflationary account of truth, this version omits ‘is true’ (e.g., Daniel and Nic 2014). Yet both versions amount to pragmatic (near-)equivalents. 13 Schemes, Critical Questions, and Complete Argument Evaluation 473 (1b′) Argument scheme ‘from a position to know’; applied level, instance 2 Harry is in a position to know whether ‘Mary is the murderer’ is true. Harry asserts, “‘Mary is the murderer’ is true.” If Harry is in a position to know whether ‘Mary is the murderer’ is true, and Harry asserts ‘Mary is the murderer’, then ‘Mary is the murderer’ is true. ‘Mary is the murderer’ is true. (1c′) Argument scheme ‘from a position to know’; applied level, instance 3 Harry is in a position to know whether ‘Mary is the murderer’ is true. Harry asserts “‘Mary is the murderer’ is true.” Harry is an honest (trustworthy, reliable) source. ‘Mary is the murderer’ is true. As these examples show, at the applied level one simply replaces ‘a’ by ‘Harry’, and ‘A’ by ‘Mary is the murderer’. At the theoretical level, moreover, 1(b) externalizes the logical inference underlying 1(a). Finally, 1(c′) states information—viz. Harry is an honest (trustworthy, reliable) source—that, if it is false, expresses exceptions to the inference in 1(b′), as do (1b–1c). (We return to this in Sect. 2.2). Evaluating a given scheme instance requires a general normative yardstick, one not limited to specific schemes or instances. Only if the theoretical level provides such normativity, or reasonableness, can this normativity transfer unproblematically from the theoretical to the applied level. This constraint forces us to recognize a third level: the meta-level. Its instances are the logical forms (1a″–c″), which are themselves yet more abstract than the instances at the theoretical level. As desired, these meta-level logical forms are sufficiently general to transfer their own evident reasonableness from the meta-level to the theoretical level. (1a″) Argument scheme ‘from a position to know’; meta-level, instance 1 Premise(s) Conclusion (1b″) Argument scheme ‘from a position to know’; meta-level, instance 2 Premise(s) If premise(s), then conclusion Conclusion 13 474 (1c″) S. Yu, F. Zenker Argument scheme ‘from a position to know’; meta-level, instance 3 Premise(s) Absence of exception(s) Conclusion Insofar as the meta-level is a logical construction, one must accordingly treat an argument scheme as a logical structure. This structure one can study independently of dialectical or rhetorical considerations, because such considerations will affect neither a scheme’s structure nor its components. They rather pertain to the criteria for evaluating a scheme instance, for instance with regard to being internally consistent, audience-accepted, commitment-incurring, burden-of-proof discharging, etc. (see Sect. 6.2). 2.2 A Logical Account of Argument Schemes The two basic features of our account thus are an inference rule and exceptions to it. Argument schemes, after all, “can be transformed into instances of logical inference rules by adding the connection between premises and conclusion as a conditional premise” (Prakken 2005, 307; see Walton 1996, 2016, 323–325). Treating schemes as logical constructs therefore entails that “a procedure for evaluating arguments primarily takes the form of a [non-monotonic] logic” (Prakken 2010, 1).8 For instance, the inference rule in (2)—containing ‘usually’—is the defeasible modus ponens rule. (2) Defeasible modus ponens rule P If P then usually Q Therefore (presumably), Q Saturating (2) to the ‘from a position to know’-scheme can either yield (3), containing a defeasible inference rule, or (4), containing a generalised conditional premise that must itself be defeasible (Prakken 2010, 7f.). The choice between (3) and (4), of course, is a matter of taste rather than of substance. 8 Prakken (2010, 5) submits that a scheme not only includes “defeasible inference rules, i.e., elements of a reasoning method.” He rather finds that a scheme can be “a reasoning method in itself” (ibid.), because it features “abstractions of more complex lines of reasoning, which may not always be naturally reduced to reasoning with defeasible inference rules” (ibid., 13). What thus amounts to an identification of the method with its elements, he illustrates with the modern version of Peirce’s abductive reasoning (Yu and Zenker 2018)—an identification we reject. For it raises questions (e.g., “How many reasoning methods are there; what separates them?”) that Prakken himself leaves unaddressed. 13 Schemes, Critical Questions, and Complete Argument Evaluation 475 (3) Argument scheme ‘from a position to know’; defeasible inference rule a is in a position to know whether A is true. a asserts that A is true. A is true. (4) Argument scheme ‘from a position to know’; generalised conditional premise a is in a position to know whether A is true. a asserts that A is true. If a is in a position to know if A is true, and a asserts that A is true, then A is true. A is true. When formulating the inference rule, another matter of taste is whether to include a dedicated premise stating the absence of exceptions,9 as in (5). (5) Argument scheme ‘from a position to know’; inference-rule account with exception-premise(s) a is in a position to know whether A is true. a asserts that A is true. a is an honest (trustworthy, reliable) source. A is true. Moving beyond considerations of mere taste, evaluating a defeasible argument completely always presupposes that one makes its implicit parts explicit (Ennis 1982, 66; Gough and Tindale 1985, 99; van Eemeren and Grootendorst 1992, 141; Yu and Zenker 2019). At the applied level, this demands listing an inference’s exceptions. At the same time, however, it is unclear whether all actual and possible exceptions can be listed (Zenker 2009). Even if all premises of (5) are true, for instance, one cannot safely infer ‘A is true’, because a might lie. To give another example, the premise ‘a is generally a reliable source’ does not imply ‘a speaks the truth now’. Otherwise, one could deduce ‘A is true’ from ‘a is generally a reliable source’ and ‘a asserts that A’ alone. (As this sidesteps the premise ‘a is in a position to know whether A is true’, however, it also makes it questionable that one treats the ‘from a position to know’-scheme.) The gap in meaning between ‘generally’ and ‘now’ thus implies implicit parts, which to make explicit is an unsatisfied need that rightly fosters doubt whether a list of applied CQs can ever be complete (see note 9). 9 For any scheme, adding inference-invalidating exceptions to the premises turns undercutting attacks— directed at the inference in (3)—into premise attacks in (5), for one can always transform a scheme’s CQs into additional premises (Prakken 2010, 9; Hoffmann 2018, 233). Logically equivalent, both methods differ in convenience. 13 476 S. Yu, F. Zenker In developing a logical account that fits with any argument scheme, the forgoing provides strong reasons against operating at the applied level. Instead, we opt for a meta-level version of a defeasible conditional premise, as in (6). (6) Meta-level logical structure for any argument scheme Premise(s) If premise(s), then conclusion Therefore, conclusion As (6) abstracts from (2), one may omit ‘usually’ (contained in the ‘if–then’ conditional’s saturated version), because this term becomes redundant at the meta-level. An argument scheme’s logical structure now looks deductive. Far from providing clarity, however, a deductive structure rather obscures the conditional’s defeasible character that the ‘if–then’ premise expresses, one that pivots on substantive rather than logical information. We therefore proceed to complement our logical account with a substantive account. 3 A Substantive Account of Argument Schemes 3.1 The Pragma‑Dialectical Theory Pragma-dialecticians view argumentation as “a verbal, social, and rational activity aimed at convincing a reasonable critic of the acceptability of a standpoint by putting forward a constellation of propositions justifying or refuting the proposition expressed in the standpoint” (van Eemeren and Grootendorst 2004, 1). As the authors rightly note, logic alone offers but limited insights into how the acceptability of an argument’s premises transfers to a claim. For such transfers “to achieve the interactional effect that the listener accept[s] [the speaker’s] standpoint,” speakers rely not on logic but rather “on a ready-made argumentation scheme”—or argument scheme—that is, “a more or less conventionalized way of representing the relation between what is stated in the argument and what is stated in the standpoint” (van Eemeren and Grootendorst 1992, 96; their italics). Argument schemes thus represent kinds of relations between premises, or reasons, and a standpoint, or claim. As the theory’s basic kinds, moreover, Pragmadialecticians distinguish relations based on symptomaticity, similarity, or causality (Garssen 1994, 1997). As an example, if “someone tries to convince his interlocutor by pointing out that something is symptomatic of something else,” then acceptability is transferred by “a relation of concomitance” (van Eemeren and Grootendorst 1992, 96f.). In such cases, “[t]he argumentation is presented as if it [were] an expression, a phenomenon, a sign or some other kind of symptom of what is stated in the standpoint” (ibid., 97). As is the case with the other two kinds, this relation cites substantive rather than formal content. 13 Schemes, Critical Questions, and Complete Argument Evaluation 477 3.2 A Substantive Account’s Benefits As a logical premise, the ‘if–then’ premise is central to both deductive inferences such as modus ponens, as well as to inductive ones such as the statistical syllogism. Insofar as the three basic relations specify the ‘if–then’ premise’s substance, moreover, suitable replacement in (6) yields: (7) Combined argument scheme at the meta-level Premise(s). The relation R holds between the referents of premise(s) and conclusion. Therefore, conclusion. This specifies what the ‘if–then’ premise means. Like a logical account, also a substantive account allows one to distinguish three levels. Given the similarity relation, for instance, one can readily obtain three increasingly abstract specifications: (8a) Example, similarity relation, applied level “The method I propose worked last year (and this problem is similar to last year’s), so the method will work again.” (van Eemeren and Grootendorst 1992, 97) (8b) Example, similarity relation, theoretical level “For [problem] X, [method] Y is valid because [, f]or [problem] Z, [method] Y is valid and X is comparable [because it is relevantly similar] to Z.” (van Eemeren and Kruiger 1987, 73f.) (8c) Example, similarity relation, meta-level For X, Y is valid because, for Z, Y is valid; and the relation R connects X and Z. In (8a), the similarity between last year’s and this year’s problem shall support that the method is valid for this year’s problem, a conclusion that is grounded in the method’s proven validity for a similar problem. Strictly speaking, if the acceptability-grounding similarity relation, R, holds true, it cannot in any good sense hold between (parts of) premises and the conclusion, but must rather hold between the two problems, on one hand, and between the methods’ validity for each problem, on 13 478 S. Yu, F. Zenker the other.10 Of course, where the distinction between the linguistic and the ontic— between words and things—is negligible, one may abstract R yet further: (9a) Example, similarity relation, applied level The method I propose worked last year (and the method’s proven problemsolving ability for last year’s problem is similar to the method’s problemsolving validity for this year’s problem, because the problems are similar), so the method will work again. (9b) Example, similarity relation, theoretical level Premise(s). Therefore, conclusion. (9c) General relation, meta-level Premise(s). Therefore, conclusion. Like the meta-level treatment of the ‘from a position to know’-scheme in (1a″–c″),11 (9c) abstracts fully from the relation being one of similarity. It is this scheme, therefore, that applies to all types of substantive relations. Having thus represented an argument scheme’s structure, we can turn to its substantive aspect. 3.3 Defining Argument Scheme A consensus-definition of ‘argument scheme’ being absent, our own definition pivots on three choices. These relate to the level of abstraction, to the substantive versus logical distinction, and to the object of study. First, we define ‘argument scheme’ at the meta-level, to which both the theoretical and the object level are subordinate. The ‘argument from a position to know’-scheme, for instance, exemplifies the concept 10 As Wagemans (2019, 61) observes, “the common term [that is shared between the premises] functions as the fulcrum of the leverage of acceptability from the premise to the conclusion.” A transfer of acceptability that is itself achieved through speakers’ use of natural language argument will trivially depend on linguistic information. The transfer’s own reasonableness, however, may non-trivially depend on ontic information, viz. on how the world is. This holds although—on the linguistic side—one can distinguish, at the same abstract level, two additional similarity-levels. In 8(a), at one level, a similarity relation holds between the entire premise and the conclusion—viz. between ‘the method’s problem-solving validity for last year’s problem’ and ‘the method’s problem-solving validity for this year’s problem’. At a second level, an additional similarity relation holds between this year’s and last year’s problem, now treated as parts of the premise and the conclusion. (We thank an anonymous reviewer for this observation.). 11 One can alternatively treat the ‘from a position to know’-scheme as in (7). Considering the witness as an authority lets the scheme feature a symptomatic relation (Garssen 2001, 92). 13 Schemes, Critical Questions, and Complete Argument Evaluation 479 ARGUMENT SCHEME, yet the scheme itself remains distinct from the concept.12 Second, argument schemes do not merely cite substantive information that a logical account abstracts “away,” they also cite such information at structural positions that a logical account helps identify. A logical and a substantive account are therefore complementary. Third, if one were to define an argument scheme at the meta-level in terms of the substantive relation alone, then one would capture merely the ‘if–then’ premise. However, one could obviously not saturate this scheme any further to (re-) construct an argument at the applied-level. We therefore define as follows: Definition of ‘argument scheme’: S is an argument scheme if, and only if, S is a meta-level argument with at least one premise and a conclusion, where the transfer of the premise(s)’s acceptability to the conclusion grounds in a substantive—as opposed to a logical—relation, R, that holds between the scheme’s sentences, their parts, or their referents. This captures the combined meta-level account of argument scheme in (7). We view (7) as embodying an argument scheme’s structure and its substance. Having thus defined the noumenon of argument scheme, we can turn to its evaluation, a task for which the Toulmin model proves sufficient. 4 Argument Evaluation on the Toulmin Model 4.1 The Toulmin Model Viewing arguments as organisms, Toulmin (1958 [2003])13 observed that different fields use distinct argument evaluation criteria, but the same argument layout, or model (Conley 1990, 294–296; Hitchcock and Verheij 2006; van Eemeren et al. 2014, 203–256; Zenker 2018). In fact, Toulmin’s model became so influential as to widely “replace the old concepts of ‘premises’ and ‘conclusion’ with the new concepts of ‘claim’, ‘data’, ‘warrant’, ‘model qualifier’, ‘rebuttal’ and ‘backing’” (van Eemeren et al. 2014, 204). Figure 1 illustrates this model with what probably is Toulmin’s best-known example (Toulmin 2003, 97). In Fig. 1, the starting point is the claim (C) ‘Harry is a British subject’; the data element (D) is ‘Harry was born in Bermuda’ (see ibid., 90). The warrant (W)—the relevant instance of any “general, hypothetical statements, which can act as bridges, and authorize the sort of step to which our specific argument commits us” (ibid., 91)—is: ‘A man born in Bermuda will generally be a British subject’. Alas, this warrant “would possess neither authority nor currency” without a backing (B), which 12 By analogy, the infamous bird Tweety and its genus—penguin—both exemplify the concept BIRD. Yet both are distinct from the concept itself. The level at which one defines BIRD thus is more abstract than the level where one defines penguin or Tweety. In other words, Tweety, penguin, and BIRD are housed at the applied, the theoretical, and the meta-level. 13 Page numbers cited for Toulmin (1958) refer to Toulmin (2003). 13 480 S. Yu, F. Zenker Harry was born in Bermuda. So, presumably, Since A man born in Bermuda will generally be a British subject. Harry is a British subject. Unless Both his parents were aliens/he has become a naturalized American/… On account of The following statutes and other legal provisions: … Fig. 1 Toulmin’s “Harry is a British subject”-example Toulmin abbreviates as: “[t]he following statues and other legal provisions: …” (ibid., 96f.). Since a warrant can feature “exceptional conditions which might be capable of defeating or rebutting the warranted conclusion,” the rebuttal (R) may be instantiated, for instance, by “[b]oth his parents were aliens,” or by “[h]e has become a naturalized American” (ibid., 94, 106). Accordingly, a qualifier (Q)—e.g., ‘presumably’—must indicate “the strength conferred by the warrant on this step” (ibid., 94), which thus specifies the claim. We will return to this model in Sect. 4.3. 4.2 Evaluation Criteria Virtually all approaches to argument evaluation seek to supply general norms. For instance, the RSA criteria—relevance, sufficiency, acceptability—demand that a good argument’s premises are acceptable, relevant to the conclusion, and that they support it sufficiently (Johnson and Blair 1994, 54f.). So do Govier’s (2001) ARGconditions, as well as Freeman’s (1991) “basic dialectical questions […] a rational judge would ask in drawing out an argument from a proponent” (ibid., 37f.; see his 1985, 38f.) Johnson (2000) adds premise truth as a fourth criterion, yielding the RSAT criteria.14 The RSAT criteria are interrelated: premise sufficiency presupposes premise relevance. Both in turn presuppose premise acceptability (Biro and Siegel 1992; 14 Comparing truth to acceptability, Johnson (2000, 336f.) favors including the truth criterion, so that truth can ground acceptability (ibid., 197f.). For criticisms, see Blair (2011, 94), van Rees (2001, 236), van Eemeren et al. (2014, 383), and Bondy (2010). 13 Schemes, Critical Questions, and Complete Argument Evaluation 481 Godden and Zenker 2018), while the truth criterion may conflict with the acceptability criterion (Pinto 1994).15 In specifying how the criteria apply, Johnson (2000) suggests that for an argument to achieve rational persuasion, it is necessary (but insufficient) that, next to the argument’s illative core, analysts also address its dialectical tier—for instance by asking such CQs as ‘how well does the arguer deal with standard objections and criticism?’; ‘how well does the argument address alternative positions, as well as its consequence or implications?’ (ibid., 207f.). Analysts may need various CQs to render the RSAT criteria applicable; yet none of the authors cited here claim that the criteria are complete. Indeed, the specification and the completeness problem—how to detail all dialectical obligations to address objections to the illative core (ibid., 327–333); are the illative core- and dialectical tier-criteria exhaustive (ibid., 333–336)—have both resisted a satisfactory solution. Rather than discuss the criteria’s scope, independence, or consistency, therefore, our immediate task is to address their completeness. 4.3 Making Toulmin’s Model More Precise Various scholars already use the Toulmin model in argument evaluation (e.g., Freeley and Steinberg 2009; Grennan 1997; Hastings 1962; Kienpointner 1992; Toulmin et al. 1984). Particularly Verheij (2003, 2006) and Prakken (2010) use it for a complete evaluation (see Sect. 5.2). Because the model prescribes how an argument’s structural parts should function, it offers a useful sense in which the model is normatively complete: an argument is good (or valid) if, and only if, all model components are fully explicit and fulfill their functions. Thus, to evaluate an argument completely is to evaluate whether all its components function well. Some scholars have suggested equating an argument scheme with the warrant (e.g., Grennan 1997; Hitchcock 2006; see van Eemeren 2018, 171). As Marraud (2018, 325) observes, however, even if argument schemes are normally classified according to their warrants, nothing forces us to identify both. At any rate, this would run counter to understanding a warrant as a genuine part of an argument scheme (see Sect. 3.3). If one instead treats only the substantive relation R as a warrant, then Toulmin’s model is already sufficiently general to subsume any specific argument. A decisive reason to nevertheless make the model yet more precise is Toulmin’s inconsistent stance as to whether warrants must be more general than ‘if D, then C’ (see Hitchcock 2003, 74; and our Sect. 5.2). In addressing this question, we now turn to the three central components of Toulmin’s model: warrant, backing, and qualifier. 15 “If […] the premises have satisfied the sufficiency criterion, then […] they are true” (Johnson 2000, 342), suggesting this order: acceptability, relevance, truth, sufficiency (ibid., 341–343). “[T]he easiest determination,” he finds, “is that of relevance” (ibid., 342), while acceptability is easier to apply than relevance or truth. 13 482 S. Yu, F. Zenker 4.4 Warrant, Backing, and Qualifier Regarding the warrant, Toulmin (1958, 91) states that warrants “may normally be written very briefly (in the form ‘If D, then C’).” This form answers to the How do you get there-question. But the warrant is neither a kind of premise, because “a premise is that from which an argument starts”—which instead suggests “Toulmin’s grounds [or data]” (Hitchcock 2003, 71)—nor is the warrant an implicit premise; it is rather “not a premise at all” (ibid., 72). Moreover, one cannot readily interpret the warrant as a specific saturation of the indicative conditional ‘if D, then C’. For this “misses the generality of warrants, which is one of their key features” (ibid., 73). This keeps from always considering the warrant as an explicit ‘if–then’ premise. We view the warrant as the element that expresses the argument’s substantive relation. With Hitchcock (2003), we thus assume that the warrant differs functionally from the data element, and that a complete argument evaluation must make both parts explicit.16 Where the warrant ‘If D, then C’ is overt, then, we view it as an explicit premise, otherwise as an implicit one. (This characterization is somewhat simpler than Hitchcock’s own.) Regarding the backing, some scholars view “the relation between backing and warrant [as] the same as the relation between datum and claim” (Verheij 2006, 192). As we now explain, this equation is problematic. Toulmin treats D and B as facts, whereas he treats W as a rule (Toulmin 1958; esp. 90, 91, 98). Specifically, the D-to-C inference is fact-to-claim; the B-to-W inference is fact-to-rule. The content of C, Toulmin left unconstrained (C can be a rule or a fact), while the B-to-W inference is a context-specific transformation of the ‘if–then’ premise. (A transformation in opposite direction is what Goodnight (1993, 51) calls ‘legitimation inference’: the specific selection of backing material as pertinent to a context.) To appreciate the problem in equating the B–W relation with the D–C relation, consider the wellworn, yet instructive “Petersen is a Swede”-example (Toulmin 1958, 102): (10) D–W–C version (D) Petersen is a Swede. (W) A Swede can be taken almost certainly not to be a Roman Catholic. So, (C) Petersen is almost certainly not a Roman Catholic. (11) D–B–C version (D) Petersen is a Swede. (B) The proportion of Roman Catholics Swedes is minute. So, (C) Petersen is almost certainly not a Roman Catholic. 16 Leaving the warrant implicit indicates that Toulmin (1958) had broadly ignored argument evaluation. Only in his later textbook (Toulmin et al. 1984, esp. 46, 56) did he made the warrant explicit (see Hitchcock 2003, 71f.). 13 Schemes, Critical Questions, and Complete Argument Evaluation 483 In (10) and (11), warrant and backing may seem to express two distinct reasons why few Swedes are Roman Catholics, in virtue of the backing expressing “a statistical report, [while the other reason expresses] an inference-warrant” (Toulmin 1958, 102). Both reasons nevertheless converge in content. Indeed, the warrant’s content (“almost certainly not property x”)—read as an inference rule—mirrors the backing’s content (“population with minute proportion of property x-bearers”) read as a factual distribution. In this sense, the warrant formulates a doxastic rule, the backing a doxastic fact. ‘Doxastic’ means that one believes the respective contents. (One might change ‘belief’ to ‘commitment’.) Far from denying a backing-to-warrant inference, this identifies two ways of expressing the same state of affairs. If the backing-to-warrant inference is simple, it may behave as the transformation between (10) and (11). Other B-to-W inferences can be far more complex. For instance, one could treat the backing as a rule’s legal source and the warrant as its legal interpretation. On this very matter, however, the philosophy of law offers no consensus. In legal “hard cases,” indeed, a B-to-W inference may very well trigger insufficiency, inconsistency, or indeterminacy, or raise axiological issues—regarding natural or positivistic law (Peczenik 2009, 18)—on which arguers themselves need not be coherent. So, as we transform (10) into (11), we assume that warrant and backing can be co-transformed. The qualifier refers to “the degree of force which our data confer on our claim in virtue of our warrant” (Toulmin 1958, 93)—as in: ‘Presumably, Harry is a British subject’. Some scholars treat ‘presumably’ as “[…] a part of the sentence that expresses the claim supported by the data” (Verheij 2006, 189). Others hold that “[a] modality[expression such as ‘presumably’] is not a component either of the premises or of the conclusion of an argument[, but instead] serves to modify the claim that the premises support the conclusion” (Freeman 1985, 154; italics added). Between these two options, the following seeks to offer a midway approach. Clearly, one may challenge the data themselves, for instance as being “fake,” biased, or incomplete. Moreover, the qualifier’s strength depends on the warrant’s force and the probability that data are faithful to the empirical reality generating them (aka the errorrate). For instance, if W and D are independent, and if W’s force without D is 90%, and the error-rate is 10%, then the warrant’s force reduces from 90% to 90% × 90% = 81%. So, poor data necessarily weaken the qualifier. While this speaks for keeping the claim’s content distinct from its modality, the modality shall nevertheless associate to the claim itself. Our midway approach is to combine the qualifier and the claim into a new claim. It conveys the overall strength of the transition from “D, and ‘if D then C’”, to ‘C’, given W and B. To distinguish this construction from Toulmin’s own qualifier, call it qualifier-inside-a-claim. In sum, the warrant is the ‘if D, then C’-premise that expresses the substantive relation between D and C (aka. the associated conditional; see Sect. 5.2). The B-toW inference transforms substantive (rather than logical) information from fact-level to rule-level. Finally, a qualifier that is a part of a claim differs from the qualifier expressing the warrant’s force. With this precisification of Toulmin’s model as background, we turn to related work that informs our own CQ-list (Sect. 6). 13 484 S. Yu, F. Zenker 5 CQs as Argument Attacks or Rebuttals 5.1 Three Attack Types Prakken (2005, 303) submits that “the argument-scheme approach and the way it has been employed in AI & Law respects some of the main lessons to be learnt from Toulmin’s The Uses of Argument.” His own version of a complete CQ-list identifies CQs dialectically as “pointers to counterarguments” (Prakken 2005, 306; 2010, 6). His list rests on the insight that “arguments can only be attacked [either] on their premises” or “on their applications of [deductive or] defeasible inference rules” (Prakken 2010, 3; see Pollock 1987, 1995; Vreeswijk 1997; Besnard and Hunter 2008; Bondarenko et al. 1997). A defeasible attack has two sub-types: the comparatively weaker undercutter targets the inference from premises to conclusion; the stronger rebuttal targets premises or conclusion directly. Thus, “arguments can be attacked in three ways: on their premises, on their inference and on their conclusion” (Prakken 2010, 3). The three attack-types motivate associating three kinds of CQs to a scheme, namely CQs addressing (i) “whether a premise is true, […] [ii] whether there is an exception (undercutter) to the scheme[,] and […] [iii] whether there is an argument with a contradictory conclusion (rebuttal)” (ibid., 8f.). Because the three attack-types assign a distinct role to each CQ, call this the three role CQ-version. 5.2 Five Rebuttal Types Viewing argumentation schemes as “a kind of generalized rules of inference […] express[ing] that, given certain [accepted] premises, a specific conclusion can be drawn” (Verheij 2003, 167), Verheij observes that schemes are often “defeasible, concrete and contingently valid, i.e., valid in certain contexts or under certain circumstances” (ibid.). Since a CQ’s purpose is “to question the dialectical relevance of an argument based on the scheme” (ibid., 173), he views rebuttals as functional elements that “involve conditions of exception for the argument” (2006, 194). Verheij thus distinguishes rebuttals as per what one may argue against: (11) Five elements one may argue against 1. The data, D 2. The claim, C 3. The warrant, W 4. The associated conditional ‘if D, then C’ expressing the bridge from D to C 5. The associated conditional ‘if W, then if D, then C’ expressing the bridge between W and ‘if D, then C’ From this, one straightforwardly obtains five types of rebuttal (Fig. 2). 13 Schemes, Critical Questions, and Complete Argument Evaluation 485 Fig. 2 Five types of rebuttals (Verheij 2006, 195) The arrows in Fig. 2 denote the associated conditional, itself representing the linguistic realization of what Verheij calls ‘primitive implication’ (ibid., 178, esp. note 4). Symbolized as ‘~>’, the truth of this conditional’s antecedent provides a reason for, and thus supports, the consequent exclusively in virtue of a premise’s substance (rather than its logical form). ‘D~>C’ thus expresses a “relation between D and C that is not captured by the truth values of D and C alone” (ibid., 178). Unlike the “notorious material conditional of standard logic” (ibid., 178), the associated conditional “only validates [the formally valid inference] Modus ponens” (ibid., 187). The truth of ~> does therefore not follow from the falsity of its antecedent, nor does the truth of ~> require a true antecedent.17 For instance, granted that ‘Harry was born in Bermuda ~> Harry is a British subject’ is true, the associated conditional remains true even if its antecedent is defeated, i.e., if it turns out that Harry was not born in Bermuda. Marked in Fig. 2 with lines ending in an ‘x’, moreover, type-1, 2, 3 rebuttals attack respectively the data, the claim, and the warrant, whereas type-4 and type-5 rebuttals attack the modus ponens inference. A rebuttal of type-5, for instance, targets: ‘If W, then if D, then C’, in form: W~>(D~>C) (ibid., 191). Similarly, a type-4 rebuttal targets D~>C. Toulmin and Verheij differ in how they treat the warrant. To Verheij (ibid., 192), a warrant justifies the connection between D and C (justification interpretation); to Toulmin (1958, 91), by contrast, a warrant functions as a “bridge” and merely connects D and C (connection interpretation).18 As the item that shoulders the justification, Verheij’s warrant thus plays the functional role that Toulmin assigns to the backing. Both authors chose different formulations: Verheij’s warrant appears in a rule statement; Toulmin’s backing appears in a fact statement (see Sects. 4.3, 4.4). 17 We thank an anonymous reviewer for clarifying the nuances of this construction, which earlier versions of this paper had misrepresented severely. 18 Remaining somewhat vague as to his own preferred interpretation, Toulmin (2003, 91; italics added) states that warrants “can act as bridges, and authorize the sort of step to which our particular argument commits us. These may normally be written very briefly (in the form ‘If D, then C’) […].” The term ‘authorize’ may suggest that the warrant justifies the D-to-C transition. Yet the bulk of Toulmin’s writing instead indicates that he adopts the comparatively weaker connection-interpretation. 13 486 S. Yu, F. Zenker 5.3 From Five Types to Three Type-1 and type-2 rebuttals feature distinct contents that attack the data, respectively the claim. Though type-3, 4, and 5 rebuttals are structurally distinct, each type directly or indirectly attacks the modus ponens relation. For instance, the type-3 rebuttal ‘A man born in Bermuda is normally French’ targets ‘A man born in Bermuda is normally a British subject’ (W). The type-4 rebuttal ‘Harry has become a naturalized American’ targets the (saturated) associated conditional ‘if D, then C’, thus targeting the connection between ‘Harry was born in Bermuda’ (D) and ‘Harry is a British subject’ (C). Finally, the type-5 rebuttal ‘Harry’s parents are aliens’ targets ‘A man born in Bermuda will generally be a British subject’ (W) by providing a reason against applying W. Both a type-3 and a type-5 rebuttal thus attack the connection between D and C. A type-4 rebuttal attacks ‘If D, then C’ directly, and a type-5 or type-3 rebuttal attacks the justification of ‘If D, then C’ directly, and hence attacks ‘If D, then C’ indirectly. Therefore, type-3 and type-5 rebuttals are structurally distinct but functionally equivalent variants of a type-4 rebuttal. Verheij models an argument that transfers an evaluation from accepted premises (marked with ‘!’) to claims about issues (marked with ‘?’) (Fig. 3) as a two-step modus ponens derivation (Fig. 4). This model includes, first, a justification “from the warrant statement […] to the arrow representing the associated conditional, that if Harry was born in Bermuda, he is a British subject”—thus from ‘W, W~>(D~>C)’ to ‘D~>C’. Second, it includes a justification from data to claim—thus from ‘D, D~>C’ to ‘C’. (Hence, attacking ‘W’ and attacking ‘W~>(D~>C)’ are two options of attacking D~>C.) Finally, in ‘If W, then if D, then C’ (type-5), the ‘if W’-part specifies ‘If D, then C’ (type-4) by providing a condition under which ‘If D, then C’ holds. One can thus specify ‘If D, then C’ either to ‘If W, then if D, then C’ or to ‘If not W, then if D, then C’, which therefore makes type-5 a functional specification of type-4. By further integrating this fivefold typology, one retains three types, such that a type-3 and a type-5 rebuttal are both functional specifications of a type-4 Fig. 3 Data, claim and warrant-model (Verheij 2006, 191) Fig. 4 Two-step modus ponens derivation (Verheij 2006, 192) 13 Schemes, Critical Questions, and Complete Argument Evaluation Fig. 5 Three kinds of attacks, leading to three kinds of CQs 487 D Attack 1 A relation of type X holds between D and C. Attack 2 C Attack 3 rebuttal. The CQs associated to type-3 and type-5 rebuttals should therefore be subCQs to those for a type-4 rebuttal (see below). This allows our own CQ-list to use the three-role version, where one attacks arguments on their premises, inferences, or conclusions. 6 A Complete CQ‑List 6.1 Premise, Inference, and Conclusion Attacks A CQ-list’s primary purpose being that of enabling a systematic argument evaluation, a complete evaluation requires a (preferably short) finite CQ-list.19 Although analysts are free to ask CQs in any order, the specific order we propose lets us develop a scheme’s CQs step-by-step, level-by-level, starting at the meta-level. This list is meta-level complete insofar as sub-CQs arise only at the theoretical or applied level. (This is similar to how Pragma-dialecticians arrange argumentative patterns (van Eemeren 2016, 2017) and to how Macagno, Walton and Tindale (2017) detail analogical arguments via inferential structures and defeasibility conditions.) Restricted to an attack’s three targets—premises, inference, conclusion—we retain the following components of Toulmin’s model, here combing a logical with a substantive account of argument schemes according to modus ponens (Fig. 5). Attack-1 and attack-2 both imply an attack-3 because by attacking D, or the D–C relation, one attacks C indirectly, without offering a direct counter-reason. The three kinds yield a straightforward meta-level CQ-list: CQ-1 for attack 1, CQ-2 for attack 2, and CQ-3 for the direct attack 3. As will become clear soon, one should first ask CQ-1, followed by CQ-2, and next CQ-3. 19 When treated instead as pointers to counterarguments (see Sect. 5.1), CQs would be assertives rather than interrogatives. For the same premise, moreover, the number of pointers can exceed the number of necessary CQs. In case of ‘a is an honest source’, for instance, the necessary CQ simply is: ‘Is a in fact an honest source?’ Yet multiple pointers can direct us towards counter-arguments. For instance: ‘Liars are prone to dishonesty’, or ‘An ill-hearted person can be a dishonest source’, ‘As can an interest-conflicted person’, etc. Hence, if one saw the main purpose of a CQ-list in generating these pointers, then one could readily associate several pointers to a single CQ. To avoid such multiplicity, we define our CQs extensionally (in Sects. 6.2, 6.3). 13 488 S. Yu, F. Zenker 6.2 Towards a Complete Meta‑Level CQ‑List We first present the basic CQs and their evaluative answers, adding further CQs and sub-CQs in Sect. 6.3. Our CQs all use the term ‘correct’; of course, one may substitute with ‘(rhetorically) acceptable’, ‘(logically or factually) true’, ‘(epistemologically or doxastically) plausible’, etc. CQ-1. Are the data correct? This CQ evaluates the content of the data element. CQ-2. Is ‘If D, then C’ correct? Once saturated, this conditional expresses the focal substantive relation. The corresponding CQ, then, is: “Does the (content of the) ‘D therefore C’-relation hold?” Evaluating the D-to-C-relation regularly requires more information than arguers offer or analysts can access readily. For instance, in “[a]s Daniel is an American […] he is sure to be concerned about the costs” (van Eemeren and Grootendorst 1992, 97), acceptability would transfer from data to claim, insofar as a symptomatic relation were to hold between Daniel being American and being inclined to care about the costs. Upon assuming that Daniel is American, however, one may nevertheless specify this relation in various ways: “Americans generally care (lots) about money” (ibid.), ‘Young Americans care (lots) …’, or ‘Male Americans …’, etc. Yet the right choice is not obvious. Absent information to the contrary, one should interpret D’s predicate (“is American”) as naming the intended category. For acceptability to transfer, then, this category must include D’s subject (Daniel). Indeed, since argument evaluation presupposes that one can know the intended category, the first step is to determine it. If D’s intended category implies C’s predicate necessarily, because D’s subject necessarily belongs to the intended category, then D’s subject implies C’s predicate logically, and hence ‘D implies C’ holds. (One may interpret ‘necessary implication’ epistemically or ontically; regardless, ‘knowing-p’ entails ‘p’, but not vice versa.) By contrast, if D’s intended category fails to imply C’s predicate necessarily, then one must test whether D’s subject belongs to the intended category’s exception class. If so, then D’s subject fails to imply C’s predicate logically. In this case, one arrives at the following sub-CQs: CQ-2.1 “What is the intended category of D’s subject?” CQ-2.2 “What is the content of the substantive relation between D and C (represented by the ‘if–then’ conditional)?” CQ-2.3 “Is the relation between the intended category of D’s subject and C’s predicate claimed to hold necessarily?” Finally, if one answers CQ-2.3 negatively: CQ-2.4 “Does D’s subject belong to the focal exception class?” In this way, CQ-2 narrowly examines only (the content of) the inference ‘if D, then C’. For instance, instead of category membership—as in “Daniel is American”— arguers may assert state of affairs such as “The weather is fine,” and conclude: “let’s go for a hike.” Assume the weather is fine. The evaluation would proceed as follows: to the arguer, suppose, fine weather is but one among other enabling conditions such 13 Schemes, Critical Questions, and Complete Argument Evaluation 489 as “money in the bank,” “time on one’s hand,” or “energy to spare,” etc. (Where such conditions are negated, they express inference-exceptions.) Since the relation between the weather and the hike is contingent rather than necessary, evaluation thus proceeds with the above sub-CQ-2.1 to CQ-2.4. For contingent relations generally, one cannot fix all enabling conditions that count towards the arguer’s intended category. CQ-3. Is the claim correct? Evaluating an argument’s claim implies evaluating whether all relevant data have been considered fully. This makes CQ-3 broader than CQ-1 (correctness of data), and presupposes having specified the ‘D-to-C’ relation by answering CQ-2. If analysts can offer additional relevant data, D, then further pro- or con-arguments relate to the claim. To evaluate them, and then compare the strength of pro- and con-arguments, one must iterate the evaluative process. We have so far shown that our CQ-list applies specifically to the Harry-example, which cites a symptomatic relation between premise and claim. This leaves the relations of similarity and causality effectively untreated. Two anonymous reviewers therefore rightly expressed doubt that our CQ-list is general. For reasons of limited space, we can only offer an indirect response: one can regiment any argument scheme to the form: ‘x has property F, so x has property G’ (Hitchcock 2017, 296). It follows that all object-level arguments are meta-level sign arguments. Put differently, the symptomatic relation reduces both the similarity and the causality relation. Hence, if our model evaluates sign arguments completely, and we believe it does, then it evaluates any argument completely. 6.3 Full Meta‑Level CQ‑List The full version of our CQ-list (including sub-CQs) is the following: CQ-1 Is D correct? • If not, or if no clear answer is forthcoming, then the evaluation process stops, resulting in a negative evaluation. • If yes, then the evaluation process continues. CQ-2.1 What is the intended category of D’s subject? • If no clear answer comes forth, the process stops with a negative evaluation. • Otherwise, the evaluation continues. CQ-2.2 What is the content of the ‘D therefore C’-relation? • If no clear answer comes forth, the process stops with a negative evaluation. • Otherwise, the evaluation process continues. 13 490 S. Yu, F. Zenker CQ-2.3 Does the relation between D’s intended category and C’s predicate hold necessarily? • If yes, then the process stops with a positive evaluation. • If not, or if no clear answer comes forth, then the evaluation continues. CQ-2.4 Does D’s subject belong to an exception-class of its intended category (as per CQ-2.1)? • If yes, then the process stops with a negative evaluation. • If not, then the process stops with a positive evaluation. • If no clear answer comes forth, then the evaluation continues. CQ-3.1 Can one offer additional relevant data (D) besides D? • If no clear answer comes forth, the process stops with a negative evaluation. • If yes, then add these data, and return to CQ-1. • If no, then the evaluation continues. CQ-3.2 Are there other arguments against the claim?20 • If yes, then evaluate these arguments (pros versus cons). • If no, then the process stops with a positive evaluation. The evaluation process stops if analysts find the argument good, or bad; or if lack of information makes this impossible (e.g., for CQ-1, one may not know whether D is correct). Having thus defined our CQs extensionally, we can proceed to apply them.21 6.4 Applying the CQs Our CQ-list assumes that D is explicit. In a realistic argumentative context, this is implausible. The Harry-example, for instance, might read: ‘A man born in Bermuda will be a British subject, so Harry is a British subject’. This leaves D implicit. Generally, analysts must make choices when they specify implicit elements, and these specifications can easily differ in use value. For instance, Verheij (2006, 190) distinguishes three warrant-versions: (i) ‘A man born in Bermuda will generally be a British subject’; (ii) ‘If a person was born in Bermuda, then generally that person is a British subject’; (iii) ‘If Harry was born in Bermuda, then generally he is a British subject.’ Verheij (ibid., 190f.) treats (i) as the warrant’s ordinary language 20 CQ-3.1 sits before CQ-3.2, because the reverse order may require asking CQ-3.2 twice, namely once again if the answer to CQ-3.1 is positive. One should consider CQ-3.1 before CQ-3.2 also because the answer “fewer than all relevant data were considered” normally amounts to a reason against the claim. 21 Walton and Sartor (2013, 111) state that argument evaluation “needs to take into account the attitudes of dialogue partners as well as normative models of dialogue and communicative activity types, in particular social and cultural settings.” Of course, such considerations may influence one’s evaluative criteria, as well as the answers to the CQs themselves. Yet they will not determine a meta-level CQ-list’s content. 13 Schemes, Critical Questions, and Complete Argument Evaluation 491 C Logical C specifies minimum Pragmatic optimum Associated W conditional D C justifies Pragmatic optimum W D~>C D D Fig. 6 Two D–W–C models Fig. 7 D–W–B–C model C Associated conditional specifies justifies Pragmatic optimum W informs D B expression, (ii) as “the formal explication of the bridge-like connection,” and (iii) as “the associated conditional” expressing that a particular D implies a particular C. Given the pragmatic optimum versus logical minimum-distinction (van Eemeren and Grootendorst 1992, 98f.), (iii) thus expresses the logical minimum, (i) expresses the pragmatic optimum, and (ii) can be a pragmatic optimum in at least some contexts.22 To Verheij, the pragmatic optimum justifies the associated conditional formally, while Pragma-dialecticians proceed in the opposite direction: the pragmatic optimum specifies the logical minimum. In specifying the logical minimum to the pragmatic optimum, then, one invariably justifies how one arrives at the pragmatic optimum in a specific context. This entails that one obtains two D–W–C models (Fig. 6). 22 The logical minimum is “a modus ponens-like inference from premises to a conclusion” (van Eemeren and Grootendorst 1992, 97). The pragmatic optimum requires that arguers make the logical minimum “as informative as possible without ascribing unwarranted commitments to the speaker and [while] formulating it in a colloquial way that fits in with the rest of the argumentative discourse” (ibid., 64). 13 492 S. Yu, F. Zenker According to modus ponens, the logical minimum is valid even if D is false. Similarly, when D is false, the associated conditional D~>C can be correct, as long as D supports C. Per CQ-1, instead, if D is false, then argument evaluation stops with a negative result. Further, a D-to-C transition differs from a B-to-C transition: the first justifies the transition, while the second transforms this justification according to a context (see Sect. 4.3). Thus, in Fig. 7, the backing informs the pragmatic optimum. This we can articulate further in Fig. 8. Given a context, one may specify a doxastic fact-formulation into a doxastic ruleformulation (see the W-to-B transformation; Sect. 4.3). As an instance of Fig. 8, then, see Fig. 9, which connects both doxastic elements (using a line without arrowhead) to represent that information is neither added nor deleted. C If D, then C; or: the specifies relation R holds justifies Doxastic rule-formulation of the relation between D and C. between D and C. informs D Doxastic fact-formulation of the relation between D and C Fig. 8 Argument components Harry is a British subject. If Harry was born in Bermuda, then Harry is a British subject. Or: a A man born in Bermuda is British. symptomatic relation holds between “Harry was born in Bermuda” and “Harry is a British subject.” Harry was born in Bermuda. Fig. 9 The components of Toulmin’s Harry-example 13 All men born in Bermuda are British. Schemes, Critical Questions, and Complete Argument Evaluation Table 1 Possible argumentcomponent combinations 493 (1) D, so C (2) W, so C (3) B, so C (4) D, W, so C (5) D, B, so C (6) W, B, so C (7) D, W, B, so C 6.5 Standardization Before concluding, we review the seven possible combinations of argument components that may appear in natural language (Table 1). By rearranging and/or supplementing these components, one can standardize (1)–(7) to the form ‘D, W, so C’. In fact, ‘D, so C’ already suffices to apply our CQlist. The following illustrates this with the Harry-example: 1. D, so C (“Harry was born in Bermuda. So, Harry is a British subject.”) To this form, the CQ-list is already applicable. 2. W, so C (“A man born in Bermuda is a British subject. So, Harry is a British subject.”) To obtain a data element, one must transform W into a doxastic factformulation. The warrant’s ontological formulation is the original backing: ‘All men born in Bermuda are British subjects’. Considering this backing as a dataelement, one can treat ‘W, so C’ as (1).23 3. B, so C (“All men born in Bermuda are British subjects, so Harry is a British subject.”) One may treat (3) as ‘D, so C’—which puts one back at (1)—or may transform B into W—which puts one back at (2). 4. D, W, so C (“Harry was born in Bermuda, and a man born in Bermuda is a British subject; so Harry is a British subject.”) It is easy to distinguish D and W; one can apply ‘D, so C’ as per (1), or ‘W, so C’ as per (2). 5. D, B, so C (“Harry was born in Bermuda, and all men born in Bermuda are British subjects; so Harry is a British subject.”) Both B and D are fact-formulations. Without further assumptions, distinguishing their roles proves challenging. After all, B’s doxastic rule-formulation can represent the relation between D and C, but D’s doxastic rule-formulation cannot represent the relation between B and C. Of course, one may assume some fact-formulation as B, and then test whether it represents the relation between the other fact-formulation and C. If so, then one should treat it as B, otherwise as D. At last, then, one can distinguish D and B, and thus apply ‘D, so C’ as per (1) or, respectively, ‘B, so C’ as per (3). 23 The warrant connects C to D (rather than to B). Since D and B both constitute a fact-formulation, our model applies insofar as a fact-formulation serves as a premise. Thus, one need not formulate the original data, but can use the backing as a new D, itself transformed from W. Where the original D is unknown, the transformation reflects an evaluation based on additional known information. 13 494 S. Yu, F. Zenker When evaluating the form ‘D, B, so C’—e.g., “Harry was born in Bermuda, and all men born in Bermuda are British subjects; so Harry is a British subject”—one cannot distinguish the premises’ roles immediately, because both premises state facts. One therefore needs something like the following test: the rule-formulation of “All men born in Bermuda are British subjects” is “A man born in Bermuda is a British subject.” This reflects the relation between “Harry was born in Bermuda” and “Harry is a British subject.” So B is: “All men born in Bermuda are British subjects.” By contrast, the rule-formation of “Harry was born in Bermuda” is “If a man is Harry, then he was born in Bermuda.” But this cannot suitably connect “All men born in Bermuda are British subjects” with “Harry is a British subject.” Therefore, one cannot justify “Harry is a British subject” with “All men born in Bermuda are British subjects.” It follows that “Harry was born in Bermuda” is not a B.24 6. W, B, so C (“A man born in Bermuda is a British subject, and all men born in Bermuda are British subjects; so Harry is a British subject.”) It is easy to distinguish W and B; the first is a rule-formulation, the other is a fact-formulation. Hence, one can apply ‘W, so C’ as per (2), or ‘B, so C’ as per (3). 7. D, W, B, so C (“Harry was born in Bermuda, and a man born in Bermuda is a British subject, and all men born in Bermuda are British subjects; so Harry is a British subject.”) One can treat this form either as ‘D, so C’ as per (1), or as ‘W, so C’ as per (2), or as ‘B, so C’ as per (3). Given argument components that correspond to D, W, B, C, then, the foregoing applies to any argument, and so facilitates a complete meta-level evaluation. 7 Conclusion Treating ARGUMENT SCHEME as a logical concept, we have sought to capitalize on the distinction between an argument scheme’s levels, separating an applied level, from a theoretical, and a meta-level. If this approach to evaluating natural language arguments completely does succeed, then this would be so because we proceeded from the outset at the meta-level. This showed that one can abstract all elements that a complete evaluation must address. Although this result is fully consistent with a logical account, we also saw why a substantive account must complement it, and how a specification of Toulmin’s model can ground this hybrid account. Having applied our complete CQ-list so far only exemplarily, future research should relate our meta-level CQs to the applied level in order to 24 We thank an anonymous reviewer for pointing out an alternative rule-formation for “Harry was born in Bermuda,” namely “If every man born in Bermuda has a certain property, then Harry has it.” This alternative rule-formulation can connect “All men born in Bermuda are British subjects” with “Harry is a British subject.” However, it not only is a less natural formulation, but also involves a second-order generalization. 13 Schemes, Critical Questions, and Complete Argument Evaluation 495 obtain specific criteria for a given scheme. This should advance the construction of an exhaustive list of applied CQs. Acknowledgements We thank Minghui Xiong, as well as two anonymous reviewers for this journal, for their extensive and at times painstakingly detailed comments, all of which served to improve previous versions of this paper. SY acknowledges support from the Major Program of the National Social Science Foundation of China (No. 14ZDB013). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/. References Besnard, P., and A. Hunter. 2008. Elements of argumentation. Cambridge, MA: MIT Press. Biro, J.I., and H. Siegel. 1992. Normativity, argumentation and a doxastic theory of fallacies. In Argumentation illuminated, ed. F.H. van Eemeren, R. Grootendorst, J.A. Blair, and C.A. Willard, 85–103. Amsterdam: Sic Sat. Blair, J.A. 2001. Walton’s argumentation schemes for presumptive reasoning: A critique and development. Argumentation 15: 365–379. Blair, J.A. 2011. Groundwork in the theory of argumentation. New York: Springer. Bondarenko, A., P. Dung, R. Kowalski, and F. Toni. 1997. An abstract, argumentation-theoretic approach to default reasoning. Artificial Intelligence 93: 63–101. Bondy, P. 2010. Truth and argument evaluation. Informal Logic 30: 142–158. Conley, T.M. 1990. Rhetoric in the European tradition. Chicago, IL: University of Chicago Press. Daniel, S., and D. Nic. 2014. The deflationary theory of truth. In The Stanford Encyclopedia of Philosophy (Fall 2014 Edition), ed. Edward N. Zalta. https://plato.stanford.edu/archives/fall2014/ entries/truth-deflationar y/. Accessed 18 Feb 2020. Ennis, R. 1982. Identifying implicit assumptions. Synthese 51: 61–86. Freeley, A.J., and D.L. Steinberg. 2009. Argumentation and debate: Critical thinking for reasoned decision making, 12th ed. Boston: Wadsworth Cengage Learning. Freeman, J.B. 1985. Dialectical situations and argument analysis. Informal Logic 7: 151–162. Freeman, J.B. 1991. Dialectics and the macrostructure of arguments: A theory of argument structure. Berlin and New York: Foris Publications. Garssen, B. 1994. Recognizing argumentation schemes. In Studies in pragma-dialectics, ed. F.H. van Eemeren and R. Grootendorst, 105–111. Amsterdam: Sic Sat. Garssen, B. 1997. Agumentatieschema’s in Pragma-dialectisch Perspectief. Een Theoretisch en Empirisch Onderzoek [Argument Schemes from a pragma-dialectical Perspective: A Theoretical and Empirical Overview]. Dissertation. Amsterdam: IFOTT. Garssen, B. 2001. Argument schemes. In Crucial concepts in argumentation theory, ed. F.H. van Eemeren, 81–99. Amsterdam: Amsterdam University Press. Godden, D., and F. Zenker. 2018. A probabilistic analysis of argument cogency. Synthese 195: 1715–1740. Goodnight, G.T. 1993. Legitimation inferences: An additional component for the Toulmin model. Informal Logic 15: 41–52. Gough, J., and C. Tindale. 1985. ‘Hidden’ or ‘missing’ premises. Informal Logic 7: 99–106. Govier, T. 2001. A practical study of argument, 5th ed. Belmont, CA: Wadsworth. (1st ed. 1985). 13 496 S. Yu, F. Zenker Grennan, W. 1997. Informal logic: Issues and techniques. Montreal: McGill-Queen’s University Press. Hahn, U., and J. Hornikx. 2016. A normative framework for argument quality: Argumentation schemes with a Bayesian foundation. Synthese 193: 1833–1873. Hastings, A.C. 1962. A reformulation of the modes of reasoning in argumentation. Dissertation. Northwestern University, Evanston, IL. Hitchcock, D. 2003. Toulmin’s warrants. In Anyone who has a view. Theoretical contributions to the study of argumentation, ed. F.H. van Eemeren, J.A. Blair, C.A. Willard, and A.F. Snoeck Henkemans, 69–82. Dordrecht: Kluwer. Hitchcock, D. 2006. Good reasoning on the Toulmin model. In Arguing on the Toulmin model. New essays in argument analysis and evaluation, ed. D. Hitchcock and B. Verheij, 203–218. Netherlands: Springer. Hitchcock, D. 2017. On reasoning and argument: Essays in Informal Logic and Critical Thinking. Berlin: Springer. Hitchcock, D., and B. Verheij (eds.). 2006. Arguing on the Toulmin model. New essays in argument analysis and evaluation. Netherlands: Springer. Hitchcock, D.L., and J.H.M. Wagemans. 2011. The pragma-dialectical account of argument schemes. In Keeping in touch with pragma-dialectics, ed. E.T. Feteris, B.J. Garssen, and A.F. Snoeck Henkemans, 185–205. Amsterdam: Benjamins. Hoffmann, M. 2016. Reflective argumentation: A cognitive function of arguing. Argumentation 30: 365–397. Hoffmann, M. 2018. The elusive notion of “argument quality”. Argumentation 32: 213–240. Johnson, R.H. 2000. Manifest rationality: A pragmatic theory of argument. Mahwah, NJ: Lawrence Erlbaum Associates. Johnson, R.H., and J.A. Blair 1994. Logical self-defense (U.S. edition). New York: McGraw-Hill. Kienpointner, M. 1992. Alltagslogik. Struktur und Funktion von Argumentationsmustern [Everyday Logic. Structure and Function of Argument Patterns]. Stuttgart-Bad Cannstatt: Frommann-Holzbog. Lumer, C. 2011. Argument schemes—An epistemological approach. In Argumentation: Cognition and community. Proceedings of the 9th international conference of the Ontario Society for the Study of Argumentation (OSSA), ed. F. Zenker, 1–32. Windsor, ON (CD ROM). https://scholar.uwindsor.ca/ cgi/viewcontent.cgi?referer=&httpsredir=1&article=1016&context=ossaarchive. Accessed 18 Feb 2020. Macagno, F., D. Walton, and C. Tindale. 2017. Analogical arguments: Inferential structures and defeasibility conditions. Argumentation 31: 221–243. Marraud, H. 2018. Arguments from ostension. Argumentation 32: 309–327. Peczenik, A. 2009. On law and reason. Dordrecht: Springer. Perelman, C., and L. Olbrechts-Tyteca. 1958. La nouvelle rhétorique. Traité de l’argumentation. Paris: Presses Universitaires de France. [Perelman, C., and Olbrechts-Tyteca, L. 1969. The New Rhetoric. A Treatise on Argumentation (trans: Wilkinson, J., and Weaver, P.). Notre Dame: University of Notre Dame Press]. Pinto, R.C. 1994. Logic, epistemology and argument appraisal. In New essays in informal logic, ed. R.H. Johnson and J.A. Blair, 116–124. Windsor, ON: Informal Logic. Pollock, J.L. 1987. Defeasible reasoning. Cognitive Science 11: 481–518. Pollock, J.L. 1995. Cognitive carpentry. A blueprint for how to build a person. Cambridge, MA: MIT Press. Prakken, H. 2005. AI & law, logic and argument schemes. Argumentation 19: 303–320. Prakken, H. 2010. On the nature of argument schemes. In Dialectics, dialogue and argumentation. An examination of Douglas Walton’s theories of reasoning and argument, ed. C.A. Reed and C. Tindale, 167–185. London: College Publications. http://www.cs.uu.nl/groups/IS/archive/henry/schem es10.pdf. Accessed 18 Feb 2020. Rapp, C., and T. Wagner. 2013. On some Aristotelian sources of modern argumentation theory. Argumentation 27: 7–30. Rigotti, E., and S. Greco Morasso. 2006. Topics: The argument generator. In Argumentation for financial communication, Argumentum eLearning module. www.argumentum.ch. Accessed 18 Feb 2020. Rigotti, E., and S. Greco Morasso. 2009. Argumentation as object of interest and as social and cultural resource. In Argumentation and education: Theoretical foundations and practices, ed. A.N. PerretClermont and N. Müller-Mirza, 9–66. New York: Springer. 13 Schemes, Critical Questions, and Complete Argument Evaluation 497 Rigotti, E., and S. Greco Morasso. 2010. Comparing the argumentum model of topics to other contemporary approaches to argument schemes: The procedural and material components. Argumentation 24: 489–512. Rubinelli, S. 2009. Ars Topica. The classical technique of constructing arguments from Aristotle to Cicero. Netherlands: Springer. Schut, D., and J.H.M. Wagemans. 2014. Argumentatie en debat. Den Haag: Boom Lemma. Tindale, C. 2007. Fallacies and argument appraisal. Cambridge: Cambridge University Press. Toulmin, S. E. (2003; 1958). The uses of argument (Updated ed.). Cambridge: Cambridge University Press (1st ed., 1958). Toulmin, S.E., R.D. Rieke, and A. Janik. 1984. An introduction to reasoning, 2nd ed. New York: Macmillan. (1st ed., 1978). van Eemeren, F.H., and T. Kruiger. 1987. Identifying argumentation schemes. In Argumentation: Perspectives and approaches: Proceedings of the conference on argumentation 1986, ed. van Eemeren, F.H., T. Kruiger, R. Grootendorst, J.A. Blair, and C.A. Willard, 70–81. Dordrecht-Holland/Providence: Foris Publications. [reprinted in: F.H. van Eemeren (ed.), Reasonableness and effectiveness in argumentative discourse, 2015, pp. 703–712. Cham: Springer International Publishing AG.]. van Eemeren, F.H. 2016. Identifying argumentative patterns: A vital step in the development of pragmadialectics. Argumentation 30: 1–23. van Eemeren, F.H. (ed.). 2017. Prototypical argumentative patterns. Exploring the relationship between argumentative discourse and institutional context. Amsterdam/Philadelphia: John Benjamins. van Eemeren, F.H. 2018. Argumentation theory: A pragma-dialectical perspective. Cham: Springer International Publishing AG. van Eemeren, F.H., B. Garssen, E.C.W. Krabbe, F. Snoek Henkemans, B. Verheij, and J.H.M. Wagemans. 2014. Handbook of argumentation theory. Dordrecht: Springer. van Eemeren, F.H., and R. Grootendorst. 1992. Argumentation, communication, and fallacies. Hillsdale: Lawrence Erlbaum. van Eemeren, F.H., and R. Grootendorst. 2004. A systematic theory of argumentation. The pragma-dialectical approach. Cambridge: Cambridge University Press. van Rees, M.A. 2001. Review of Ralph H. Johnson (2000), Manifest rationality. A pragmatic theory of argument. Argumentation 15: 231–237. Verheij, B. 2003. Dialectical argumentation with argumentation schemes: An approach to legal logic. Artificial Intelligence and Law 11: 167–195. Verheij, B. 2006. Evaluating arguments based on Toulmin’s scheme. In Arguing on the Toulmin model. New essays in argument analysis and evaluation, ed. D. Hitchcock and B. Verheij, 181–202. Netherlands: Springer. Verheij, B. 2014. Arguments and their strength: Revisiting Pollock’s anti-probabilistic starting points. In Computational models of argument. Proceedings of the COMMA 2014, ed. S. Parsons, N. Oren, C. Reed, and F. Cerutti, 433–444. Amsterdam: IOS Press. http://dx.doi.org/10.3233/978-1-61499 -436-7-433. Vreeswijk, G. 1997. Abstract argumentation systems. Artificial Intelligence 90: 225–279. Wagemans, J.H.M. 2016. Constructing a periodic table of arguments. In Argumentation, objectivity, and bias. Proceedings of the 11th international conference of the Ontario Society for the Study of Argumentation (OSSA), ed. P. Bondy and L. Benacquista, 1–12. Windsor, ON. http://scholar.uwindsor.ca/ ossaarchive/OSSA11/papersandcommentaries/106. Wagemans, J.H.M. 2019. Four basic argument forms. Research in Language 17: 57–69. Walton, D. 1996. Argumentation schemes for presumptive reasoning. Mahwah: Lawrence Erlbaum. Walton, D. 2016. Some artificial intelligence tools for argument evaluation: An introduction. Argumentation 30: 317–340. Walton, D., C. Reed, and F. Macagno. 2008. Argumentation schemes. Cambridge: Cambridge University Press. Walton, D., and G. Sartor. 2013. Teleological justification of argumentation schemes. Argumentation 27: 111–142. Walton, D., C. Tindale, and T. Gordon. 2014. Applying recent argumentation methods to some ancient examples of plausible reasoning. Argumentation 28: 85–119. Yu, S., and F. Zenker. 2018. Peirce knew why abduction isn’t IBE. A scheme and critical questions for abductive argument. Argumentation 32: 569–587. Yu, S., and F. Zenker. 2019. A dialectical view on conduction: Reasons, warrants, and normal suasory inclinations. Informal Logic 39: 32–69. 13 498 S. Yu, F. Zenker Zenker, F. 2009. Ceteris Paribus in conservative belief revision. Berlin: Peter Lang. Zenker, F. 2018. Logic, reasoning, argumentation: Insights from the wild. Logic and Logical Philosophy 27: 421–451. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 13
https://openalex.org/W2790859354	https://europepmc.org/articles/pmc5887904?pdf=render	English	null	Co‐targeting driver pathways in prostate cancer: two birds with one stone	EMBO molecular medicine	2,018	cc-by	2,161	Co-targeting driver pathways in prostate cancer: two birds with one stone Amina Zoubeidi & Martin E Gleave more active regimens, either by combining currently approved drugs, or via design of novel biologically rational regimens to create contextual lethality based on genomic biomarker enrichment, and/or co-targeting adaptive survival pathways activated by AR pathway inhibitors. Indeed, impressive results from CHAARTED (NCT00309985), STAMPEDE (NCT00268476), and LATITUDE (NCT01715285) clinical trials highlight significant benefits of combination versus monotherapy regimens, as well as early versus late intervention in advanced prostate cancer. These clinical trials combined androgen-deprived therapy (ADT) with chemotherapy (for CHAARTED, STAM- PEDE), and ADT with abiraterone (for LATITUDE, STAMPEDE) in advanced castrate-sensitive prostate cancer patients. The studies all report a remarkably similar 40% reduction in death rate and provide precedent for future effective combination regimens across the prostate cancer treat- ment landscape. Co-targeting strategies strive to improve cancer outcomes by combining therapies under contextualized genetic and environ- mental conditions that selectively target exploitable alterations in tumor cells. Adaptive survival pathways triggered by inhibition of driver genes in the androgen receptor (AR) or PI3K/AKT pathways are of great interest, since they are among the most frequently altered in castrate- resistant prostate cancer (CRPC). Unfortu- nately, negative feedback loops exist between the AR and PI3K/AKT pathways such that targeting AR leads to activation of PI3K/AKT signaling, while PI3K/AKT path- way inhibition leads to increased AR tran- scriptional activity. Hence, targeting both pathways provides an opportunity for conditional lethality and a high therapeu- tic index. In this issue of EMBO Molecular Medicine, Yan et al (2018) present an elegant study showing that histone deacetylase 3 (HDAC3) acts as a common upstream activator of both AR and AKT signaling pathways, and use HDAC3 inhibi- tors as a monotherapy to co-target two major pathways driving CRPC growth. prostate cancers harbor PTEN alterations and 10% harbor SPOP mutations. Both pheno- types are associated with activated AR and PI3K/AKT pathways despite the fact that, in early treatment-naı¨ve localized disease, SPOP mutations are mutually exclusive with genomic alterations in PI3K/AKT pathway. Interestingly, these alterations can co-occur in CRPC. Expression of mutant SPOP activates PI3K/AKT pathway and upregulates AR signaling, maintaining AR transcriptional activity against PI3K/AKT-mediated negative feedback, effectively activating the two most common driver pathways critical in prostate cancer. Hence, combined blockade of these pathways may delay treatment resistance and significantly improve patient outcome. News & Views Co-targeting driver pathways in prostate cancer: two birds with one stone The study by Carver et al (2011) was the first to demonstrate that the AR and PI3K pathways co-regulate one another via recip- rocal negative feedback, such that inhibition of one activates the other. Mechanistically, inhibition of the PI3K pathway increased AR signaling in PTEN-deficient prostate cancer in part via relief of negative feedback to HER kinases; conversely, AR antagonism relieves feedback inhibition of AKT by reducing FKBP5-mediated stability of the phosphatase PHLPP. While tumor cells can adapt and survive when either single path- way is inhibited, combined inhibition of PI3K/AKT and AR signaling using the PI3K/ mTOR inhibitor BEZ235 and the AR antago- nist enzalutamide (ENZ) significantly delayed castrate-resistant LNCaP tumor progression. Similar data were reported by Thomas et al (2013) and Toren et al (2015); increased AR transcriptional activity observed using monotherapy with the AKT inhibitor AZD5363 was overcome by combining AZD5363 with ENZ, resulting in Castrate-resistant prostate cancer most often progress with an activated AR despite potent AR pathway suppression, supported by a vast network of pro-survival genes and growth factor pathways, including the PI3K/ AKT pathway. Indeed, the AR (via amplifi- cation, mutation, variants) and PI3K/AKT (via Pten loss) pathways are the two most frequently genomically altered pathways in CRPC. While clinical responses are common with AR pathway inhibitors, responses using PI3K inhibitors are rare in preclinical models or patients with CRPC. Monotherapy with AR or AKT inhibitors results in reciprocal cross- talk activation that supports emergence of acquired resistance (Carver et al, 2011; Mulholland et al, 2011). Genomically, 70% of The Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada. E-mail: m.gleave@ubc.ca DOI 10.15252/emmm.201808928 \| Published online 23 March 2018 EMBO Mol Med (2018) 10: e8928 See also: Y Yan et al (April 2018) T he discovery that castration-resistant prostate cancer (CRPC) most often remains fueled by androgen receptor (AR) signaling ushered in the development and clinical integration of highly potent AR pathway inhibitors. These agents have undoubtedly benefited patients with CRPC, but are merely palliative as resistance inevi- tably emerges. There is a need to develop T ª 2018 The Authors. Published under the terms of the CC BY 4.0 license ª 2018 The Authors. Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine 10: e8928 \| 2018 1 of 3 Co-targeting driver pathways Amina Zoubeidi & Martin E Gleave EMBO Molecular Medicine AKT Ac AKT P P Ub Ub Ub Ub Ub Ac HDAC3 Ac HDAC3 AKT inactivated AKT activated CYTOPLASM NUCLEUS Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Transcriptional activation AR expressed Relaxed chromatin Ac Ac Ac Transcriptional repression AR repressed Condensed chromatin HDAC3 inhibitor © EMBO Figure 1. HDAC3 inhibitor effects on AKT and AR pathways. HDAC3 integrates major signaling pathways in prostate cancer: the androgen receptor and the AKT pathways. Targeting HDAC3 using a small-molecule inhibitor or siRNA inhibits AKT phosphorylation and HDAC3 binding to APPL1 in the cytoplasm, while it represses AR transcription via histone deacetylation and condensed chromatin in the nucleus. HDAC inhibition decreases AR full-length and splice variant mRNA levels in the PTEN loss and SPOP mutant prostate cancer models. HDAC3 inhibitor shows efficacy in these two prostate cancer subtypes that share activated AR and AKT pathways. This preclinical proof of principle is encouraging and will help guide bench-to-bedside transla- tion to integrate HDAC3 inhibitor into clini- cal trials. Key issues include evaluation as monotherapy or as part of a combination regimen with an approved AR pathway inhi- bitor, as well as biomarker enrichment (e.g., PTEN and/or SPOP mutated, AR amplified) to include those more likely to respond. Targeting AKT and AR was more profound when combined with castration (ADT) in prostate cancer (Toren et al, 2015). Beyond the focus on AR and AKT pathways, HDAC3 inhibition may also enhance activity of PARP inhibitors (Ha et al, 2014) or chemotherapy (Long et al, 2017), supporting potential combinations in DNA repair-deficient cancers, or when docetaxel is indicated. EMBO Mol Med (2018) 10: e8928 See also: Y Yan et al (April 2018) In summary, the data presented by Yan et al (2018) define a mechanism-based target- ing of HDAC3 upstream of two key genomic altered pathways in prostate cancer and provide preclinical proof of principle to guide inhibitor development toward the clinic. References Figure 1. HDAC3 inhibitor effects on AKT and AR pathways. HDAC3 integrates major signaling pathways in prostate cancer: the androgen receptor and the AKT pathways. Targeting HDAC3 using a small-molecule inhibitor or siRNA inhibits AKT phosphorylation and HDAC3 binding to APPL1 in the cytoplasm, while it represses AR transcription via histone deacetylation and condensed chromatin in the nucleus. Carver BS, Chapinski C, Wongvipat J, Hieronymus H, Chen Y, Chandarlapaty S, Arora VK, Le C, Koutcher J, Scher H et al (2011) Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer. Cancer Cell 19: 575 – 586 which synergistically delayed CRPC LNCaP tumor progression in vivo (Matsumoto et al, 2013). synergistic inhibition of cell proliferation and induction of apoptosis, and delayed CRPC tumor growth in vivo. These data provided preclinical proof of principle to support evaluation of combination AZD5363 and ENZ in the clinic (NCT02525068). Another approach to co-targeting AR and AKT pathways focussed on stress chaperone proteins activated by AR pathway inhibitors, coordinated by a feed-forward loop involv- ing p90rsk-mediated phospho-activation of YB-1 with subsequent induction of CLU and AKT/MAPK activity. Co-targeting the AR (with ENZ) and molecular chaperone CLU (with OGX-011) repressed ENZ-induced acti- vation of AKT and MAPK pathways, acceler- ated AR degradation and repressed AR transcriptional activity through mechanisms involving decreased YB-1-regulated expres- sion of the AR co-chaperone, FKBP52, Ha K, Fiskus W, Choi DS, Bhaskara S, Cerchietti L, Devaraj SG, Shah B, Sharma S, Chang JC, Melnick AM et al (2014) Histone deacetylase inhibitor treatment induces ‘BRCAness’ and synergistic lethality with PARP inhibitor and cisplatin against human triple negative breast cancer cells. Oncotarget 5: 5637 – 5650 The data presented by Yan et al (2018) describe an elegant approach to co-target these pathways by inhibiting histone deacetylase 3 (HDAC3), an upstream regula- tor for both AR and AKT pathways, effec- tively eliminating two birds with one stone (Fig 1). HDAC3, which is upregulated in prostate cancer (Weichert et al, 2008), facili- tates lysine-63-chain polyubiquitination and phosphorylation of AKT in prostate cancer cells, a non-nuclear effect mediated by AKT deacetylation at lysine 14 and 20 residues and HDAC3 interaction with the scaffold protein APPL1. Targeted inhibition of HDAC3 blocks interaction with APPL1, decreasing AKT acetylation and thereby inhibiting AKT phosphorylation. EMBO Molecular Medicine 10: e8928 \| 2018 3 of 3 References In addition, Long J, Fang WY, Chang L, Gao WH, Shen Y, Jia MY, Zhang YX, Wang Y, Dou HB, Zhang WJ et al (2017) Targeting HDAC3, a new partner protein of AKT in the reversal of chemoresistance in acute myeloid leukemia via DNA damage response. Leukemia 31: 2761 – 2770 Matsumoto H, Yamamoto Y, Shiota M, Kuruma H, Beraldi E, Matsuyama H, Zoubeidi A, Gleave M (2013) Cotargeting androgen receptor and clusterin delays castrate-resistant prostate cancer progression by inhibiting adaptive stress response and AR stability. Can Res 73: 5206 – 5217 Long J, Fang WY, Chang L, Gao WH, Shen Y, Jia MY, Zhang YX, Wang Y, Dou HB, Zhang WJ et al (2017) Targeting HDAC3, a new partner protein of AKT in the reversal of chemoresistance in acute myeloid leukemia via DNA damage response. Leukemia 31: 2761 – 2770 acute myeloid leukemia via DNA damage response. Leukemia 31: 2761 – 2770 Matsumoto H, Yamamoto Y, Shiota M, Kuruma H, Beraldi E, Matsuyama H, Zoubeidi A, Gleave M (2013) Cotargeting androgen receptor and clusterin delays castrate-resistant prostate cancer progression by inhibiting adaptive stress response and AR stability. Can Res 73: 5206 – 5217 2 of 3 EMBO Molecular Medicine 10: e8928 \| 2018 ª 2018 The Authors Amina Zoubeidi & Martin E Gleave Co-targeting driver pathways Amina Zoubeidi & Martin E Gleave Co-targeting driver pathways EMBO Molecular Medicine Mulholland DJ, Tran LM, Li Y, Cai H, Morim A, Wang S, Plaisier S, Garraway IP, Huang J, Graeber TG et al (2011) Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth. Cancer Cell 19: 792 – 804 Thomas C, Lamoureux F, Crafter C, Davies BR, Beralidi E, Fazli L, Kim S, Thaper D, Gleave ME, Zoubeidi A (2013) Synergistic targeting of PI3K/ AKT-pathway and androgen-receptor axis significantly delays castration-resistant prostate cancer progression in vivo. Mol Cancer Ther 12: 2342 – 2355 Mulholland DJ, Tran LM, Li Y, Cai H, Morim A, Wang S, Plaisier S, Garraway IP, Huang J, Graeber TG et al (2011) Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth. Cancer Cell 19: 792 – 804 Yan Y, An J, Yang Y, Wu D, Bai Y, Cao W, Ma L, Chen J, Yu Z, He Y et al (2018) Dual inhibition of AKT- mTOR and AR signaling by targeting HDAC3 in PTEN- or SPOP-mutated prostate cancer. EMBO Mol Med 10: e8478 Toren P, Kim S, Cordonnier T, Crafter C, Davies BR, Fazli L, Gleave ME, Zoubeidi A (2015) Combination AZD5363 with enzalutamide significantly delays enzalutamide-resistant prostate cancer in preclinical models. Eur Urol 67: 986 – 990 Thomas C, Lamoureux F, Crafter C, Davies BR, Beralidi E, Fazli L, Kim S, Thaper D, Gleave ME, Zoubeidi A (2013) Synergistic targeting of PI3K/ AKT-pathway and androgen-receptor axis significantly delays castration-resistant prostate cancer progression in vivo. Mol Cancer Ther 12: 2342 – 2355 Weichert W, Roske A, Gekeler V, Beckers T, Stephan C, Jung K, Fritzsche FR, Niesporek S, Denkert C, Dietel M et al (2008) Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy. Br J Cancer 98: 604 – 610 Weichert W, Roske A, Gekeler V, Beckers T, Stephan C, Jung K, Fritzsche FR, Niesporek S, Denkert C, Dietel M et al (2008) Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy. Br J Cancer 98: 604 – 610 License: This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and repro- duction in any medium, provided the original work is properly cited. EMBO Molecular Medicine 10: e8928 \| 2018 3 of 3 EMBO Molecular Medicine 10: e8928 \| 2018 3 of 3 ª 2018 The Authors ª 2018 The Authors
https://openalex.org/W2130929412	https://europepmc.org/articles/pmc3799451?pdf=render	English	null	Direct assessment of transcription fidelity by high-resolution RNA sequencing	Nucleic acids research	2,013	public-domain	12,835	ABSTRACT Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 104–105 per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through 103nt DNA sites assures that the RNA-seq can analyze the fidelity in a large number of the sites where errors occur. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectiv- ity for the cognate substrate, and (ii) increased backtracking of the RNA polymerase that decreases a chance of error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. This method is adoptable to a genome-wide assessment of tran- scription fidelity. Deep sequencing technologies such as RNA sequencing (RNA-seq) can analyze 1010 bases in a single run, po- tentially allowing both a genome-wide and in vitro detec- tion of transcription error rates around 105 b1 rate (7,17,18). However, conventional protocols for RNA-seq generate background errors at >105 b1 frequency during the process of cDNA library/cluster formation, sequencing/detection and the mapping of the reads (24), which has made it difﬁcult to detect transcription errors. Advanced deep sequencing techniques use tagging of indi- vidual DNA molecules by random sequences in polymer- ase chain reaction (PCR) primers to identify and ﬁlter out the PCR artifacts by counting only those error spots that persist throughout all DNA molecules carrying the same tag (25–27). This tag-based method substantially reduces randomly distributed PCR and sequencing errors of the deep DNA/RNA sequencing (25–27). A problem remain- ing in this method is that it cannot reduce the errors introduced by reverse transcriptases (RTs) that typically Direct assessment of transcription fidelity by high-resolution RNA sequencing Received June 10, 2013; Revised July 12, 2013; Accepted July 15, 2013 assumption has been made that transcription errors are randomly distributed. However, several reports have sug- gested that transcription errors exhibit strong sequence preferences (11–14). Fidelity analysis for the entire tran- scriptome has been limited by a lack of a reliable method- ology. In the past decade, extensive in vitro analyses of transcription ﬁdelity revealed several error-avoidance and error-correcting mechanisms based on biochemical assays for misincorporation of a unique NMP (12,13, 15–20) and single-molecule assays using optical trapping techniques (11,21). Typically, these experiments included limited or unbalanced substrate concentrations to detect misincorporation. These in vitro data cannot be easily extrapolated to the genetic ﬁdelity assays involving reporter genes transcribed at high in vivo concentration of substrates and in the presence of transcription factors and structural proteins compacting DNA (1,7–10,22,23). Therefore, there is an urgent need for an approach that would allow simultaneous assessment of transcription ﬁdelity in vivo and in vitro under balanced NTP concen- tration and on the same DNA sequences. Published by Oxford University Press 2013. This work is written by US Government employees and is in the public domain in the US. Published online 7 August 2013 Published online 7 August 2013 9090–9104 Nucleic Acids Research, 2013, Vol. 41, No. 19 doi:10.1093/nar/gkt698 Published by Oxford University Press 2013. This work is written by US Government employees and is in the public domain in the US. INTRODUCTION We veriﬁed the production of a homogeneous 5.7 kb RNA by agarose-gel electro- phoresis before adding DNase I (Supplementary Figure S1C). The 5.7 kb RNA was puriﬁed from the digested DNA, NTPs, abortive oligo-RNA products and proteins as shown in Supplementary Figure S1D. 0.1 mg/ml bovine serum albumin] (Supplementary Figure S1B). The reaction was stopped by heat denatur- ation for 3 min at 90C followed by DNase I (Takara Bio) treatment for 20 min at 37C. We veriﬁed the production of a homogeneous 5.7 kb RNA by agarose-gel electro- phoresis before adding DNase I (Supplementary Figure S1C). The 5.7 kb RNA was puriﬁed from the digested DNA, NTPs, abortive oligo-RNA products and proteins as shown in Supplementary Figure S1D. Library preparation RNAP holoenzyme of Escherichia coli RL-916 (the strain was a kind gift from Dr Robert Landick) containing a histidine-tagged RpoC subunit was puriﬁed as described previously (37). The GreA and GreB expression plasmids pDNL278 and pMO1.4 were kind gifts from Dr Sergei Borukov. The plasmids were transformed into E. coli strain XL1-Blue cells (Stratagene) for overexpression. The recombinant GreA and GreB were puriﬁed according to (38) with the addition of Mono Q column (GE Healthcare) chromatography. The ﬁrst DNA strand was synthesized using the transcript from the PR promoter (0.8 mg RNA synthesized in vitro or 5 mg total RNA puriﬁed from E. coli cells) and a RT PrimeScript. The RNA transcript was mixed with 1 mM dNTP and 5 mM of two speciﬁc primers (a and b, Figure 1A) that hybridizes to the RNA transcript at the most 30 portion of the DNA segments 1 and 6 (Figure 1A) of the ﬁrst PCR. A hairpin structure between the segments 1 and 2 inhibits elongation of RT on the RNA transcript to the 50 end. The mixture was incubated for 5 min at 65C. The PrimeScript, 1 PrimeScript buffer and RNase Inhibitor were added to the mixture according to the manufacturers’ instructions, and the mixture was incubated for 45 min at 42C, for 5 min at 37C with RNase H and for 15 min at 70C. The single-strand DNA product was puriﬁed with MinElute PCR puriﬁca- tion kit and eluted with 10 ml of the elution buffer. The ﬁrst PCR including the second DNA strand synthesis was performed with a DNAP PrimeSTAR Max based on the manufacturers’ instructions at 5 cycles for the RNA prep- aration in vitro and 10 cycles for the RNA preparation in vivo. We noticed that the total RNA puriﬁed from E. coli cells had a concentration of the unique transcript from the PR promoter of the pPR9 plasmid by 30-fold less than the in vitro RNA preparation. Thus, the ﬁve additional cycles make almost same ﬁnal concentrations In vivo RNA preparation Total RNA was prepared from E. coli MG1655 strain harboring pPR9 plasmid. Cells were cultured at 28C in LB medium containing ampicillin. The overnight cell culture was inoculated into the fresh medium at 1/70 (v/ v) and was incubated for 2 h at 28C (OD600 reached 0.35) and then for 2 h at 42C (OD600 reached 2.3) to induce the PR promoter (39). The cells in 200 ml culture were harvested and resuspended with a solution contain- ing 0.5% sodium dodecyl sulphate, 20 mM sodium acetate (pH 5.5) and 10 mM EDTA. The suspended cells were mixed with an equal volume of prewarmed saturated phenol (20 mM sodium acetate, 10 mM EDTA, pH 5.5) and incubated for 5 min at 60C. The mixture was centrifuged, and RNA and DNA were precipitated with ethanol from the supernatant. The pellet was dissolved in DNase I buffer with 10 U of DNaseI and incubated for 30 min. RNA was separated from the digested DNA by acidic phenol extraction followed by G-50 Micro column (GE Healthcare) puriﬁcation and then precipitated with ethanol. The pellet was dissolved in diethylpyrocarbonate- treated water and used for cDNA synthesis. Reagents NTPs, oligonucleotides and DNA puriﬁcation kits were purchased from GE Healthcare, Integrated DNA Technologies and Qiagen, respectively. NTPs used in the misincorporation assay (Figure 5 and Supplementary Figure S5) were further puriﬁed as described previously (17). The high ﬁdelity RT PrimeScript and the DNAP PrimeSTAR Max used for the cDNA preparation were purchased from Takara Bio. INTRODUCTION Transcription inﬁdelity by RNA polymerases (RNAPs) has been proposed to contribute to genome instability (1) and heritable phenotypic changes (2,3), which may affect aging (4) and carcinogenesis (5,6). To date, assess- ment of transcription ﬁdelity in vivo has been performed with reporter genes targeting a small number of sequences with a limited spectrum of errors (1,7–10). To extrapolate this limited ﬁdelity analysis to a genome-wide scale, an Nucleic Acids Research, 2013, Vol. 41, No. 19 9091 have lower ﬁdelity than DNA polymerases (DNAPs) used for PCR (28,29). More recently, a deep-sequencing method was developed involving analysis of mismatches in overlapping read pairs to identify the artifact errors, but not the RT errors (30). Thus, so far there is no an approach suitable for discriminate RNA errors from the RNA-seq artifacts. Here, we present a high-resolution RNA-seq method based on a remarkable sequencing depth of 106 accompanied by several technical improve- ments reducing background errors to 105 and 104 levels. This technique enables statistically reliable detection of changes in transcription ﬁdelity in vitro and in living cells, despite the presence of the artifact errors. This meth- odology may also be instrumental in addressing contro- versial noncanonical posttranscriptional RNA-editing (31–35), identiﬁcation of genomic ‘hotspots’ for transcrip- tion errors and their contribution to the genetic diversity of viral populations (27,29,30,36). have lower ﬁdelity than DNA polymerases (DNAPs) used for PCR (28,29). More recently, a deep-sequencing method was developed involving analysis of mismatches in overlapping read pairs to identify the artifact errors, but not the RT errors (30). Thus, so far there is no an approach suitable for discriminate RNA errors from the RNA-seq artifacts. Here, we present a high-resolution RNA-seq method based on a remarkable sequencing depth of 106 accompanied by several technical improve- ments reducing background errors to 105 and 104 levels. This technique enables statistically reliable detection of changes in transcription ﬁdelity in vitro and in living cells, despite the presence of the artifact errors. This meth- odology may also be instrumental in addressing contro- versial noncanonical posttranscriptional RNA-editing (31–35), identiﬁcation of genomic ‘hotspots’ for transcrip- tion errors and their contribution to the genetic diversity of viral populations (27,29,30,36). 0.1 mg/ml bovine serum albumin] (Supplementary Figure S1B). The reaction was stopped by heat denatur- ation for 3 min at 90C followed by DNase I (Takara Bio) treatment for 20 min at 37C. In vitro RNA preparation The pPR9 plasmid containing lambda phage PR promoter and fd phage terminator was used for the DNA template (Supplementary Figure S1A). The transcribed region is composed of refampicin-resistant rpoB gene that contains a 1546G!T mutation, and partial rplL and rpoC genes of E. coli. The plasmid DNA was puriﬁed by Qiagen mini-prep kit and phenol/chloroform/ isoamylalcohol (25:24:1). The residual phenol that may affect transcription was removed by solvent extraction with diethyl ether. For transcription reaction, 400 nM holoenzyme in the absence or presence of 12 mM GreA and 4 mM GreB was incubated with 1 mM NTP and 2 nM the plasmid DNA for 15 min at 37C in transcription buffer [TB; 20 mM Tris–HCl, pH 7.9, 5 mM MgCl2 (or 1 mM MnCl2), 1 mM 2-mercaptoehanol, 0.1 M KCl, 9092 Nucleic Acids Research, 2013, Vol. 41, No. 19 9092 Internal control 1st read 2nd read Internal control 5 5 3 3 cDNA cDNA ~20 b~73 b ~63 b ~20 b Barcode Adapter Adapter p A B C D Figure 1. Experimental setup for transcription-error analysis. (A) Schematic representation of reverse transcription and two PCR steps used to produce barcoded cDNA libraries. The ﬁve libraries were made from each of the RNA samples corresponding to the ﬁve transcription conditions (Mg2+, Mn2+, GreAB/Mg2+ and GreAB/Mn2+ for in vitro and E.coli cell). The RT primers ‘a’ and ‘b’ (the green arrowheads) replace transcription errors with the chemical oligonucleotide synthetic errors during reverse transcription step. Similarly, in a course of PCR, the ﬁrst PCR primers (green and yellow lines) replace (green lines) or dilute by >10-fold (yellow lines) transcription errors in the corresponding regions to which these primers hybridize (shown by empty boxes). A six-bases barcode (purple line) and Illumina-speciﬁc sequencing adapters (orange and red lines) are introduced to the libraries during ﬁrst and second PCR steps. (B) The cDNA and internal control regions in the PCR fragment used for Illumina paired-end sequencing. The lengths and directions of the ﬁrst and the second sequencing reads are indicated. Both sequencing reads contain 20 bases of the primer-hybridizing regions where transcription errors are signiﬁcantly depleted during cDNA preparation (internal controls). All colors are the same as in panel A, except that the DNA regions lacking the original mRNA are white-shaded. (C) Scatter plot of transition-error rates for Mn2+ and Mg2+ RNA products in vitro. In vitro RNA preparation Positions in cDNA and internal control are indicated by red and blue colors. The diagonal dotted lines represent y = 2 x (upper), y = x (middle) and y = 1/2 x (lower). Correlation coefﬁcient (R) of the two samples with or without cutoff value >3 104 b1 is shown. (D) Transition-error rates in the second read (lower) of the paired-end sequencing are higher than those in the ﬁrst read (upper). Transition-error rates averaged by the ﬁve different RNA preparations and the six sequence segments (see panel A) are plotted against DNA positions with the standard deviations. Red line indicates the cutoff value. 9092 Nucleic Acids Research, 2013, Vol. 41, No. 19 rimental setup for transcription-error analysis. (A) Schematic representation of reverse transcription and two PCR steps used to produce barcoded cDNA libraries. The ﬁve ade from each of the RNA samples corresponding to the ﬁve transcription conditions (Mg2+, Mn2+, GreAB/Mg2+ and GreAB/Mn2+ for in vitro and E.coli cell). The RT primers green arrowheads) replace transcription errors with the chemical oligonucleotide synthetic errors during reverse transcription step. Similarly, in a course of PCR, the ﬁrst PCR and yellow lines) replace (green lines) or dilute by >10-fold (yellow lines) transcription errors in the corresponding regions to which these primers hybridize (shown by empty ases barcode (purple line) and Illumina-speciﬁc sequencing adapters (orange and red lines) are introduced to the libraries during ﬁrst and second PCR steps. (B) The cDNA and regions in the PCR fragment used for Illumina paired-end sequencing. The lengths and directions of the ﬁrst and the second sequencing reads are indicated. Both sequencing 20 bases of the primer-hybridizing regions where transcription errors are signiﬁcantly depleted during cDNA preparation (internal controls). All colors are the same as in panel A, DNA regions lacking the original mRNA are white-shaded. (C) Scatter plot of transition-error rates for Mn2+ and Mg2+ RNA products in vitro. Positions in cDNA and internal cated by red and blue colors. The diagonal dotted lines represent y = 2 x (upper), y = x (middle) and y = 1/2 x (lower). Correlation coefﬁcient (R) of the two samples with or value >3 104 b1 is shown. (D) Transition-error rates in the second read (lower) of the paired-end sequencing are higher than those in the ﬁrst read (upper). Ternary elongation complex formation and biochemical transcription assays of cDNA libraries derived from the in vitro and in vivo RNA preparations. One-tenth total reaction volume of the single strand DNA puriﬁed by the Qiagen kit and each primer pair including a barcode and the inner Illumina sequence adapters in the 50 tails (Figure 1A and Supplementary Table S5) were used for the PCR. This PCR ampliﬁed the six different DNA segments comprising the cDNA (transcript) and the internal control (primer) (Figure 1A and B) for the ﬁve libraries with respective barcodes. We conﬁrmed that no ﬁrst PCR product was obtained in each primer pair when RNA solution without RT was used as a template. The six DNA segments obtained from each reaction tube of the ﬁrst PCR were mixed and puriﬁed by the Qiagen kit, and eluted with 10 ml of the elution buffer. The second PCR was performed with one-ﬁfth total reaction volume of the obtained PCR products, a primer pair containing the outer sequencing adapters in the 50 tails (Supplementary Table S5), and the same PCR enzyme as the ﬁrst PCR at 6 cycles. The presence of the full-length Illumina sequence adapter and barcode sequence (Illumina TruSeq Index 1–5) in each of the ﬁve cDNA libraries was conﬁrmed by Sanger sequencing. The ternary elongation complexes (TECs) carrying 50-labelled RNAs (see Supplementary Table S6) were assembled and immobilized on Ni2+-NTA agarose (Qiagen) in TB as described previously (42,43). Five to ten picomoles of RNAP was incubated with 7.5–15 pmols of the preannealed RNA–DNA hybrid in 25–50 - ml volume for 10 min at room temperature. Next, 15–50 pmols of the nontemplate DNA strand (NDS) were added for 10 min. The immobilized TEC9s were washed with TB containing 1 M KCl. The TECs were eluted from Ni2+- NTA agarose by TB(-MgCl2) with 100 mM imidazole as described previously (17) and diluted with TB(-MgCl2). TEC18s were obtained from TEC9s by walking on the template of 170 G and 474 G sequences (37). To allow the 1 or 2 steps of walking, the 14 G and 17 G on the 170 G and 474 G sequences, respectively, were substituted with C (see Figures 4A, 5B and Supplementary Figure S3A). The typical concentration of TEC is 1 nM (44). All reactions were performed in TB at 37C. The reactions were stopped by gel-loading buffer (5 M urea; 25 mM EDTA ﬁnal concentrations). The RNA products were analyzed as described previously (17). In vitro RNA preparation Transition-error by the ﬁve different RNA preparations and the six sequence segments (see panel A) are plotted against DNA positions with the standard deviations. Red line indicates the cutoff D Internal control 1st read 2nd read Internal control 5 5 3 3 cDNA cDNA ~20 b~73 b ~63 b ~20 b Barcode Adapter Adapter p B D C B A Nucleic Acids Research, 2013, Vol. 41, No. 19 9093 Ternary elongation complex formation and biochemical transcription assays Details about the experimental setups for misincorporation, mismatch-extension, RNA cleavage and NTP competition assays are described in the corres- ponding ﬁgures or supplementary ﬁgures. Exonuclease III footprinting The rear-end Exonuclease III (ExoIII) footprinting was performed as described previously (17,44). TECs were assembled on the 50 end-labeled template DNA strand and the unlabeled NDS (Supplementary Table S6). The reaction was started by mixing 15 ml TB containing 10 U of ExoIII (New England Biolabs) with 15 ml of the elong- ation complex at 30C. To prevent digestion of the NDS by ExoIII for the rear-end footprinting, the NDS carries phosphorothioate bond at the 30 end. The active state (pretranslocated state) and backtracked states of TECs were determined by shifting the boundaries of RNAP due to stepwise extension of RNA in TECs (17,44). Data analysis for the sequencing The initial data processing, including reads separation by the barcodes and the generation of fastq ﬁles, was per- formed with the CASAVA software (Illumina). A single large fastq format ﬁle of high quality reads (Phred score Q 30, see Supplementary Table S1) was split into about 10 smaller ﬁles by using a shell script splitReads.sh (http://code.google.com/p/perm/downloads/detail?name =splitReads.sh) to use SAMtools mpileup –A commands for the following error analysis in the sequence reads (see below). The obtained reads were aligned and mapped to the pPR9 plasmid DNA sequences (1056 bp) using a program Bowtie 0.12.7 that does not allow insertion and deletion in the alignment (40). We chose Bowtie parameter that allows three mismatches. To calculate error rates, we counted the numbers of 4 bases A, T, G, C, and N (not determined) in each position of the mapped reads by using the program SAMtools 0.1.18 (41) with supplemental use of a Perl script, parse_samtools_mpileup.pl (a kind gift from Dr Wei Shao). Each type of error rates per position was determined as the number of sequence reads with a particu- lar type of base-substitution divided by the number of the reads with the reference base in each DNA position. Illumina sequencing Quantiﬁcations of the numbers of ampliﬁable molecules in the libraries were performed by qPCR using a Library Quantiﬁcation Kit (KK4824, Kapa Biosystems) and Agilent 2100 Bioanalyzer. The cluster generation on a paired-end ﬂow cell and sequencing were performed with cBot and HiSeq 2000, respectively, according to the user guides of Illumina. The summary of sequencing data is shown in Supplementary Table S1. Raw sequencing data and processed data are available for download at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GS E46479. Strategy for the assessment of transcription ﬁdelity by RNA-seq Importantly, we used these outer segments (shown by green and yellow lines, Figure 1A and B) as internal controls to compare error rates in these sequences with those in the embedded cDNA segment carrying intact transcription errors (Figure 1B, blue lines). Base substitu- tion errors made during synthesis of primer DNA are reported at 104–105 b1 rates (based on the manufac- turer information), which is consistent with our data (see Supplementary Figure S2 and Table S4). We obtained 191 099 124 reads with high base-calling quality [Phred score Q 30 (46)] by the paired-end sequencing (Supplementary Table S1). Each sequenced read included the cDNA and the internal control sequence (Figure 1B). The uniquely mapped sequence reads covered 1056 bp with an average 3 106 read depth (Supplementary Table S2). To assess types and rates of RNA/DNA changes per position, we excluded insertion and deletion errors to avoid reads misalignment (47) during bioinformatic analysis. In the mapped sequence reads, we found a few positions with abnormally high background of transversions A!C (ﬁrst read) and G!T (second read) with 102 or 103 b1 frequency, which are unlikely due to transcription errors. These errors are probably caused by the relatively close emission spectra of the corresponding ﬂuorophores and their incomplete separation by optical ﬁlters in the Illumina platform at these particular positions (24). These rare positions have been ignored. p The RNA samples for the RNA-seq were generated by transcription of pPR9 plasmid (39) by E. coli RNAP in vitro and in vivo. The plasmid contains an 5.7-kb fragment of E. coli rpoBC operon that is transcribed from a strong lambda phage PR promoter and terminated at an fd phage transcription terminator (Supplementary Figure S1A). A multi-round transcription by the puriﬁed RNAP holoenzyme generated 1015 RNA molecules with a uniform length of 5.7 kb (Supplementary Figure S1 B- D). The reference transcription reaction was performed in a TB (42) with 5 mM MgCl2 to determine the standard error rate. To reduce ﬁdelity (the error-prone condition), we replaced Mg2+ with Mn2+ (48–50). To increase ﬁdelity (the error-proof condition), we added GreA/GreB proteins (51) for proofreading activity. We kept a balanced high concentration of NTPs (1 mM) in all con- ditions to avoid forced nonphysiological misincor- poration, although the actual concentrations of NTPs in vivo may not be uniform and vary under different growth conditions (52). Strategy for the assessment of transcription ﬁdelity by RNA-seq The strategy is based on two key assumptions: (i) combined error rates of RT and DNAPs used for the RNA-seq can be reduced to 105 b1 range by using high-ﬁdelity RT and DNAPs. The estimated error rates are based on information provided by the manufacturers, which are consistent with our data (see Supplementary Table S3), (ii) multi-subunit RNAPs generate errors with sequence preferences different from those of the structurally unrelated RTs/DNAPs as suggested previ- ously (11–14,36,45). We did not use the tag-based error- correction method to reduce artifact errors because this approach cannot identity/correct RT errors and typically 9094 Nucleic Acids Research, 2013, Vol. 41, No. 19 9094 increases the number of PCR cycles owing to the loss of the original templates for PCR in a course of the DNA tagging (25–27). Instead, we signiﬁcantly reduced the number of PCR cycles to minimize the DNAP errors during the library production (see ‘Materials and Methods’ section). To identify the sequence sites dominated by transcription errors, we used error-prone and error-proof transcription conditions in vitro to increase and decrease transcription errors, respectively. In this system, transcription error rates are changed in a controlled manner for the sequencing reads, whereas the artifact errors remain constant. Detection of transcription errors should be possible at sequence sites favoring tran- scription and disfavoring the artifact errors even when the averaged enzymatic artifact rates exceed those of tran- scription. Widespread existences of such sites through the analyzed sequences should be also statistically evaluated. We also reduced the nonenzymatic sequencing errors caused by incorrect base-calling (46) and misalign- ment (47) of the Illumina reads by setting an appropriate ﬁlter eliminating these artiﬁcial error hotspots (see below). Finally, we signiﬁcantly increased the read depth to average 3 106 to cover the predicted 105 b1 rate for transcription errors. cycle and the remaining 4 cycles of the ﬁrst PCR, chemical synthetic errors in the DNA primers replace and steadily dilute transcription errors by 2-fold in mRNA segments to which these primers hybridize. Thus, transcription errors in the corresponding cDNA segments were replaced or 16- fold reduced by 5 cycles of the ﬁrst PCR (Figure 1A, the empty boxes). Consequently, contribution of transcription errors in these segments becomes negligible in the ﬁnal cDNA libraries compared with the rest of cDNA. Strategy for the assessment of transcription ﬁdelity by RNA-seq For the in vivo ﬁdelity measure- ment, we puriﬁed total RNA from the wild-type E. coli strain harboring the same pPR9 plasmid after 2 h induc- tion of the PR promoter at 42C (39). We plotted transition error rates for the cDNA se- quences in the standard Mg2+ transcription condition against the error-prone Mn2+ condition (Figure 1C, red dots). We compared this plot with the corresponding plot derived from the internal controls where transcription errors were replaced or diluted with the oligo DNA-syn- thetic errors (Figure 1C, blue dots). If there are no differ- ences between the Mg2+/Mn2+sets (indicating a failure in detection of transcription errors), the data should fall along the y = x line as is observed for the internal control positions (R > 0.9). In contrast, for the cDNA pos- itions, the plots of the lower error rates were localized in the y > x area (R < 0.9 for the 3 104 b1 rates). Two-tailed F-test for the Mg2+/Mn2+ RNA samples con- ﬁrmed that the error rates 3 104 b1 are not equally distributed in cDNA [P = 2 104 (n = 540)] as opposed to their equal distribution in the internal controls [P = 0.5 (n = 125)]. Notably, the transition errors occurring at >3 104 b1 rates were primarily observed in the second read that required an additional strand synthesis step (Figure 1D). Therefore, these errors mostly derived from the artifact of paired-end sequencing. We used this information to set a cutoff value of 3 104 b1 error rate in our statistical analysis of transcription errors. We established a method for preparing ﬁve different cDNA libraries each with its own barcode for Illumina sequencing (Figure 1A). Each 6-nt barcode allows multi- plexing all ﬁve in vitro and in vivo preparations in a single sequencing analysis. The 50 fragment of the 5.7 kb RNA transcripts was reverse transcribed, and the product was subjected to PCR reactions that generated six 200 bp segments (Figure 1A). The primers contained a speciﬁc barcode for each of the ﬁve starting preparations and the inner Illumina-sequencing adapters (Figure 1A). The second step of PCR generated the ﬁnal cDNA libraries for the Illumina sequencing by using the ﬁrst-step PCR product as a template and primers containing the outer sequencing adapters in the 50 tails (Figure 1A). In the ﬁrst Nucleic Acids Research, 2013, Vol. Strategy for the assessment of transcription ﬁdelity by RNA-seq 41, No. 19 9095 a signiﬁcant difference in means of the two samples (P < 0.05). We observed slight Mn2+-dependent increase in C!T(U) transition rate (P = 0.09) and no difference in A!G transition (P = 0.7). Because the detected mean rate of A!G transition was the lowest among the four types of transitions (Supplementary Figure S2), the A!G transcription errors appeared to be masked by the arti- facts even in the error-prone conditions for RNAP. Note that the internal control showed no signiﬁcant effect of Mn2+ on any type of transition error (Figure 2, left column). Thus, the RNA-seq detected Mn2+- The high-resolution RNA-seq detects changes in transition error rates in vitro The high-resolution RNA-seq detects changes in transition error rates in vitro The high-resolution RNA-seq detects changes in transition error rates in vitro Next, we separately compared each type of transition error between the two in vitro RNA samples representing the standard and the error-prone transcription conditions (Mg2+/Mn2+ plot, Figure 2, left column). We observed an up to 2-fold Mn2+-dependent increase in errors for G!A and T(U)!C transitions in a majority of cDNA positions in the error range from 3 104 to 6 105 b1. A nonparametric t-test between the two samples provided Figure 2. Scatter plots of transition-error rates. The error rates per position in the cDNA and internal control are plotted for error-prone/standard (left column), error-prone/error-proof (middle column) and moderate-error-proof/error-proof (right column) sets of conditions as shown on the top. The error rates 3 104 b1 were used for the statistical analysis. P value of two-tailed nonparametric t-test for the two samples is shown. For the cDNA, n = 132 (G!A), n = 142 (C!T), n = 104 (T!C) and n = 162 (A!G). For the internal control, n = 39 (G!A), n = 26 (C!T), n = 30 (T!C) and n = 30 (A!G). Figure 2. Scatter plots of transition-error rates. The error rates per position in the cDNA and internal control are plotted for error-prone/standard (left column), error-prone/error-proof (middle column) and moderate-error-proof/error-proof (right column) sets of conditions as shown on the top. The error rates 3 104 b1 were used for the statistical analysis. P value of two-tailed nonparametric t-test for the two samples is shown. For the cDNA, n = 132 (G!A), n = 142 (C!T), n = 104 (T!C) and n = 162 (A!G). For the internal control, n = 39 (G!A), n = 26 (C!T), n = 30 (T!C) and n = 30 (A!G). Nucleic Acids Research, 2013, Vol. 41, No. 19 9096 dependent increase in three types of transcription errors made by E. coli RNAP in vitro at the 105–104 b1 rates. rates might be caused by a modest cellular stress during shift to 42C for induction of PR promoter of the rpoBC gene (see ‘Materials and Methods’ section), decrease of intrinsic ﬁdelity of RNAP for certain types of errors at elevated temperature or due to a spontaneous cytosine deamination before or during RNA puriﬁcation from E. coli cells. Comparison of transition-error rates in vitro and in vivo It is broadly assumed that RNAP has similar intrinsic ﬁdelity in vivo and in vitro (7,17). However, in vitro ﬁdelity assessed by single NMP misincorporation assay does not account for error propagation to full-length RNA. Moreover, the in vitro ﬁdelity, deﬁned as a ratio of kpol/Kd for cognate and noncognate NTP (17,18), does not take into account for proofreading activity of RNAP that requires backtracking of the enzyme. The in vivo ﬁdelity could also be affected by local DNA struc- tures, DNA damage and promoter strength of the gene (1,54). Therefore, the in vitro ﬁdelity is not exactly related to the in vivo ﬁdelity. y To evaluate these differences, we used the RNA-seq to compare the error rates for the same RNA produced either in vitro or in vivo. Scatter plots visualized the differ- ences in transition-error rates between in vivo sample and in vitro Mg2+ samples ± GreA/B (Figure 3). In the cDNA positions, we observed signiﬁcant differences between the in vivo and the standard (+Mg2+) in vitro samples: C!T(U) transitions were overrepresented in the in vivo samples (P < 0.05), whereas G!A and T(U)!C transi- tions were underrepresented (P 0.05) (Figure 3). For G!A and T(U)!C transitions, addition of GreA/B to the in vitro reaction reduced the differences between the in vivo and in vitro samples (Figure 3). This result indicates an extensive RNA proofreading by GreA/B in the living cells as was suggested previously (55). Thus, our data suggest that transcription in the wild-type E. coli cells con- taining functional GreA/B proteins has similar ﬁdelity as transcription in vitro in the presence of Gre factors. In each cluster, we aligned the 9-nt sequences located immediately upstream from the G!A error site (Figure 4A). This was based on the assumption that the catalytic properties of RNAP are mainly determined in 9- bp RNA–DNA hybrid of a TEC (58). Interestingly, the RNA–DNA hybrid sequences for Mn2+-insensitive errors were strongly enriched with short A/T(U) tracts (group A in Figure 4A) as opposed to the more balanced sequence content of the sites affected by Mn2+ and GreA/B (the representative Mn2+-sensitive group F in Figure 4A). Backtracking controls mismatch extension We performed a hierarchical clustering analysis (57) of the error rates in all positions used for the statistical analysis of the errors at the lower than the threshold value, 3 104 b1. This analysis connects by a series of branches the DNA positions and the ﬂuctuation patterns/levels of error rates depending on transcription conditions. Thus, this analysis identiﬁes DNA positions exhibiting the similar error-rate proﬁles under the standard in vitro, error-prone Mn2+, error-proof GreA/B and the in vivo conditions. We chose G!A error because of its highest sensitivity to Mn2+ and GreA/B (Figure 2). The G!A errors were clustered into major C–G and minor A, B groups where the error rates were increased and not affected by Mn2+, respectively (Figure 4A). The majority of the Mn2+-sensitive errors in groups C–G was also reduced by GreA/B (Figure 4A). This signiﬁcant overlap strongly indicates transcription origin of these errors, which were susceptible to the chemical and protein factors speciﬁcally targeting transcription ﬁdelity. We further argue that the Mn2+-insensitive errors belonged to the RNA-seq artifacts that become more prominent at the sequences exhibiting relatively low transcription error rates. Alternatively, these se- quences might generate ‘true’ transcription errors with an intrinsic resistance to Mn2+. Note, that the averaged error rate in group A (1.1 104) was lower than in group B (1.8 104), suggesting that transcription errors from the former group are more diluted with the artifact errors. In each cluster, we aligned the 9-nt sequences located immediately upstream from the G!A error site (Figure 4A). This was based on the assumption that the catalytic properties of RNAP are mainly determined in 9- bp RNA–DNA hybrid of a TEC (58). Interestingly, the RNA–DNA hybrid sequences for Mn2+-insensitive errors were strongly enriched with short A/T(U) tracts (group A in Figure 4A) as opposed to the more balanced sequence content of the sites affected by Mn2+ and GreA/B (the representative Mn2+-sensitive group F in Figure 4A). A/U-rich sequences in the RNA–DNA hybrid have been shown to promote RNAP backtracking on DNA (59) as The high-resolution RNA-seq detects changes in transition error rates in vitro Although the in vivo frequency of a spontan- eous deamination of cytosines in DNA is known to occur at 109 order (56), the corresponding rate for the RNA is unknown. We also observed minor increases in the error rates for G!A and C!T(U) transitions in the internal controls for the in vivo sample compared with the standard in vitro sample (Figure 3). This was likely due to the errors introduced during the DNA oligonucleotides synthesis rather than the DNAP errors during PCR [see Supplementary Figure S2, the error rates for the oligo- DNA synthesis are slightly higher (by 1 104) than those of transcription for all four types of transition]. y To further validate the difference of the error rates in the Mn2+/Mg2+ samples, we added GreA/B to our standard reaction (Mg2+). GreA/B are expected to reduce errors by its proofreading activity (15,53). As expected, GreA/B ampliﬁed the differences in the rates of G!A, T(U)!C and C!T(U) transitions between the Mn2+ and Mg2+ samples (Figure 2, middle column), and signiﬁcantly reduced the means of the three transition-error rates in Mg2+ and Mn2+ samples (Figure 2, right column), indicating that we detected proofreading activity of GreA/B in both Mg2+ and Mn2+ conditions. GreA/B did not affect the error rates in the internal control. A!G transitions were also not affected by GreA/B in the cDNA and in the internal control regions, again suggesting the artifact origin of the majority of A!G errors. In a fraction of cDNA positions for the other three transition types, we also did not observe signiﬁcant changes in the error rates between the error-prone and error-proof tran- scription conditions (Figure 2). Comparison of transition-error rates in vitro and in vivo A/U-rich sequences in the RNA–DNA hybrid have been shown to promote RNAP backtracking on DNA (59) as The increased rate of C!T(U) transition in the in vivo sample was insensitive to GreA/B, suggesting that these errors may be introduced by DNAP during the ﬁve add- itional cycles of the ﬁrst PCR used only for cDNA syn- thesis with the in vivo RNA sample. The same increased background may dilute G!A and T(U)!C errors for the in vivo sample. The detected difference in C!T(U) error Nucleic Acids Research, 2013, Vol. 41, No. 19 9097 er plot of transition-error rates for in vivo and in vitro Mg2+ samples with (left) or without (right) ntrol (bottom). All symbols are the same as in Figure 2. The cutoff for the error rates is applied for tw scatter plots. The n for the t-test is same as in Figure 2. Figure 3. Scatter plot of transition-error rates for in vivo and in vitro Mg2+ samples with (left) or without (right) GreA/B in the cDNA (top) and internal control (bottom). All symbols are the same as in Figure 2. The cutoff for the error rates is applied for two-tailed nonparametric t-test, but not for the scatter plots. The n for the t-test is same as in Figure 2. 9098 Nucleic Acids Research, 2013, Vol. 41, No. 19 A B C D Figure 4. Mn2+-sensitivity and frequency of G!A errors depend on propensity of RNAP to backtrack at the error site. (A) Hierarchical clustering was performed with MeV v4.7.0. G!A error rates exceeding 3 104 b1 at 132 DNA positions are used to generate the clustering diagram. Each error rate is subtracted by the mean of ﬁve different RNA preparations to distinguish the error rate difference among the transcription conditions per position. Clusters A–G are indicated by boxes. The 10-nt DNA sequences (nontranscribed strand, 50-to-30 direction) where the G!A error occurred at the 30 RNA end are shown. The number on the left side of each sequence indicates position of G residue analyzed by the RNA-seq. Two sequences from clusters A and F (170 G and 474 G, respectively) that were used for biochemical analyses are underlined. (B) G!A transition rates at the positions 170 G and 474 G in the ﬁve RNA preparations analyzed by the RNA-seq. Comparison of transition-error rates in vitro and in vivo (C) Schematic representations of reversible backtracking of TEC18 bearing the 10-nt sequence of 170G or 474G from +10 to +19 position, where +1 is 50 end of the RNA. The access of Exo III form the rear end of RNAP is also shown. (D) ExoIII footprinting of the TEC18A and TEC18C. The reaction scheme is shown on the top. The rear-end boundaries of RNAP in the active and backtracked states are shown. The bottom panel shows the 18-nt RNA transcripts in the TECs. The capital letter following the number indicates the base of the 30 RNA and the RNA length in TEC. AMP-misincorporation at the position 19 is marked by asterisk. B D A B D A C C Figure 4. Mn2+-sensitivity and frequency of G!A errors depend on propensity of RNAP to backtrack at the error site. (A) Hierarchical clustering was performed with MeV v4.7.0. G!A error rates exceeding 3 104 b1 at 132 DNA positions are used to generate the clustering diagram. Each error rate is subtracted by the mean of ﬁve different RNA preparations to distinguish the error rate difference among the transcription conditions per position. Clusters A–G are indicated by boxes. The 10-nt DNA sequences (nontranscribed strand, 50-to-30 direction) where the G!A error occurred at the 30 RNA end are shown. The number on the left side of each sequence indicates position of G residue analyzed by the RNA-seq. Two sequences from clusters A and F (170 G and 474 G, respectively) that were used for biochemical analyses are underlined. (B) G!A transition rates at the positions 170 G and 474 G in the ﬁve RNA preparations analyzed by the RNA-seq. (C) Schematic representations of reversible backtracking of TEC18 bearing the 10-nt sequence of 170G or 474G from +10 to +19 position, where +1 is 50 end of the RNA. The access of Exo III form the rear end of RNAP is also shown. (D) ExoIII footprinting of the TEC18A and TEC18C. The reaction scheme is shown on the top. The rear-end boundaries of RNAP in the active and backtracked states are shown. The bottom panel shows the 18-nt RNA transcripts in the TECs. The capital letter following the number indicates the base of the 30 RNA and the RNA length in TEC. AMP-misincorporation at the position 19 is marked by asterisk. Comparison of transition-error rates in vitro and in vivo 19 9099 170 G as opposed to the 474 G sequence is responsible for the relatively lower error rate detected by the RNA-seq (see Supplementary text and Supplementary Figure S3). 170 G as opposed to the 474 G sequence is responsible for the relatively lower error rate detected by the RNA-seq (see Supplementary text and Supplementary Figure S3). cleavage was consistent with the ExoIII footprinting data (Figure 4D), showing backtracking of this complex at 6 bp distance. We propose that backtracking after the misincorporation generated a substrate for the cleavage in this complex with or without GreA/B. At the longest 6–24 min incubation time with noncognate ATP, Mn2+ also appeared to decrease extension of the 30 error (19 A* product) with the next cognate substrate (21 A* and 22 A** products) (Figure 5C and E). One would expect these opposite effects of Mn2+ on error correction and ex- tension to counteract one another leading to a net zero impact of Mn2+ to ﬁdelity. However, at the shorter 10–90 s, where an impact of the slow intrinsic RNA cleavage was negligible, Mn2+ stimulated rather than inhibited the error extension (Figure 5C and E). This effect was conﬁrmed in the experiment with the TEC Next, we tested if backtracking on 474 G sequence affects the G!A misincorporation in the presence of Mn2+ as was indicated by the clustering analysis. To mimic the G!A misincorporation at 474 G site during processive elongation, we measured the rate of AMP- misincorporation in TEC18C in the presence of Mg2+ or Mn2+ (Figure 5A and B). As expected, Mn2+-sensitive TEC18C misincorporated AMP more rapidly in the presence of Mn2+ than in the presence of Mg2+ (Figure 5C). Remarkably, we detected high level of an endonucleolytic RNA cleavage at 7-nt upstream of the 30 RNA end in the presence of Mg2+, and to a substantially less extent in the presence of Mn2+ (Figure 5C). This A B E C D Figure 5. Effects of backtracking on the efﬁciencies of mismatch extension (ME) and intrinsic transcript cleavage, and their dependences on Mn2+. (A) Reaction scheme for AMP misincorporation followed by ME. (B) RNA and downstream nontemplate DNA sequences in the TECs with long (18 nt) and short (8 nt) transcripts used in the assay. (C) Incubations of TEC18C/474 G with the noncognate ATP in the presence of Mg2+ or Mn2+. Comparison of transition-error rates in vitro and in vivo that digests DNA from the rear-end of RNAP (Figure 4C) produces the expanded rear-end boundary(ies) of the backtracked state(s), which converts to a boundary of the active state on prolonged incubation with the nuclease (44). We observed two differences in backtrack- ing at the 170 G and 474 G sequences (Figure 4D). TEC18A/170G was equilibrated between the active state and 1–10 bp stably backtracked states within 90 s of incu- bation with ExoIII, whereas TEC18C/474 G was equilibrated primarily in the active state with a minor 6- bp backtracked state. Thus, backtracking from the active state was more strongly induced or stabilized in TEC18A/ 170G compared with TEC18C/474 G as was predicted from the difference in their A/T(U) sequence contents (Figure 4A). Interestingly, AMP misincorporation in TEC18C/474 G (mimicking the G!A error during processive elongation) did not cause RNAP to advance 1 bp forward on the DNA (Figure 4D). This result indi- cates that the TEC19A remains in a 1 bp backtracked state after the misincorporation, which is consistent with the previous ﬁndings on the effect of misincorporation on backtracking (15,21). We concluded that the higher back- tracking potential and thus better proofreading on the one of the mechanisms increasing RNAP ﬁdelity (21). Thus, we assumed that a relatively low frequency of Mn2+-insensitive transcription errors was related to increased backtracking of RNAP. To address backtracking as a potential error-correcting mechanism during processive elongation, we arbitrarily selected one sequence from each group: 170G (Mn2+-in- sensitive group A, relatively lower error rate) and 474 G (Mn2+-sensitive group F, relatively high error rate) (Figure 4B) and analyzed RNAP backtracking at these sequences by ExoIII footprinting (17,44,60). A dynamic pattern of DNA digestion by ExoIII provides information of distance and stability for individual backtracked states of RNAP. The TEC was assembled with a 9-nt RNA hybridized to the DNA template containing 170 G or 474 G sequence with a modiﬁcation that is required for the TEC walking (see ‘Materials and Methods’ section) (37). The 9-nt RNA was elongated to 18-nt length with NTPs, making TEC18A (corresponding to the 170 G sequence) or 18C (corresponding to the 474 G sequence), which has the new 30RNA end located imme- diately 50 of the site where G!A error was detected (Figure 4C). When RNAP reversibly backtracks, ExoIII Nucleic Acids Research, 2013, Vol. 41, No. Error rate depends on the nucleotide at the 30 end of the transcript p p g g To prove that backtracking was the major error correc- tion mechanism at the 474 G site, we assembled a version of TEC18C/474 G but reducing the RNA length from 18 to 8 nt by removing 10 nt from the 50 end (TEC8C, Figure 5B). The shortening of the nascent RNA has been shown to prevent backtracking (44). Interestingly, elimination of backtracking dramatically enhanced G!A error and extension of the error with the next cognate AMP at this sequence (Figure 5C and D). The efﬁciency and the rate of mismatch extension increased 4 - and >10-fold, respectively, in TEC8C compared with the original TEC18C in the presence of Mg2+ or Mn2+ (Figure 5E). Thus, backtracking followed by error correc- tion by an intrinsic RNA cleavage represents a major mechanism for control of the G!A error at the 474 G site during processive transcription. The slow rate of the intrinsic cleavage at the 474 G site indicated that a sub- stantial fraction of the 30RNA misincorporation are not able to propagate to the full-length transcript due to back- track pausing of RNAP after misincorporation. Applying We noted that not all short A/T tracts followed by a 30 guanine residue identiﬁed by the RNA-seq exhibit low frequency of G!A errors, suggesting that high propensity for backtracking may not be the only parameter to control transcription ﬁdelity (12). In search of another ﬁdelity par- ameter embedded into sequence context, we aligned the sequences surrounding the G!A sites composed of the top 10% of the either lowest or highest error rate group, each of which was displayed by sequence logo (62,63). This analysis revealed a strong preference for adenine in n 1 position for the low error rate sites and cytosine in the same position for high error rate sites when n is a position for the error (Figure 6A). This analysis also revealed sequence preferences for n1 and n2 DNA pos- itions for the other types of transition errors (Supplementary Figure S4). Next, we tested if a residue in the DNA or the RNA in n 1 position affects RNAP misincorporation rate. Figure 6. A 30 residue in the nascent transcript determines the G!A error rate. (A) DNA logo derived from a sequence alignment around the dG residues coding for the low or high G!A error rate. Comparison of transition-error rates in vitro and in vivo Arrows indicate the original 18-nt RNA, misincorporation (marked by asterisks) and ME. (D) 50 RNA shortening to 8-nt length in TEC18C (making TEC8C) increases ME. (E) Quantiﬁcation of the ME (% of the total fraction in each detection time) from the panels C and D. The curves represent the single-exponential ﬁt of the data; apparent rate constants (k) are shown. Note for lager k in ‘Long, Mg2+’ compared with ‘Long, Mn2+’ condition: This difference is due to the intrinsic transcript cleavage of 19 A* product of misincorporation, which occurs substantially faster in Mg2+ compared with Mn2+. The faster cleavage in Mg2+ leads to apparent earlier than expected saturation of the ME reaction under these conditions. Although the plotting of ME appeared to follow single exponential kinetics, they result from a superposition of 3 different processes of 19 A* misincorporation, 19 A* cleavage and 19 A* extension with the next cognate NMP. B B A A C C E E E D D Figure 5. Effects of backtracking on the efﬁciencies of mismatch extension (ME) and intrinsic transcript cleavage, and their dependences on Mn2+. (A) Reaction scheme for AMP misincorporation followed by ME. (B) RNA and downstream nontemplate DNA sequences in the TECs with long (18 nt) and short (8 nt) transcripts used in the assay. (C) Incubations of TEC18C/474 G with the noncognate ATP in the presence of Mg2+ or Mn2+. Arrows indicate the original 18-nt RNA, misincorporation (marked by asterisks) and ME. (D) 50 RNA shortening to 8-nt length in TEC18C (making TEC8C) increases ME. (E) Quantiﬁcation of the ME (% of the total fraction in each detection time) from the panels C and D. The curves represent the single-exponential ﬁt of the data; apparent rate constants (k) are shown. Note for lager k in ‘Long, Mg2+’ compared with ‘Long, Mn2+’ condition: This difference is due to the intrinsic transcript cleavage of 19 A* product of misincorporation, which occurs substantially faster in Mg2+ compared with Mn2+. The faster cleavage in Mg2+ leads to apparent earlier than expected saturation of the ME reaction under these conditions. Although the plotting of ME appeared to follow single exponential kinetics, they result from a superposition of 3 different processes of 19 A* misincorporation, 19 A* cleavage and 19 A* extension with the next cognate NMP. 9100 Nucleic Acids Research, 2013, Vol. 41, No. Comparison of transition-error rates in vitro and in vivo 19 9100 the RNA-seq to nascent transcripts, isolated from back- tracked elongation complexes of RNAP containing a 30 error, by the previously established NET-seq method warrants addressing the effect of mismatch extension on the detected error rates (61). disregarding the cleavage activity described below. Thus, Mn2+ appeared to decrease transcription ﬁdelity on 474 G sequence by suppressing intrinsic RNA proofreading activity in the backtracked complex and by promoting ex- tension of the error with the next cognate NMP, which allowed the error propagation into a full-length RNA. Error rate depends on the nucleotide at the 30 end of the transcript Top lowest 10% (left) and top highest 10% (right) of all G!A error rates (<1 103) averaged by ﬁve different RNA preparations are used for the analysis. The residue frequencies from n 2 to n+1 (G!A error occurs at n site) were plotted with WebLogo (63). Y-axis is not shown as typical log base 2, but it represents the actual number to depict the residue types. (B) DNA/RNA scaffold for testing the effect of dC!dA substitution in the n 1 site of DNA. TEC18C (n 1 = C) and TEC18A (n 1 = A) on the 474 G sequence are shown. (C) Biochemical G!A error rates in TEC18C or 18 A as determined by NTP competition assay (see text for more details) (64,65). (D) Time course of AMP misincorporation for GMP in TEC18C or TEC18A. The curves represent the double exponential (TEC18C) or single expo- nential (TEC18A) ﬁt of the data; apparent rate constants (k) are shown. The slower misincorporation rate obtained from the double-exponential ﬁtting curve for TEC18C data was related to the intrinsic cleavage of 30 RNA in this complex. Figure 6. A 30 residue in the nascent transcript determines the G!A error rate. (A) DNA logo derived from a sequence alignment around the dG residues coding for the low or high G!A error rate. Top lowest 10% (left) and top highest 10% (right) of all G!A error rates (<1 103) averaged by ﬁve different RNA preparations are used for the analysis. The residue frequencies from n 2 to n+1 (G!A error occurs at n site) were plotted with WebLogo (63). Y-axis is not shown as typical log base 2, but it represents the actual number to depict the residue types. (B) DNA/RNA scaffold for testing the effect of dC!dA substitution in the n 1 site of DNA. TEC18C (n 1 = C) and TEC18A (n 1 = A) on the 474 G sequence are shown. (C) Biochemical G!A error rates in TEC18C or 18 A as determined by NTP competition assay (see text for more details) (64,65). (D) Time course of AMP misincorporation for GMP in TEC18C or TEC18A. The curves represent the double exponential (TEC18C) or single expo- nential (TEC18A) ﬁt of the data; apparent rate constants (k) are shown. Error rate depends on the nucleotide at the 30 end of the transcript The slower misincorporation rate obtained from the double-exponential ﬁtting curve for TEC18C data was related to the intrinsic cleavage of 30 RNA in this complex. Figure 6. A 30 residue in the nascent transcript determines the G!A error rate. (A) DNA logo derived from a sequence alignment around the dG residues coding for the low or high G!A error rate. Top lowest 10% (left) and top highest 10% (right) of all G!A error rates (<1 103) averaged by ﬁve different RNA preparations are used for the analysis. The residue frequencies from n 2 to n+1 (G!A error occurs at n site) were plotted with WebLogo (63). Y-axis is not shown as typical log base 2, but it represents the actual number to depict the residue types. (B) DNA/RNA scaffold for testing the effect of dC!dA substitution in the n 1 site of DNA. TEC18C (n 1 = C) and TEC18A (n 1 = A) on the 474 G sequence are shown. (C) Biochemical G!A error rates in TEC18C or 18 A as determined by NTP competition assay (see text for more details) (64,65). (D) Time course of AMP misincorporation for GMP in TEC18C or TEC18A. The curves represent the double exponential (TEC18C) or single expo- nential (TEC18A) ﬁt of the data; apparent rate constants (k) are shown. The slower misincorporation rate obtained from the double-exponential ﬁtting curve for TEC18C data was related to the intrinsic cleavage of 30 RNA in this complex. Nucleic Acids Research, 2013, Vol. 41, No. 19 9101 We used a previously developed NTP competition assay monitoring a single NMP misincorporation in the presence of a mixture of a cognate and noncognate NTP (64,65). We compared the G!A error rates in TEC18C/ 474 G and TEC18A/474 G carrying C18 (n 1)!A sub- stitution in DNA (Figure 6B, C and Supplementary Figure S5 A–C). As predicted by the sequence logo (Figure 6A), the C18 (n 1)!A substitution decreased the biochemical G19!A error rate in both Mg2+ and Mn2+ on 474 G sequence (Figure 6C) without affecting RNAP backtracking and intrinsic transcript cleavage (Supplementary Figure S5 D and E). Error rate depends on the nucleotide at the 30 end of the transcript The n 1 mutation also caused a 10-fold reduction of the rate of G19!A transcription error in a single AMP misincorporation assay lacking the cognate GTP (Figure 6D) Interestingly, the n 1 mutation stimulated AMP misincorporation without a strong effect on the cognate GMP incorporation (data not shown). This difference suggests that a chemical nature of the 30 RNA–DNA base pair plays a major role in binding or addition of a noncognate NTP with only minor contribution to the same processes with a cognate substrate. opportunity for application of the RNA-seq for evalu- ation of transition-type transcription errors in E. coli cells harboring viable greA/greB deletions, mutations in RNAP subunit that reduce ﬁdelity in vitro (19,66) and in the wild-type cells under different growth conditions including biological stresses/DNA damages (25,67). Although we did not detect any obvious hotspots (29) for the RNAP errors within the tested sequence, our results do not exclude that these hotspots exist genome- wide. The transversion errors by RNAP seem to occur with lower rates than transitions hindering their detec- tion by the RNA-seq (Supplementary Figure S6). The transversions appear to favor conversion to thymine or adenine rather than to cytosine or guanine (Supplementary Figure S6), suggesting that RNAP has preferences for transversion errors that are similar to RT and/or DNAP. / Although transcription ﬁdelity has been extensively studied by a single NMP incorporation in elongation complexes deprived of NTP substrates, the mechanism of ﬁdelity control during processive transcription awaits development of an appropriate methodology. Our new approach that combines RNA-seq and biochemical analyses of transcription errors propagating to the full- length RNA revealed two sequence-speciﬁc mechanisms used during processive transcription under physiological NTPs concentration: (i) NTP selection related to the chemical nature of DNA–RNA base pair immediately upstream from the error site, and (ii) postincorporation error correction by the intrinsic transcript cleavage in the backtracked RNAP (Figure 7). A recent biochemical study suggested that a noncognate NTP is rejected from RNAP by formation of a stressed sugar–phosphate backbone in the template DNA strand, which involves DISCUSSION The authors claimed transcription rather than an artiﬁcial origin of these errors by showing that the rate was increased to 1.7 103/bp in the mutant cell lacking Rpb9 subunit of RNAP II. Rpb9 is linked to transcription ﬁdelity based on the results of in vitro misincorporation assays (49). Surprisingly, deletion of yeast DST1 gene coding for RNA proofreading factor TFIIS (GreA/B analog) had almost no effect on the error rate in vivo (9). Once again, this result was different from our observation of a major impact of GreA/B proteins on transcription ﬁdelity in E. coli. A source of these differences requires additional investigation. It is worth mentioning that transcription errors at 105 error rate may have a deleterious effect on genome stabil- ity by inducing a prolonged stalling of RNAP at multiple sites across >105bp transcribed region in a genome. The irreversibly arrested TEC should block DNA replication and subsequent rounds of transcription leading to double- strand DNA breaks and cessations of gene expression (64,70). The prolonged RNAP stalling is exempliﬁed by almost irreversible loss of RNAP catalytic activity after an NMP misincorporation to TEC18, which was not accompanied by dissociation of RNAP from DNA (Figure 5). This mechanism is different from the previ- ously proposed production of toxic proteins due to tran- scription errors, which requires an assumption that error rate of transcription is comparable with that of transla- tion. Transcription misreading may have an impact on cell physiology comparable with translation misread- ing owing to multi-round translation of an erroneous mRNA molecule. q g The future application of the RNA-seq will allow moni- toring genome-wide transcription ﬁdelity under different growth conditions and in different cell types. Would 105 read depth (instead of 106 read depth used here) that covers 105 b1 frequency be sufﬁcient for detection of changes in transition-type transcription errors? This depth reduction could allow determination of ﬁdelity against 105 bases of transcriptome by a single Illumina sequencing analysis and lead to a signiﬁcant cost reduc- tion. We positively answer this question by showing that, for a voluntarily chosen position 578 of the rpoBC tran- script, G!A transition-error rate was not signiﬁcantly affected by a 10-fold decrease of the depth to 2 105 (Supplementary Figure S7). At this reduced depth, we suc- cessfully detected the responses of the error rates to Mn2+ and GreA/B in vitro. DISCUSSION We speculate that 30 rA-dT and 30 rC-dG base pairs could have an unequal stacking potential to differently affect the noncognate NTP rejec- tion. Further analysis is warranted for generalizing this sequence-speciﬁc mechanism for the NTP selection. When the preincorporation NTP selection fails, the enhanced backtracking that interferes with an extension of the 30 RNA mismatch provides an additional time to proofread the error by RNA cleavage. In this scenario, trans-acting factors that promote backtracking like DNA-bound proteins or nucleosomes may increase ﬁdelity, whereas factors that interfere with backtracking like trailing RNAP, ribosomes (in prokaryotes) or second- ary structure in the nascent RNA may decrease ﬁdelity. in vitro transcription samples and 16 cycles for the in vivo sample. However, we found that the in vivo sample had signiﬁcantly lower G!A and T(U)!C errors than the standard (Mg2+) in vitro sample, indicating no signiﬁcant contribution of the PCR artifacts to the types of transcrip- tion errors detected in this work (Figure 3 and Supplementary Figure S2). This suggests that such an increase in the PCR cycles appeared not to dilute tran- scription errors beyond the detection limit. Our data also clearly indicate that the most signiﬁcant technical im- provement allowing reduction of the artifact transition errors in the Illumina platform is a suppression of the errors during the second read of the paired-end sequencing, which typically occur with >3 104 b1 fre- quency (Figure 1D). The tag-based method (25–27) and overlapping read pairs method (30) have a strong poten- tial in identiﬁcation of the sequencing errors. Additional improvement of the RNA-seq bioinformatics has the po- tential to discriminate between transcription frame-shift errors (not analyzed in this work) and insertion/deletions artifacts associated with the Illumina platform and reads mapping. This approach may enable detection of physiologically relevant transcription slippage at short homopolymeric tracts and dinucleotide repeats broadly present in transcribed genes and contributing to slippage-associated diseases in humans (23,69). 5 y y y In eukaryotic transcription, Nesser et al. previously analyzed transcription errors throughout 450-bp cDNA of CAN1 transcript in yeast by Sanger sequencing (9). Their work reported a much higher 1.3 103/bp rate of substitutions compared to the rate observed for E. coli RNAP in our study and the rates determined for yeast RNAP II in vitro (17). DISCUSSION Our work provides the ﬁrst evidence that RNA-seq can assess physiological transcription error rates even in the presence of artifact errors. We directly detected changes in the transition-error rates in the range of 4 105 to 3 104 b1 (Figures 2 and 3). These limits identify a lower baseline of the standard transition-error rates at 105 order or less. Our ﬁndings that GreA/B increases the ﬁdelity of processive transcription in vitro to a level of the ﬁdelity in vivo (Figures 2 and 3) provide an ample Figure 7. Multiple pathways for control of RNAP ﬁdelity. Transcription error rate is determined by the 30 RNA–DNA base pair in TEC (preincorporation substrate selection) and by backtracking propensity of RNAP (postincorporation proofreading). The 30 RNA–DNA base pair controls misincorporation rate of a noncognate substrate (indicated by an asterisk). The DNA sequences such as A/T-rich tracts and protein factors that promote backtracking increase ﬁdelity by decreasing extension of the 30 RNA error with the next cognate NMP (shaded). The error is corrected by the intrinsic or Gre-assisted transcript cleavage in backtacked TEC. The irreversible backtrack arrest of TEC carrying the 30 RNA error may derive from the inefﬁcient transcript cleavage in the backtracked complex (the dead-end pathway). Figure 7. Multiple pathways for control of RNAP ﬁdelity. Transcription error rate is determined by the 30 RNA–DNA base pair in TEC (preincorporation substrate selection) and by backtracking propensity of RNAP (postincorporation proofreading). The 30 RNA–DNA base pair controls misincorporation rate of a noncognate substrate (indicated by an asterisk). The DNA sequences such as A/T-rich tracts and protein factors that promote backtracking increase ﬁdelity by decreasing extension of the 30 RNA error with the next cognate NMP (shaded). The error is corrected by the intrinsic or Gre-assisted transcript cleavage in backtacked TEC. The irreversible backtrack arrest of TEC carrying the 30 RNA error may derive from the inefﬁcient transcript cleavage in the backtracked complex (the dead-end pathway). Nucleic Acids Research, 2013, Vol. 41, No. 19 9102 angling of a 30 RNA–DNA base pair to align the NTP for catalysis (16). The authors argued that the angling may weaken stacking of the 30 base pair and ribose contacts with a noncognate NTP to induce its preferential rejection from the active center without a signiﬁcant effect on the properly paired cognate NTP. ACCESSION NUMBERS GEO number: GSE46479. SUPPLEMENTARY DATA Supplementary Data are available at NAR online. Supplementary Data are available at NAR online. DISCUSSION Thus, the 105 read depth appears to be sufﬁcient to assess increases in transition errors from the basal level across an entire transcriptome with caution that the sensitivity to transcription error is varied by mRNA levels among different genes. Another potential issue for the genome-wide RNA-seq is the additional PCR cycles and barcoding bias (68), accompanied by the adapter ligation to cDNA during the library preparation. Our cDNA preparation included 11 cycles of PCR for the REFERENCES 22. Wagner,L.A., Weiss,R.B., Driscoll,R., Dunn,D.S. and Gesteland,R.F. (1990) Transcriptional slippage occurs during elongation at runs of adenine or thymine in Escherichia coli. Nucleic Acids Res., 18, 3529–3535. 1. Strathern,J.N., Jin,D.J., Court,D.L. and Kashlev,M. (2012) Isolation and characterization of transcription ﬁdelity mutants. Biochim. Biophys. Acta, 1819, 694–699. 2. Gordon,A.J., Halliday,J.A., Blankschien,M.D., Burns,P.A., Yatagai,F. and Herman,C. (2009) Transcriptional inﬁdelity promotes heritable phenotypic change in a bistable gene network. PLoS Biol., 7, e44. 23. Baranov,P.V., Hammer,A.W., Zhou,J., Gesteland,R.F. and Atkins,J.F. (2005) Transcriptional slippage in bacteria: distribution in sequenced genomes and utilization in IS element gene expression. Genome Biol., 6, R25. 3. Goldsmith,M. and Tawﬁk,D.S. (2009) Potential role of phenotypic mutations in the evolution of protein expression and stability. Proc. Natl Acad. Sci. USA, 106, 6197–6202. 24. Kircher,M., Heyn,P. and Kelso,J. (2011) Addressing challenges in the production and analysis of illumina sequencing data. BMC Genomics, 12, 382. 4. Paoloni-Giacobino,A., Rossier,C., Papasavvas,M.P. and Antonarakis,S.E. (2001) Frequency of replication/transcription errors in (A)/(T) runs of human genes. Hum. Genet., 109, 40–47. 25. Schmitt,M.W., Kennedy,S.R., Salk,J.J., Fox,E.J., Hiatt,J.B. and Loeb,L.A. (2012) Detection of ultra-rare mutations by next- generation sequencing. Proc. Natl Acad. Sci. USA, 109, 14508–14513. 5. Rodin,S.N., Rodin,A.S., Juhasz,A. and Holmquist,G.P. (2002) Cancerous hyper-mutagenesis in p53 genes is possibly associated with transcriptional bypass of DNA lesions. Mutat. Res., 510, 153–168. 26. Kinde,I., Wu,J., Papadopoulos,N., Kinzler,K.W. and Vogelstein,B. (2011) Detection and quantiﬁcation of rare mutations with massively parallel sequencing. Proc. Natl Acad. Sci. USA, 108, 9530–9535. 6. Hubbard,K., Catalano,J., Puri,R.K. and Gnatt,A. (2008) Knockdown of TFIIS by RNA silencing inhibits cancer cell proliferation and induces apoptosis. BMC Cancer, 8, 133. 27. Jabara,C.B., Jones,C.D., Roach,J., Anderson,J.A. and Swanstrom,R. (2011) Accurate sampling and deep sequencing of the HIV-1 protease gene using a Primer ID. Proc. Natl Acad. Sci. USA, 108, 20166–20171. 7. Blank,A., Gallant,J.A., Burgess,R.R. and Loeb,L.A. (1986) An RNA polymerase mutant with reduced accuracy of chain elongation. Biochemistry, 25, 5920–5928. 28. Ji,J.P. and Loeb,L.A. (1992) Fidelity of HIV-1 reverse transcriptase copying RNA in vitro. Biochemistry, 31, 954–958. 8. Taddei,F., Hayakawa,H., Bouton,M., Cirinesi,A., Matic,I., Sekiguchi,M. and Radman,M. (1997) Counteraction by MutT protein of transcriptional errors caused by oxidative damage. Science, 278, 128–130. 29. Hu,W.S. and Hughes,S.H. (2012) HIV-1 reverse transcription. Cold Spring Harb. Perspect. Med., 2, a006882. 30. Chen-Harris,H., Borucki,M.K., Torres,C., Slezak,T.R. and Allen,J.E. (2013) Ultra–deep mutant spectrum proﬁling: improving sequencing accuracy using overlapping read pairs. BMC Genomics, 14, 96. 9. Nesser,N.K., Peterson,D.O. and Hawley,D.K. FUNDING Fellowship from JSPS (to M.I. in part); Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (to M.K). Funding for open access charge: The Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. Fellowship from JSPS (to M.I. in part); Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (to M.K). Funding for open access charge: The Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. 18. Roghanian,M., Yuzenkova,Y. and Zenkin,N. (2011) Controlled interplay between trigger loop and Gre factor in the RNA polymerase active centre. Nucleic Acids Res., 39, 4352–4359. 19. Holmes,S.F., Santangelo,T.J., Cunningham,C.K., Roberts,J.W. and Erie,D.A. (2006) Kinetic investigation of Escherichia coli RNA polymerase mutants that inﬂuence nucleotide discrimination and transcription ﬁdelity. J. Biol. Chem., 281, 18677–18683. 20. Bar-Nahum,G., Epshtein,V., Ruckenstein,A.E., Raﬁkov,R., Mustaev,A. and Nudler,E. (2005) A ratchet mechanism of transcription elongation and its control. Cell., 120, 183–193. Conﬂict of interest statement. None declared. Conﬂict of interest statement. None declared. 21. Shaevitz,J.W., Abbondanzieri,E.A., Landick,R. and Block,S.M. (2003) Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature, 426, 684–687. ACKNOWLEDGEMENTS We thank D.L. Court of NCI for discussions and critical reading of the manuscript; W. Tang, T.D. Schneider and Y. Zhao of NCI and T. Sakamoto of NAIST for technical comments and support in sequence analysis; S.H. Hughes, Nucleic Acids Research, 2013, Vol. 41, No. 19 9103 Nucleic Acids Research, 2013, Vol. 41, No. 19 9103 M.L. Kireeva, S. Kakar, M. Bubunenko and J.N. Strathern of NCI for discussions and comments on the manuscript; W. Shao of NCI for the Perl script. We also thank NCI sequencing facility for Illumina sequencing and early bioinfomaic analysis. 15. Erie,D.A., Hajiseyedjavadi,O., Young,M.C. and von Hippel,P.H. (1993) Multiple RNA polymerase conformations and GreA: control of the ﬁdelity of transcription. Science, 262, 867–873. 16. Sosunova,E., Sosunov,V., Epshtein,V., Nikiforov,V. and Mustaev,A. (2013) Control of transcriptional ﬁdelity by active center tuning as derived from RNA polymerase endonuclease reaction. J. Biol. Chem., 288, 6688–6703. 17. Kireeva,M.L., Nedialkov,Y.A., Cremona,G.H., Purtov,Y.A., Lubkowska,L., Malagon,F., Burton,Z.F., Strathern,J.N. and Kashlev,M. (2008) Transient reversal of RNA polymerase II active site closing controls ﬁdelity of transcription elongation. Mol. Cell, 30, 557–566. Conﬂict of interest statement. None declared. REFERENCES (2006) RNA polymerase II subunit Rpb9 is important for transcriptional ﬁdelity in vivo. Proc. Natl Acad. Sci. USA, 103, 3268–3273. y 10. Shaw,R.J., Bonawitz,N.D. and Reines,D. (2002) Use of an in vivo reporter assay to test for transcriptional and translational ﬁdelity in yeast. J. Biol. Chem., 277, 24420–24426. 31. Ramaswami,G., Zhang,R., Piskol,R., Keegan,L.P., Deng,P., O’Connell,M.A. and Li,J.B. (2013) Identifying RNA editing sites using RNA sequencing data alone. Nat. Methods, 10, 128–132. 11. Larson,M.H., Zhou,J., Kaplan,C.D., Palangat,M., Kornberg,R.D., Landick,R. and Block,S.M. (2012) Trigger loop dynamics mediate the balance between the transcriptional ﬁdelity and speed of RNA polymerase II. Proc. Natl Acad. Sci. USA, 109, 6555–6560. 32. Li,M., Wang,I.X., Li,Y., Bruzel,A., Richards,A.L., Toung,J.M. and Cheung,V.G. (2011) Widespread RNA and DNA sequence differences in the human transcriptome. Science, 333, 53–58. 33. Lin,W., Piskol,R., Tan,M.H. and Li,J.B. (2012) Comment on ‘‘Widespread RNA and DNA sequence differences in the human transcriptome’’. Science, 335, 1302; author reply 1302. 12. Sydow,J.F., Brueckner,F., Cheung,A.C., Damsma,G.E., Dengl,S., Lehmann,E., Vassylyev,D. and Cramer,P. (2009) Structural basis of transcription: mismatch-speciﬁc ﬁdelity mechanisms and paused RNA polymerase II with frayed RNA. Mol. Cell, 34, 710–721. 34. Kleinman,C.L. and Majewski,J. (2012) Comment on ‘‘Widespread RNA and DNA sequence differences in the human transcriptome’’. Science, 335, 1302; author reply 1302. 13. Kashkina,E., Anikin,M., Brueckner,F., Pomerantz,R.T., McAllister,W.T., Cramer,P. and Temiakov,D. (2006) Template misalignment in multisubunit RNA polymerases and transcription ﬁdelity. Mol. Cell, 24, 257–266. 35. Pickrell,J.K., Gilad,Y. and Pritchard,J.K. (2012) Comment on ‘‘Widespread RNA and DNA sequence differences in the human transcriptome’’. Science, 335, 1302; author reply 1302. 14. Rosenberger,R.F. and Hilton,J. (1983) The frequency of transcriptional and translational errors at nonsense codons in the lacZ gene of Escherichia coli. Mol. Gen. Genet., 191, 207–212. 36. Abram,M.E., Ferris,A.L., Shao,W., Alvord,W.G. and Hughes,S.H. (2010) Nature, position, and frequency of mutations 9104 Nucleic Acids Research, 2013, Vol. 41, No. 19 9104 made in a single cycle of HIV-1 replication. J. Virol., 84, 9864–9878. 54. Nakano,T., Ouchi,R., Kawazoe,J., Pack,S.P., Makino,K. and Ide,H. (2012) T7 RNA polymerases backed up by covalently trapped proteins catalyze highly error prone transcription. J. Biol. Chem., 287, 6562–6572. 37. Kashlev,M., Nudler,E., Severinov,K., Borukhov,S., Komissarova,N. and Goldfarb,A. (1996) Histidine-tagged RNA polymerase of Escherichia coli and transcription in solid phase. Methods Enzymol., 274, 326–334. 55. Stepanova,E., Lee,J., Ozerova,M., Semenova,E., Datsenko,K., Wanner,B.L., Severinov,K. and Borukhov,S. (2007) Analysis of promoter targets for Escherichia coli transcription elongation factor GreA in vivo and in vitro. J. Bacteriol., 189, 8772–8785. y 38. REFERENCES Borukhov,S. and Goldfarb,A. (1996) Puriﬁcation and assay of Escherichia coli transcript cleavage factors GreA and GreB. Methods Enzymol., 274, 315–326. 56. Drake,J.W. (1991) A constant rate of spontaneous mutation in DNA-based microbes. Proc. Natl Acad. Sci. USA, 88, 7160–7164. 39. Kashlev,M.V., Bass,I.A., Lebedev,A.N., Kaliaeva,E.S. and Nikiforov,V.G. (1989) [Deletion-insertion mapping of the region non-essential for functioning of the beta-subunit of Escherichia coli RNA polymerase]. Genetika, 25, 396–405. coli RNA polymerase]. Genetika, 25, 396–405. 57. Eisen,M.B., Spellman,P.T., Brown,P.O. and Botstein,D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA, 95, 14863–14868. 40. Langmead,B., Trapnell,C., Pop,M. and Salzberg,S.L. (2009) Ultrafast and memory-efﬁcient alignment of short DNA sequences to the human genome. Genome Biol., 10, R25. 58. Komissarova,N. and Kashlev,M. (1998) Functional topography of nascent RNA in elongation intermediates of RNA polymerase. Proc. Natl Acad. Sci. USA, 95, 14699–14704. 41. Li,H., Handsaker,B., Wysoker,A., Fennell,T., Ruan,J., Homer,N., Marth,G., Abecasis,G. and Durbin,R. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics, 25, 2078–2079. 59. Komissarova,N., Becker,J., Solter,S., Kireeva,M. and Kashlev,M. (2002) Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination. Mol. Cell, 10, 1151–1162. 42. Kireeva,M.L., Lubkowska,L., Komissarova,N. and Kashlev,M. (2003) Assays and afﬁnity puriﬁcation of biotinylated and nonbiotinylated forms of double-tagged core RNA polymerase II from Saccharomyces cerevisiae. Methods Enzymol., 370, 138–155. 60. Artsimovitch,I., Chu,C., Lynch,A.S. and Landick,R. (2003) A new class of bacterial RNA polymerase inhibitor affects nucleotide addition. Science, 302, 650–654. 43. Komissarova,N., Kireeva,M.L., Becker,J., Sidorenkov,I. and Kashlev,M. (2003) Engineering of elongation complexes of bacterial and yeast RNA polymerases. Methods Enzymol., 371, 233–251. 61. Churchman,L.S. and Weissman,J.S. (2011) Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature, 469, 368–373. 44. Imashimizu,M., Kireeva,M.L., Lubkowska,L., Gotte,D., Parks,A.R., Strathern,J.N. and Kashlev,M. (2013) Intrinsic Translocation Barrier as an Initial Step in Pausing by RNA Polymerase II. J. Mol. Biol., 425, 697–712. 62. Schneider,T.D. and Stephens,R.M. (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res., 18, 6097–6100. 63. Crooks,G.E., Hon,G., Chandonia,J.M. and Brenner,S.E. (2004) WebLogo: a sequence logo generator. Genome Res., 14, 1188–1190. 45. Sousa,R. (1996) Structural and mechanistic relationships between nucleic acid polymerases. Trends Biochem. Sci., 21, 186–190. 64. Walmacq,C., Cheung,A.C., Kireeva,M.L., Lubkowska,L., Ye,C., Gotte,D., Strathern,J.N., Carell,T., Cramer,P. and Kashlev,M. (2012) Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage. Mol Cell, 46, 1–12. 46. Ewing,B. and Green,P. REFERENCES (1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res., 8, 186–194. 47. Grant,G.R., Farkas,M.H., Pizarro,A.D., Lahens,N.F., Schug,J., Brunk,B.P., Stoeckert,C.J., Hogenesch,J.B. and Pierce,E.A. (2011) Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq uniﬁed mapper (RUM). Bioinformatics, 27, 2518–2528. 65. Kireeva,M.L., Opron,K., Seibold,S.A., Domecq,C., Cukier,R.I., Coulombe,B., Kashlev,M. and Burton,Z.F. (2012) Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase. BMC Biophys., 5, 11. 48. Niyogi,S.K. and Feldman,R.P. (1981) Effect of several metal ions on misincorporation during transcription. Nucleic Acids Res., 9, 2615–2627. 66. Nedialkov,Y.A., Opron,K., Assaf,F., Artsimovitch,I., Kireeva,M.L., Kashlev,M., Cukier,R.I., Nudler,E. and Burton,Z.F. (2013) The RNA polymerase bridge helix YFI motif in catalysis, ﬁdelity and translocation. Biochim. Biophys. Acta, 1829, 187–198. 49. Walmacq,C., Kireeva,M.L., Irvin,J., Nedialkov,Y., Lubkowska,L., Malagon,F., Strathern,J.N. and Kashlev,M. (2009) Rpb9 subunit controls transcription ﬁdelity by delaying NTP sequestration in RNA polymerase II. J. Biol. Chem., 284, 19601–19612. 67. Saxowsky,T.T., Meadows,K.L., Klungland,A. and Doetsch,P.W. (2008) 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells. Proc. Natl Acad. Sci. USA, 105, 18877–18882. 50. Imashimizu,M., Tanaka,K. and Shimamoto,N. (2011) Comparative study of cyanobacterial and E. coli RNA polymerases: misincorporation, abortive transcription, and dependence on divalent cations. Genet. Res. Int., 2011, 572689. 68. Alon,S., Vigneault,F., Eminaga,S., Christodoulou,D.C., Seidman,J.G., Church,G.M. and Eisenberg,E. (2011) Barcoding bias in high-throughput multiplex sequencing of miRNA. Genome Res., 21, 1506–1511. pendence on divalent cations. Genet. Res. Int., 2011, 572 51. Borukhov,S., Sagitov,V. and Goldfarb,A. (1993) Transcript cleavage factors from E. coli. Cell., 72, 459–466. 52. Petersen,C. and Moller,L.B. (2000) Invariance of the nucleoside triphosphate pools of Escherichia coli with growth rate. J. Biol. Chem., 275, 3931–3935. 69. Atkins,J.F. and Bjork,G.R. (2009) A gripping tale of ribosomal frameshifting: extragenic suppressors of frameshift mutations spotlight P-site realignment. Microbiol. Mol. Biol. Rev., 73, 178–210. 53. Orlova,M., Newlands,J., Das,A., Goldfarb,A. and Borukhov,S. (1995) Intrinsic transcript cleavage activity of RNA polymerase. Proc. Natl Acad. Sci. USA, 92, 4596–4600. 70. Dutta,D., Shatalin,K., Epshtein,V., Gottesman,M.E. and Nudler,E. (2011) Linking RNA polymerase backtracking to genome instability in E. coli. Cell, 146, 533–543.
https://openalex.org/W2971288684	https://europepmc.org/articles/pmc7851030?pdf=render	English	null	Underlying disease determines the risk of an open abdomen treatment, final closure, however, is determined by the surgical abdominal history	European journal of trauma and emergency surgery	2,019	cc-by	5,504	Abbreviations DCS Damage control surgery ACS Abdominal compartment syndrome EAF Entero-atmospheric fistula TAC Temporary abdominal closure DCS Damage control surgery ACS Abdominal compartment syndrome EAF Entero-atmospheric fistula TAC Temporary abdominal closure DCS Damage control surgery ACS Abdominal compartment syndrome EAF Entero-atmospheric fistula TAC Temporary abdominal closure DCS Damage control surgery ACS Abdominal compartment syndrome EAF Entero-atmospheric fistula TAC Temporary abdominal closure * Steffi Karhof S.Karhof@umcutrecht.nl European Journal of Trauma and Emergency Surgery (2021) 47:113–120 https://doi.org/10.1007/s00068-019-01205-2 European Journal of Trauma and Emergency Surgery (2021) 47:113–120 https://doi.org/10.1007/s00068-019-01205-2 ORIGINAL ARTICLE ORIGINAL ARTICLE Underlying disease determines the risk of an open abdomen treatment, final closure, however, is determined by the surgical abdominal history Steffi Karhof1 · Mark Haverkort1 · Rogier Simmermacher1 · Falco Hietbrink1 · Luke Leenen Received: 29 April 2019 / Accepted: 10 August 2019 / Published online: 26 August 2019 © The Author(s) 2019 1 Department of Surgery, University Medical Centre Utrecht, Utrecht, The Netherlands Abstract Introduction Temporary abdominal closure is frequently used in several situations such as abbreviated surgery in damage control situations or when closing is impossible due to organ distention or increased abdominal pressure. The ultimate goal is to eventually close the fascia; however, little is known about factors predicting abdominal closure. The purpose of this study was to identify characteristics associated with the need for open abdomen as well as indicating the possibility of delayed fascial closure after a period of open abdominal treatment. Methods A retrospective review of all patients that underwent midline laparotomy between January 2008 and December 2012 was performed. Both factors predicting open abdominal treatment and possibility to close the fascia afterwards were identified and analyzed by univariate and multivariate analyses. i Results 775 laparotomies in 525 patients (60% male) were included. 109 patients (21%) had an open abdomen with a mortal- ity rate of 27%. Male gender and acidosis were associated with open abdominal treatment. In 54%, the open abdomen could be closed by delayed fascial closure. The number of laparotomies both before and during temporary abdominal treatment was associated with failure of closure. Conclusion In this study, male sex and physiological derangement, reflected by acidosis, were independent predictors of open abdominal treatment. Furthermore, the success of delayed fascial closure depends on number of abdominal surgical procedures. Moreover, based on our experiences, we suggest to change modalities early on, to prevent multiple fruitless attempts to close the abdomen. Keywords Open abdominal treatment · Temporary abdominal closure · Open abdomen · Delayed fas abdomen open after an index operation; first of all, leaving an abdomen open can be necessary in critically ill patients as part of damage control surgery (DCS). This abbreviated surgery limits operating time in favor of physiological recov- ery in ICU. Another reason for leaving the abdomen open in seriously ill patients is the development of abdominal com- partment syndrome (ACS), where distention of organs, from any origin, mostly due to resuscitation, can lead to increased intra-abdominal pressure, often preventing closure of the abdominal wall for mechanical reasons. Besides that, there are patients in whom it is not desirable to close an abdomen, because of local abdominal disease, for example, in patients in whom an early second look is warranted to re-evaluate the intra-abdominal contents. Introduction Temporary abdominal closure (TAC) as a regular step in sur- gical treatment has increasingly been accepted over the past decades. There are three main indications for leaving the Historically, patients with a septic abdomen who under- went a planned re-laparotomy because of alleged gross con- tamination used to be another indication of open abdomi- nal treatment [1–6]. However, a randomized clinical trial (0121 114 S. Karhof et al. data including gender, age, BMI, ASA classification, comor- bidities including history of smoking or alcohol abuse, and indication for laparotomy were recorded, as well as inter- ventional data (timing, closure technique, open abdominal treatment including its indication and duration of TAC). Furthermore, physiological data such as pH, lactate and base deficit were collected. Mortality and morbidity, e.g., development of entero-atmospheric fistula (EAF) or ventral hernias, were evaluated for all patients during follow-up at our hospital. CT scans were not part of standard postopera- tive care. Data on morbidities were collected either during hospital stay or during follow-up in clinic, with a standard follow-up of 6 weeks after surgery and further visits at phy- sician’s discretion. comparing on-demand with planned re-laparotomy strategy in patients with severe peritonitis did not reveal any dif- ferences on death or major peritonitis-related morbidity between both groups [7]. Hence, there is no reason anymore for leaving the abdomen open after abdominal sepsis when the fascia can be immediately closed. If temporary closure is warranted, there are several tech- niques to temporarily close the abdomen, although consen- sus is lacking [1, 4, 8–10]. Ideally, TAC techniques should, amongst other features, protect the bowel and avoid further fascial edge retraction, prevent ACS, limit contamination, prevent third space fluid losses, avoid formation of adhe- sions, provide support for ventilation and should be cost effective [2, 4]. Final goal of temporary abdominal closure after cessation of its pathophysiologic origin is delayed pri- mary closure within a reasonable window rather than delib- erately ending up in a ventral abdominal hernia [1]. Previous studies suggest closing the abdomen within 8 days, since after this period, a significant increase in complications has been found [1, 3]. Little is known about the factors that influ- ence and predict successful delayed primary closure [11], although previously it was shown that duration of treatment as well as the physiological state of the patient influences outcome in this patient population [12]. Materials and methods A retrospective study of all patients who underwent a lapa- rotomy in our department of surgery in a University teach- ing hospital between January 2008 and December 2012 was performed. University Medical Center Utrecht is a large teaching hospital and the only Level-1 trauma center in the province of Utrecht. It covers the central region of the Neth- erlands with a relatively small, but densely populated service area of 1500 km2 and approximately 1.3 million residents. Surgical technique of temporary abdominal closure In our hospital, a modified version of the vacuum pack to temporarily cover the open abdomen is used. This method was first described by Barker et al. [13] and uses multiple gauzes or a sterile surgical gown, wrapped in OpsiteTM (Smith & Nephew Inc, St. Petersburg, FL, USA) adhesive film to cover the abdominal contents. Over this pack, two holed drains are placed which is then covered with another Opsite adhesive film. The drains are connected with a Y piece and attached to a negative pressure suction pump cre- ating a vacuum. Typically, after 24–72 h the VAC-pack will be removed for second-look surgery and possible fascial closure. When it is not possible or not favorable to close the abdomen, the same TAC technique will be used again. When delayed primary closure is not deemed feasible in the near future, a mesh to temporarily cover the abdomen can be used. This decision, including the timing to do so, is based on the clinical situation and at the surgeon’s discretion. In trauma patients a dedicated flowchart is used to aid in delayed fascial closure after damage control surgery (Fig. 1). The goal of this study was to identify patient and lapa- rotomy characteristics associated with both the need for an open abdominal treatment and characteristics indicating the possibility of delayed fascial closure, after a period of open abdominal treatment. Introduction Also, the indication of abdominal surgery and the reason for the open abdomen are associated with the odds to eventually close the abdo- men, with previous studies suggesting abdominal sepsis has worse outcome on closure rates [5, 6]. Definitions of abdominal closure Abdominal closure was divided into three categories. When the fascia was closed at the first intervention it was defined as ‘primary closure’, when the fascia was closed after a period of TAC, it was defined as ‘delayed fascial closure’ and when closure of the fascia was not possible and the wound healed over a mesh, it was called ‘secondary heal- ing’ (or definitive open abdomen). All planned and emergency laparotomies by midline incision in adult patients were included. All laparoscopic procedures (approximately 500 annually) performed dur- ing the same period were not considered in this analysis; however, converted laparoscopic procedures to a laparotomy were included. Exclusion criteria were all other abdominal incisions than midline. Data were collected from computer- ized medical records through ICD codes. Demographical 3 Statistical analysis All statistical analyses were conducted using IBM SPSS Sta- tistics 22.0 (IBM Corporation, Armonk, NY, USA). All con- tinuous non-parametric variables were reported in medians 3 3 Underlying disease determines the risk of an open abdomen treatment, final closure, however,… 115 Fig. 1 Algorithm to cover the open abdomen Fig. 1 Algorithm to cover the open abdomen with interquartile ranges. Discrete variables were displayed as proportions. Bivariate analysis was performed using the χ² and Fisher’s exact test, respectively. Kruskal–Wallis one- way analysis of variance test was applied for comparisons between more than two independent groups. All variables that were tested in univariate analysis were also evaluated in a multivariate logistic regression analysis. These variables were analyzed with both forward and backward stepwise selection to identify independent risk factors for failure to close the abdomen. These outcomes were presented as odds ratios and 95% confidence interval. p values below 0.05 were considered statistically significant. Table 1 Patient demographics All data are expressed in median (IQR) or absolute numbers (%) a Unknown in 14 patients Patient demographics N (%), total n = 525 Sex, male (%) 313 (60) Age 61 (48–70) BMI 24.4 (21.7–27.7) ASA classification 2 (2–3) Comorbidities Diabetes (total n = 514), yes (%) 72 (14) Cardiovascular (total n = 511), yes (%) 197 (39) Pulmonary (total n = 520), yes (%) 83 (16) History of abdominal surgery (total n = 512), yes (%) 267 (52) Total patients with comorbidities (%)a 371 (73) Smoking (total n = 339), yes (%) 107 (32) Alcohol abuse (total n = 341), yes (%) 115 (34) Table 1 Patient demographics Results Between January 2008 and December 2012, a total of 525 patients underwent 775 laparotomies. The majority of patients were male (60%) with a median age of 61 years (Table 1). Three hundred seventy-one patients (71%) had at least one co-morbidity. Most patients had previous abdominal surgery (267; 52%) or cardiovascular history (197; 39%). The indication for laparotomy varied. The most common cause for surgery was gastro-intestinal in 593 patients (76.5%), followed by 122 patients (15.7%) after trauma and the remaining 60 patients (7.7%) had a laparotomy for vascular surgery (Table 2). There were 35 patients (7%) in whom a planned re-laparotomy was 1 3 116 S. Karhof et al. gastro-intestinal and p 0.006 for vascular surgery), male gen- der (p ≤ 0.001), previous abdominal surgery (p = 0.001), pH (p = 0.003), lactate (p ≤ 0.001) and base deficit (p = 0.001) (Table 2). When comparing all these variables (indication for laparotomy, timing of surgery, gender, previous abdomi- nal surgery, ASA classification, pH, lactate and base deficit) in a multivariate analysis, only gastro-intestinal pathology as indication for laparotomy, gender and pH remained as independent factors associated with open abdominal treat- ment. Further, male patients had a 3.4-time higher chance of open abdominal treatment than female patients (Table 2). Of all patients following laparotomy the median hospital length of stay was 23 days, with a significant longer stay for patients following TAC of 46 days (Table 3). The same goes for ICU length of stay which was 11.5 days for patients following TAC and 0 days for patients following primary closure (Table 3). Complication rates were comparable in performed: 17 patients had a second look after abdominal packing, 11 patients for evaluation of on-going ischemia after bowel ischemia and 7 patients underwent second- look surgery for abdominal sepsis. In most patients (416; 79%), the abdomen could be pri- marily closed, leaving a total of 109 patients (21%) for temporary abdominal closure (TAC). In 45 patients (41%), the abdomen was left open on account of DCS in trauma patients. In 37 patients (34%), the reason for TAC was abdominal sepsis, with the remaining 27 (25%) due to ACS (Table 2). The majority of patients (102; 93.6%) in whom the abdomen could not be primarily closed underwent sur- gery in an acute setting (Table 2). Results When comparing characteristics of the patients in whom the abdomen was primarily closed to the TAC group in univariate analysis, significant indicative parameters were indications for laparotomy (p ≤ 0.001 for trauma and 1 Table 2 Laparotomy characteristics and risk for temporary open abdomen All data are expressed in median (IQR) or absolute numbers (%) All variables with a p value < 0.05 were considered significant and are in bold NS not statistically significant, TAC temporary abdominal closure a Gastro-intestinal surgeries include surgeries for abdominal sepsis and obstruction b Values presented as median (IQR) Total N = 775 Primary closure N = 666 TAC N = 109 p value Odds ratio 95% CI Indication for laparotomy Trauma 122 80 (12%) 42 (38%) < 0.001 NS Gastro-intestinala 593 542 (81%) 51 (47%) < 0.001 0.47 0.24–0.88 Vascular 60 44 (7%) 16 (15%) 0.006 NS Timing of surgery Acute 632 530 (79.6%) 102 (93.6%) < 0.001 NS Planned 143 136 (20.4%) 7 (6.4%) Gender: male 456 372 (55.9%) 84 (77.1%) < 0.001 3.39 1.67–6.87 Previous abdominal surgery 392 355 (53.3%) 37 (33.9%) 0.001 NS ASA classificationb 2 (1) 2 (1) 2 (1) 0.451 NS pHb 7.32 (0.18) 7.34 (0.16) 7.29 (0.19) < 0.001 0.087 0.01–0.76 Lactateb 3.2 (3.95) 2.55 (2.98) 4.3 (4.2) 0.003 NS Base deficitb − 6.35 (7.7) − 5.2 (7.88) − 7.25 (7.32) 0.001 NS Table 3 Outcome parameters All variables with a p value < 0.05 were considered significant and are in bold TAC temporary abdominal closure a Values presented as median (IQR) or absolute numbers (%) Total N = 775 Primary closure N = 666 TAC N = 109 p value Hospital length of staya (days) 23 (35) 20 (29) 46 (41) < 0.001 ICU length of staya (days) 1 (9) 0 (7) 11.5 (23) < 0.001 Postoperative ventral hernia 69 56 (13%) 13 (12%) 0.5 Entero-atmospheric fistula 26 17 (4%) 9 (8%) 0.06 Mortality 331 296 (45%) 35 (32%) 0.011 Table 2 Laparotomy characteristics and risk for temporary open abdomen Total N = 775 Primary closure N = 666 TAC N = 109 p value Odds ratio 95% CI Indication for laparotomy Trauma 122 80 (12%) 42 (38%) < 0.001 NS Gastro-intestinala 593 542 (81%) 51 (47%) < 0.001 0.47 0.24–0.88 Vascular 60 44 (7%) 16 (15%) 0.006 NS Timing of surgery Acute 632 530 (79.6%) 102 (93.6%) < 0.001 NS Planned 143 136 (20.4%) 7 (6.4%) Gender: male 456 372 (55.9%) 84 (77.1%) < 0.001 3.39 1.67–6.87 Previous abdominal surgery 392 355 (53.3%) 37 (33.9%) 0.001 NS ASA classificationb 2 (1) 2 (1) 2 (1) 0.451 NS pHb 7.32 (0.18) 7.34 (0.16) 7.29 (0.19) < 0.001 0.087 0.01–0.76 Lactateb 3.2 (3.95) 2.55 (2.98) 4.3 (4.2) 0.003 NS Base deficitb − 6.35 (7.7) − 5.2 (7.88) − 7.25 (7.32) 0.001 NS Table 2 Laparotomy characteristics and risk for temporary open abdomen All data are expressed in median (IQR) or absolute numbers (%) Table 3 Outcome parameters All variables with a p value < 0.05 were considered significant and are in bold TAC temporary abdominal closure a Values presented as median (IQR) or absolute numbers (%) Total N = 775 Primary closure N = 666 TAC N = 109 p value Hospital length of staya (days) 23 (35) 20 (29) 46 (41) < 0.001 ICU length of staya (days) 1 (9) 0 (7) 11.5 (23) < 0.001 Postoperative ventral hernia 69 56 (13%) 13 (12%) 0.5 Entero-atmospheric fistula 26 17 (4%) 9 (8%) 0.06 Mortality 331 296 (45%) 35 (32%) 0.011 1 3 Underlying disease determines the risk of an open abdomen treatment, final closure, however,… Underlying disease determines the risk of an open abdomen treatment, final closure, however,… 117 Table 4 Risk factors for delayed fascial closure and secondary healing All variables with a p value < 0.05 were considered significant and are in bold All data are expressed in median (IQR) or absolute numbers (%) NS not statistically significant, TAC temporary abdominal closure Total N = 80 Delayed fascial closure N = 43 Secondary healing N = 37 p value Odds ratio 95% CI Indication for TAC DCS 36 (45.0%) 26 (60.5%) 10 (27%) 0.003 NS Abdominal sepsis 27 (33.8%) 9 (20.9%) 18 (48.6%) 0.013 ACS 17 (21.3%) 8 (18.6%) 9 (24.3%) NS Trauma 32 (40%) 22 (51%) 10 (27%) 0.028 NS Non-trauma 48 (60%) 21 (49%) 27 (73%) Surgeries before TAC 1 (2) 0 (1) 1 (2) 0.001 0.41 0.24–0.72 Surgeries during TAC 2 (3) 1 (1) 3 (4) 0.002 0.65 0.49–0.87 Total duration TAC(days) 7 (17) 3 (5) 18 (28) < 0.001 Table 4 Risk factors for delayed fascial closure and secondary healing abdominal sepsis and ten treated for ACS. Table 5 Outcome parameters following open abdomen treatment i All data are expressed in median (IQR) or absolute numbers (%) Discussion Male gender and pH, as an indicator of the severity of physi- ological derangement of the patients, at the index operation were the only significant factors associated with possible failure of abdominal wall closure in our studied population, while patients with gastro-intestinal indication for surgery were less likely to end up with an open abdomen. In addi- tion, the number of surgeries prior to TAC and the number of surgeries during TAC, but not the indication nor disease severity prior to the index laparotomy, were significant risk factors for the failure of delayed fascial closure. It is also important to distinguish between patients who have an open abdomen as a consequence of physiologi- cal derangement, and patients who have an open abdomen because of local abdominal disease. In our study popula- tion, patients with abdominal sepsis were less likely to undergo delayed fascial closure compared to patients with an open abdomen after DCS, (mainly in trauma patients) and most of abdominal sepsis patients ended with a defini- tive open abdomen. This difference in abdominal closure determined by the origin of the open abdomen has been described before by Loftus et al. [5]. They investigated a group of 224 patients that underwent open abdomi- nal treatment. In the patients who survived, the primary closure rate for trauma patients was much higher than in patients with abdominal sepsis (90% compared to 76%). Tolonen et al. [15] have investigated outcomes in patients following temporary abdominal closure for abdominal peritonitis only. In a group of 41 patients, they found a very high fascial closure rate of 92% [15]. In a system- atic review of patients treated with temporary abdominal closure for abdominal sepsis, performed by Atema et al. [6], delayed fascial closure rates were more comparable to ours with a mean delayed fascial closure rate of 50.2%. Our study results showed that the number of abdominal surgical procedures prior to and during TAC was the only significant indicator for the possibility to eventually close the abdomen (by delayed fascial closure). This has been reported before by Atema et al. [6] who demonstrated in their systematic review that fewer re-explorations and The main reasons for open abdominal treatment were DCS in trauma (45%), followed by abdominal sepsis (34%) and abdominal compartment syndrome (21%). Results When evaluat- ing multivariate analysis (both forward and backward step- wise selection) for the ability of abdominal closure, only surgical interventions before TAC and during TAC were sig- nificant predictors. We excluded total duration of TAC for the multivariate analysis since this represents more or less an outcome variable and besides that it roughly corresponds to the interventions during TAC; therefore, we chose to only include one of these two variables in multivariate analysis. These results show that the odds for secondary healing were 2.5 times higher with every previous intervention and 1.5 times higher for every intervention during open abdominal treatment (Table 4). resuscitated to prevent hypovolemia with substitution of pro- tein loss since this can lead to compromised wound healing, increase of infections and decreased survival [19–21]. Fur- ther, ventilatory problems occur frequently because an intact abdominal wall is required for adequate spontaneous ventila- tion [4]. Lastly, it is associated with local complications such as ventral hernia development due to fascial retraction and entero-atmospheric fistulas (EAF) [1–4]. i Male gender and acidosis have shown to be associated with open abdominal treatment. This has not yet been illus- trated in previous investigations. One could only speculate why male gender is prone to open abdominal treatment. Pos- sibly, since male patients are more muscular than female patients, they might have a less compliant abdominal wall. Besides that, a large part of the female population was past the child-bearing age and might have had children which means that during pregnancy the abdominal wall has been already stretched, possibly making it more compliant in case of swelling of abdominal contents. Further, patients follow- ing gastro-intestinal surgery were less likely to undergo open abdomen treatment. This could possibly be explained by the fact that surgery is usually planned in case of gastro- intestinal disease, whereas patients who have a laparotomy following trauma had it in an acute setting. Results From the remain- ing surviving 80 patients, 43 could be managed by delayed fascial closure (54%). Four of them had their abdomen closed by mesh-mediated VAC closure and in one patient the abdomen was closed using the component-separation method as described by Ramirez [16]. both groups with a mortality rate of 45% in patients fol- lowing primary closure and 32% in patients who underwent open abdomen treatment (Table 3). Twenty-nine patients died before the abdomen could be closed (27%): nine patients following DCS, ten after Fig. 2 Outcome The open abdomen of the remaining 37 patients (46%) was treated by secondary healing (Table 4). When analyzing outcome in the TAC patient group, there was a significant difference in outcome between patients after delayed fascial closure and secondary healing. Patients who underwent secondary healing had higher mortality rates, developed more often entero-atmospheric fistulas and ventral hernias than patients in whom delayed fascial closure was successful (Fig. 2; Table 5). The majority of patients who were treated with TAC in case of DCS could be managed with delayed fascial clo- sure, in contrast with patients with abdominal sepsis, since most of them continued to secondary healing following TAC (Table 4). In abdominal compartment syndrome, both delayed fascial closure and secondary healing occurred in Fig. 2 Outcome Fig. 2 Outcome Table 5 Outcome parameters following open abdomen treatment All variables with a p value < 0.05 were considered significant and are in bold All data are expressed in median (IQR) or absolute numbers (%) LOS length of stay Total N = 80 Delayed fascial closure N = 43 Secondary healing N = 37 p value Hospital LOS 46 (40) 38 (45) 56 (37) 0.013 ICU-LOS 10 (23) 9 (13) 16 (26) 0.021 Postoperative hernia 13 (16%) 1 (2.3%) 12 (33.3%) < 0.001 Entero-atmospheric fistula 7 (8%) 1 (2.3%) 6 (16.7%) 0.025 Mortality 9 (11%) 1 (2.3%) 8 (21.6%) 0.006 Table 5 Outcome parameters following open abdomen treatment 118 S. Karhof et al. half of the patients (Table 4). Patients who underwent sec- ondary healing needed more surgical interventions to finally close the abdomen compared to patients who underwent TAC for delayed fascial closure. Further, the total duration of TAC treatment was significantly longer in patients follow- ing secondary healing compared to patients who underwent successful delayed fascial closure (Table 4). Discussion All abdomi- nal sepsis patients had an open abdomen, because it could physically not be closed due to extensive swelling of the abdominal contents. None of them had their abdomen left open for a planned re-laparotomy. This is in accordance with Ruler et al. who have previously demonstrated that outcomes did not differ significantly following planned or on-demand re-laparotomy in patients with abdominal sepsis [7]. In our study, 27% of patients died before the abdomen could be closed. This high mortality rate, a reflection of the critically ill patients in whom the abdomen is frequently left open, is comparable to other studies [1, 5]. Further, an open abdomen itself is associated with multiple problems such as blood loss, fluid and electrolyte losses, respiratory problems and closure of the abdomen itself is challenging in most cases [1, 2, 4]. These critically ill patients are in a hyper- catabolic state due to fluid and protein loss, causing nutri- tional insufficiency [1]. These patients must be adequately 1 3 3 Underlying disease determines the risk of an open abdomen treatment, final closure, however,… 119 of the abdomen was possible in a small majority of the patients, with a significantly lower closure rate in patients following abdominal sepsis. However, the only independ- ent predictor of delayed fascial closure was the quantity of abdominal surgical procedures before and during TAC treat- ment. Failure to achieve delayed fascial closure increases the risk of developing a ventral hernia and entero-atmospheric fistulas. We, therefore, advise to aim for delayed fascial clo- sure as early as possible following a structured approach. In addition, we would advise to refrain from fruitless attempts at fascial closure, but rather try mesh-mediated vacuum clo- sure or accept a definitive open abdomen. A potential ventral hernia could be repaired at a later stage. shorter duration of temporary abdominal closure were associated with greater possibility of delayed fascial clo- sure as well. This difference might be related to origin of TAC as well since patients following trauma are severely injured at the time of presentation and undergo immedi- ate damage control surgery followed by open abdominal treatment. Generally, after a short period of resuscitation in ICU with only a few re-explorations, the abdomen can be closed by delayed fascial closure. Discussion This is in contrast with patients with abdominal sepsis, for example, who might have had previous abdominal surgeries and then undergo open abdominal treatment because of severely ill (contaminated) abdomen. These patients are more likely to have more re-explorations before the abdomen can finally be closed again, if it could be closed at all. Funding No funding has been provided. With the various complications that may occur following secondary healing, one should attempt to primarily close the abdomen. In our opinion, a structured approach, as listed in the flowchart, we use in our trauma department (Fig. 1), could contribute to favorable outcomes. We strive for a sec- ond look within 24–48 h and try immediately to close the fascia; if not feasible, we try again within the next 48 h. Other reports also mention this window of 48 h to close the abdomen, with increasing complication rates thereafter [2, 3]. When it becomes clear that delayed fascial closure is not feasible after two attempts, one should not continue TAC treatment, but change strategies to, for example, Ramirez technique [14], or vacuum-assisted wound closure combined with mesh-mediated fascial traction, a technique described by Petersson et al. [16]. They have introduced the combined use of mesh with the vacuum system, where the mesh is tightened with change of vacuum system every 2–3 days until the fascia could be closed again [16]. Rasilainen et al. [17] have evaluated this technique as well, with very high closure rates of 78% in 50 patients. In a recently published study by Salamone et al. [18], an even higher closure rate of 95% was found in a modified version of this technique. Compliance with ethical standards Conflict of interest Steffi Karhof, Mark Haverkort, Rogier Simmer- macher, Falco Hietbrink, Luke Leenen and Karlijn van Wessem all declare that they have no conflict of interest. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creativeco mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. References with a negative pressure wound therapy system and barker’s vac- uum packing technique. World J Surg. 2013;37(9):2018–30. 15. Tolonen M, Mentula P, Sallinen V, Rasilainen S, Bäckland M, Leppäniemi A. Open abdomen with vacuum-assisted wound clo- sure and mesh-mediated fascial traction in patients with compli- cated diffuse secondary peritonitis: a single-center 8-year experi- ence. J Trauma Acute Care Surg. 2017;82:1100–5. 9. Kirkpatrick AW, Roberts DJ, Faris PD, Ball CG, Kubes P, Tiruta C, Xiao Z, Holodinsky JK, McBeth PB, Doig CJ, Jenne CN. Active negative pressure peritoneal therapy after abbreviated laparotomy: the intraperitoneal vacuum randomized controlled trial. Ann Surg. 2015;262(1):38–46. 16. Petersson U, Acosta S, Björck M. Vacuum assisted wound closure and mesh-mediated fascial traction—a novel technique for late closure of the open abdomen. World J Surg. 2007;31:2133–7. 10. Acosta S, Björck M, Petersson U. Vacuum-assisted wound closure and mesh-mediated fascial traction for open abdo- men therapy—a systematic review. Anesthesiol Intensive Ther. 2017;49(3):139–45. 17. Rasilainen SK, Mentula PJ, Leppäniemie AK. Vacuum and mesh- mediated fascial traction for primary closure of the open abdomen in critically ill surgical patients. Br J Surg. 2012;99:1725–33. 11. Cristaudo AT, Jennings SB, Hitos K, Gunnarsson R, DeCosta A. Treatments and other prognostic factors in the management of the open abdomen: a systematic review. J Trauma Acute Care Surg. 2017;82(2):407–18. 18. Salamone G, Licari L, Guercio G, Comelli A, Mangiapane M, Falco N, Tutino R, Bagarella N, Campanella S, Porrello C, Gullo R, Cocorullo G, Gulotta G. Vacuum-assisted wound closure with mesh-mediated fascial traction achieves better outcomes than vac- uum-assisted wound closure alone: a comparative study. World J Surg. 2018;42:1679–86. 12. Beale EW, Janis JE, Minei JP, Elliott AC, Phelan HA. Predictors of failed primary closure in the trauma patient with an open abdo- men. South Med J. 2013;106:327–31. 19. Heyland DK. Nutritional support in the critically ill patients. A critical review of the evidence. Crit Care Clin. 1998;14(3):423–40. 13. Barker DE, Kaufman HJ, Smith LA, Ciraulo DL, Richart CL, Burns RP. Vacuum pack technique of temporary abdomi- nal closure: a 7-year experience with 112 patients. J Trauma. 2000;48(2):201–7. 20. Miller RS, Morris JA, Diaz JJ, Hering MB, May AK. Complica- tions after 344 damage control open celiotomies. J Trauma Inj Infect Crit Care. 2004;59(6):1365–74. 14. Ramirez OM, Ruas E, Dellon AL. “Components separation” method for closure of abdominal-wall defects: an anatomic and clinical study. Plast Reconstr Surg. 1990;86(3):519–26. 21. References 1. Coccoloni F, Biffl W, Catena F, Ceresoli M, Chiara O, et al. The open abdomen, indications, management and definitive closure. World J Surg. 2015;10:32.fl 2. Chiara O, Cimbanassi S, Biffl W, Lappeniemi A, Henry S, Scalea TM, et al. International consensus conference on open abdomen in trauma. J Trauma Acute Care Surg. 2015;80(1):173–83. 3. Coccolini F, Montori G, Ceresoli M, Catena F, Moore EE, et al. The role of open abdomen in non trauma patient: WSES consen- sus paper. World J Emerg Surg. 2017;12:39. i Our study has some limitations inherent to the retrospec- tive design of the study, conducted in a single institution. 4. van Wessem KJP. Possible devices to temporary cover the open abdomen: pros and cons. Acta Chir Belg. 2010;110:499–503. g y g Due to this retrospective nature, our long-term data were dependent upon clinic visit data with follow-up varying from several days to months. Another limitation was the heteroge- neity in treatment. There is no strict protocol in our hospital for open abdominal treatment after gastro-intestinal surgery and the decision to try to primarily close the abdomen or use a mesh is made at the surgeon’s discretion. Only in trauma patients there has been a strict protocolled approach for open abdominal treatment. 5. Loftus TJ, Jordan JR, Croft CA, Smith RS, Efron PA, Mohr AM, et al. Temporary abdominal closure for trauma and intra-abdomi- nal sepsis: different patients, different outcomes. J Trauma Acute Care Surg. 2017;82(2):345–50. 6. Atema JJ, Gans SL, Boermeester MA. Systematic review and meta-analysis of the open abdomen in temporary abdomi- nal closure techniques in non-trauma patients. World J Surg. 2015;39:912–25. 7. van Ruler O, Mahler CW, Boer KR, Reuland EA, Gooszen HG, Opmeer BC, de Graaf PW, Lamme B, Gerhards MF, Steller EP, van Till JWO, de Borgie CJAM, Gouma DJ, Reitsma JB, Boer- meester MA. Comparison of on-demand vs planned relaparotomy strategy in patients with severe peritonitis. A randomized trial. JAMA. 2007;298(8):865–73. In conclusion, our results show that male sex and disease severity, as indicated by severe acidosis, were independent predictors of open abdominal treatment, while laparotomy in the context of gastro-intestinal pathology usually ends with a primary closed fascia. Furthermore, delayed fascial closure 8. Cheatham ML, Demetriades D, Fabian TC, Kaplan MJ, Miles WS, Schreiber MA, Holcomb JB, Bochicchio G, Sarana B, Rotondo MF. Prospective study examining clinical outcomes associated 1 3 3 120 S. Karhof et al. References Rao M, Burke D, Finan PJ, Sagar PM. The use of vacuum assisted closure of abdominal wounds: a word of caution. Colorectal Dis. 2005;9:266–8. 1 3 3
W4250713953.txt	https://peerj.com/articles/cs-11v0.2/submission	en		Peer Review #2 of "MLitB: machine learning in the browser (v0.1)"	null	2,015	cc-by	10,328	Reviewing Manuscript MLiTB: machine learning in the browser Edward Meeds, Remco Hendriks, Said Al Faraby, Magiel Bruntink, Max Welling With few exceptions, the field of Machine Learning (ML) research has largely ignored the browser as a computational engine. Beyond an educational resource for ML, the browser has vast potential to not only improve the state-of-the-art in ML research, but also, inexpensively and on a massive scale, to bring sophisticated ML learning and prediction to the public at large. This paper introduces MLitB, a prototype ML framework written entirely in Javascript, capable of performing large-scale distributed computing with heterogeneous classes of devices. The development of MLitB has been driven by several underlying objectives whose aim is to make ML learning and usage ubiquitous (by using ubiquitous compute devices), cheap and effortlessly distributed, and collaborative. This is achieved by allowing every internet capable device to run training algorithms and predictive models with no software installation and by saving models in universally readable formats. Our prototype library is capable of training deep neural networks with synchronized, distributed stochastic gradient descent. MLitB offers several important opportunities for novel ML research, including: development of distributed learning algorithms, advancement of web GPU algorithms, novel field and mobile applications, privacy preserving computing, and green grid-computing. MLitB is available as open source software. PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript MLitB: Machine Learning in the Browser Edward Meeds1 , Remco Hendriks1 , Said Al Faraby1 , Magiel Bruntink1 , and Max Welling1,2,3 1 Informatics 2 Donald Institute, University of Amsterdam Bren School of Information and Computer Sciences, University of California, Irvine 3 Canadian Institute for Advanced Research ABSTRACT With few exceptions, the field of Machine Learning (ML) research has largely ignored the browser as a computational engine. Beyond an educational resource for ML, the browser has vast potential to not only improve the state-of-the-art in ML research, but also, inexpensively and on a massive scale, to bring sophisticated ML learning and prediction to the public at large. This paper introduces MLitB, a prototype ML framework written entirely in JavaScript, capable of performing large-scale distributed computing with heterogeneous classes of devices. The development of MLitB has been driven by several underlying objectives whose aim is to make ML learning and usage ubiquitous (by using ubiquitous compute devices), cheap and effortlessly distributed, and collaborative. This is achieved by allowing every internet capable device to run training algorithms and predictive models with no software installation and by saving models in universally readable formats. Our prototype library is capable of training deep neural networks with synchronized, distributed stochastic gradient descent. MLitB offers several important opportunities for novel ML research, including: development of distributed learning algorithms, advancement of web GPU algorithms, novel field and mobile applications, privacy preserving computing, and green grid-computing. MLitB is available as open source software. Keywords: Machine learning, Ubiquitous computing, Distributed computing, Client-server systems, Mobile computing, Pervasive computing, Social computing, Crowdsourcing 1 INTRODUCTION The field of Machine Learning (ML) currently lacks a common platform for the development of massively distributed and collaborative computing. As a result, there are impediments to leveraging and reproducing the work of other ML researchers, potentially slowing down the progress of the field. The ubiquity of the browser as a computational engine makes it an ideal platform for the development of massively distributed and collaborative ML. Machine Learning in the Browser (MLitB) is an ambitious software development project whose aim is to bring ML, in all its facets, to an audience that includes both the general public and the research community. By writing ML models and algorithms in browser-based programming languages, many research opportunities become available. The most obvious is software compatibility: nearly all computing devices can collaborate in the training of ML models by contributing some computational resources to the overall training procedure and can, with the same code, harness the power of sophisticated predictive models on the same devices (see Fig. 1). This goal of ubiquitous ML has several important consequences: training ML models can now occur on a massive, even global scale, with minimal cost, and ML research can now be shared and reproduced everywhere, by everyone, making ML models a freely accessible, public good. In this paper, we present both a long-term vision for MLitB and a light-weight prototype implementation of MLitB, that represents a first step in completing the vision, and is based on an important ML use-case, Deep Neural Networks. In Section 2 we describe in more detail our vision for MLitB in terms of three main objectives: 1) make ML models and algorithms ubiquitous, for both the public and the scientific community, 2) create an framework for cheap distributed computing by harnessing existing infrastructure and personal devices as novel computing resources, and 3) design research closures, software objects that archive ML models, algorithms, and parameters to be shared, reused, and in general, support reproducible research. PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript Figure 1. Overview of MLitB. (1) A researcher sets up a learning problem in his/her browser. (2) Through the internet, grid and desktop machines contribute computation to solve the problem. (3) Heterogeneous devices, such as mobile phone and tablets, connect to the same problem and contribute computation. At any time, connected clients can download the model configuration and parameters, or use the model directly in their browsing environment. In Section 3 we describe the current state of the MLitB software implementation, the MLitB prototype. We begin with a description of our design choices, including arguments for using JavaScript and the other modern web libraries and utilities. Then we describe a bespoke map-reduce synchronized event-loop, specifically designed for training a large class of ML models using distributed stochastic gradient descent (SGD). Our prototype focuses on a specific ML model, Deep Neural Networks (DNNs), using an existing JavaScript implementation (Karpathy, 2014), modified only slightly for MLitB. We also report results of a scaling experiment, demonstrating the feasibility, but also the engineering challenges of using browsers for distributed ML applications. We then complete the prototype description with a walk-through of using MLitB to specify and train a neural network for image classification. MLitB is influenced and inspired by current volunteer computing projects. These and other related projects, including those from machine learning, are presented in Section 4. Our prototype has exposed several challenges requiring further research and engineering; these are presented in Section 5, along with discussion of interesting application avenues MLitB makes possible. The most urgent software development directions follow in Section 6. 2 MLITB: VISION Our long-term vision for MLitB is guided by three overarching objectives: Ubiquitous ML: models can be training and executed in any web browsing environment without any further software installation. Cheap distributed computing: algorithms can be executed on existing grid, cloud, etc., computing resources with minimal (and possibly no) software installation, and can be easily managed remotely via the web; additionally, small internet enabled devices can contribute computational resources. Reproducibility: MLitB should foster reproducible science with research closures, universally readable objects containing ML model specifications, algorithms, and parameters, that can be used seamlessly to achieve the first two objectives, as well as support sharing of ML models and collaboration within the research community and the public at large. 2.1 Ubiquitous Machine Learning The browser is the most ubiquitous computing device of our time, running, in some shape or form on all desktops, laptops, and mobile devices. Software for state-of-the-art ML algorithms and models, on the 2/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript other hand, are very sophisticated software libraries written in highly specific programming languages within the ML research community (Bastien et al., 2012; Jia et al., 2014; Collobert et al., 2011). As research tools, these software libraries have been invaluable. We argue, however, that to make ML truly ubiquitous requires writing ML models and algorithms with web programming languages and using the browser as the computational engine. The software we propose can run sophisticated predictive models on cell phones or super-computers; for the former this extends the distributed nature of ML to a global internet. By further encapsulating the algorithms and model together, the benefit of powerful predictive modeling becomes a public commodity. 2.2 Cheap Distributed Computing The usage of web browsers as compute nodes provides the capability of running sophisticated ML algorithms without the expense and technical difficulty of using custom grid or super-computing facilities (e.g. Hadoop cloud computing (Shvachko et al., 2010)). It has long been a dream to use volunteer computing to achieve real massive scale computing. Successes include Seti@Home (Anderson et al., 2002) and protein folding (Lane et al., 2013). MLitB is being developed to not only run natively on browsers but also for scaled distributed computing on existing cluster and/or grid resources and, by harnessing the capacity of non-traditional devices, for extremely massive scale computing with a global volunteer base. In the former set-up, low communication overhead and homogeneous devices (a “typical” grid computing solution) can be exploited. In the latter, volunteer computing via the internet opens the scaling possibilities tremendously, albeit at the cost of unreliable compute nodes, variable power, limited memory, etc. Both have serious implications for the user, but, most importantly, both are implemented by the same software. Although the current version of MLitB does not provide GPU computing, it does not preclude its implementation in future versions. It is therefore possible to seamlessly provide GPU computing when available on existing grid computing resources. Using GPUs on mobile devices is a more delicate proposition since power consumption management is of paramount importance for mobile devices. However, it is possible for MLitB to manage power intelligently by detecting, for example, if the device is connected to a power source, its temperature, and whether it is actively used for other activities. A user might volunteer periodic “mini-bursts” of GPU power towards a learning problem with minimal disruption to or power consumption from their device. In other words, MLitB will be able to take advantage of the improvements and breakthroughs of GPU computing for web engines and mobile chips, with minimal software development and/or support. 2.3 Reproducible and Collaborative Research Reproducibility is a difficult yet fundamental requirement for science (McNutt, 2014). Reproducibility is now considered just as essential for high-quality research as peer review; simply providing mathematical representations of models and algorithms is no longer considered acceptable (Stodden et al., 2013b). Furthermore, merely replicating other work, despite its importance, can be given low publication priority (Casadevall and Fang, 2010) even though it is considered a prerequisite for publication. In other words, submissions must demonstrate that their research has been, or could be, independently reproduced. For ML research there is no reason for not providing working software that allows reproduction of results (for other fields in science, constraints restricting software publication may exist). Currently, the main bottlenecks are the time cost to researchers for making research available, and the incompatibility of the research (i.e. code) for others, which further increases the time investment for researchers. One of our primary goals for MLitB is to provide reproducible research with minimal to no time cost to both the primary researcher and other researchers in the community. Following (Stodden et al., 2013a), we support “setting the default to reproducible.” For ML disciplines, this means other researchers should not only be able to use a model reported in a paper to verify the reported results, but also retrain the model using the reported algorithm. This higher standard is difficult and time-consuming to achieve, but fortunately this approach is being adopted more and more often, in particular by a sub-discipline of machine learning called deep learning. In the deep learning community, the introduction of new datasets and competitions, along with innovations in algorithms and modeling, have produced a rapid progress on many ML prediction tasks. Model collections (also called model zoos), such as those built with Caffe (Jia et al., 2014) make this collaboration explicit and easy to access for researchers. However, there remains a significant time investment to run any particular deep learning model (these include compilation, library installations, platform dependencies, 3/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript GPU dependencies, etc). We argue that these are real barriers to reproducible research and choosing ubiquitous software and compute engines makes it easier. For example, during our testing we converted a very performant computer vision model (Lin et al., 2013) into JSON format and it can now be used on any browser with minimal effort.1 In a nod to the concept of closures concept common in functional programming, our approach treats a learning problem as a research closure: a single object containing model and algorithm configuration plus code, along with model parameters that can be executed (and therefore tested and analyzed) by other researchers. 3 MLITB: PROTOTYPE The MLitB project and its accompanying software (application programming interfaces (APIs), libraries, etc.) are built entirely in JavaScript. We have taken a pragmatic software development approach to achieve as much of our vision as possible. To leverage our software development process, we have chosen, wherever possible, well-supported and actively developed external technology. By making these choices we have been able to quickly develop a working MLitB prototype that not only satisfies many of our objectives, but is as technologically future proof as possible. To demonstrate MLitB on a meaningful ML problem, we have similarly incorporated an existing JavaScript implementation of a Deep Neural Network into MLitB. The full implementation of the MLitB prototype can be found on GitHub2 . 3.1 Why JavaScript? JavaScript is a pervasive web programming language, embedded in approximately 90% of web-sites (W3Techs, 2014). This pervasiveness means it is highly supported (Can I Use, 2014), and is actively developed for efficiency and functionality (JavaScript V8, 2014; asm.js, 2014). As a result, JavaScript is the most popular programming language on GitHub and its popularity is continuing to grow (Ray et al., 2014). The main challenge for scientific computing with JavaScript is the lack of high-quality scientific libraries compared to platforms such as Matlab and Python. With the potential of native computational efficiency (or better, GPU computation) becoming available for JavaScript, it is only a matter of time before JavaScript bridges this gap. A recent set of benchmarks showed that numerical JavaScript code can be competitive with native C (Khan et al., 2014). 3.2 General Architecture and Design Design Considerations The minimal requirements for MLitB are based on the scenario of running the network as public resource computing. The downside of public resource computing is the lack of control over the computing environment. Participants are free to leave (or join) the network at anytime and their connectivity may be variable with high latency. MLitB is designed to be robust to these potentially destabilizing events. The loss of a participant results in the loss of computational power and data allocation. Most importantly, MLitB must robustly handle new and lost clients, re-allocation of data, and client variability in terms of computational power, storage capacity, and network latency. Although we are agnostic to the specific technologies used to fulfill the vision of MLitB, in practice we are guided by both the requirements of MLitB and our development constraints. Therefore, as a first step towards implementing our vision, we chose technology pragmatically. Our choices also follow closely the design principles for web-based big data applications (Begoli and Horey, 2012), which recommend popular standards and light-weight architectures. As we will see, some of our choices may be limiting at large scale, but they have permitted a successful small-scale MLitB implementation (with up to 100 clients). Fig. 2 shows the high-level architecture and web technologies used in MLitB. Modern web browsers provide functionality for two essential aspects of MLitB: Web Workers (W3C, 2014) for parallelizing program execution with threads and Web Sockets (IETF, 2011) for fast bi-directional communication channels to exchange messages more quickly between server and browser. To maintain compatibility across browser vendors, there is little choice for alternatives to Web Workers and Web Sockets. These same choices are also used in another browser-based distributed computing platform (Cushing et al., 2013). 1 JavaScript Object Notation json.org/ 2 https://github.com/software-engineering-amsterdam/MLitB 4/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript On the server-side, there are many choices that can be made based on scalability, memory management, etc. However, we chose Node.js for the server application.3 Node.js provides several useful features for our application: it is lightweight, written in JavaScript, handles events asynchronously, and can serve many clients concurrently (Tilkov and Vinoski, 2010). Asynchronous events occur naturally in MLitB as clients join/leave the network, client computations are received by the server, users add new models and otherwise interact with the server. Since the main computational load is carried by the clients, and not the server, a light-weight server that can handle many clients concurrently is all that is required by MLitB. Design Overview The general design of MLitB is composed of several parts. A master server hosts ML problems/projects and connects clients to them. The master server also manages the main event loop, where client triggered events are handled, along with the reduce steps of a (bespoke) map-reduce procedure used for computation. When a browser (i.e. a heterogeneous device) makes an initial connection to the master server, a userinterface (UI) client (aka a boss) is instantiated. Through the UI, clients can add workers that can perform different tasks (e.g., train a model, download parameters, take a picture, etc). An independent data server serves data to clients using zip files and prevents the master server from blocking while serving data. For efficiency, data transfer is performed using XHR4 . Trained models can be saved into JSON objects at any point in the training process; these can later be loaded in lieu of creating new models. Figure 2. MLitB architecture and technologies. (1) Servers are Node.js applications. The master server is the main server controlling communication between clients and hosts ML projects. (2) Communication between the master server and clients occurs over Web Sockets. (3) When heterogeneous devices connect to the master server they use Web Workers to perform different tasks. Upon connection, a UI worker, or boss, is instantiated. Web Workers perform all the other tasks on a client and are controlled by the boss. See Fig. 3 for details. (4) A special data worker on the client communicates with the data server using XHR. (5) The data server, also a Node.js application, manages uploading of data in zip format and serves data vectors to the client data workers. 3 Node.js: http://nodejs.org. www.w3.org/TR/XMLHttpRequest 4 XMLHttpRequest 5/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript Master Server The master node (server) is implemented in Node.js with communication between the master and slave nodes handled by Web Sockets. The master server hosts multiple ML problems/projects simultaneously along with all clients’ connections. All processes within the master are event-driven, triggered by actions of the slave nodes. Calling the appropriate functions by slave nodes to the master node is handled by the router. The master must efficiently perform its tasks (data reallocation and distribution, reduce-steps) because the clients are idle awaiting new parameters before their next work cycle. New clients must also wait until the end of an iteration before joining a network. The MLitB network is dynamic and permits slave nodes to join and leave during processing. The master monitors its connections and is able to detect lost participants. When this occurs, data that was allocated to the lost client is re-allocated the remaining clients, if possible, otherwise it is marked as to be allocated. Data Server The data server is a bespoke application intended to work with our neural network use-case model and can be thought of a lightweight replacement for a proper image database. The data server is an independent Node.js application that can, but does not necessarily live on the same machine. Users upload data in zip files before training begins; currently, the data server handles zipped image classification datasets (where sub-directory names define class labels). Data is then downloaded from the data server and zipped files are sent to clients using XHR and unzipped and processed locally. XHR is used instead of WebSockets because they communicate large zip-files more efficiently. A redundant cache of data is stored locally in the clients’ browser’s memory. For example, a client may store 10,000 data vectors, but at each iteration it may only have the computational power to process 100 data vectors in its scheduled iteration duration. The data server uses specialized JavaScript APIs unzip.js and redis-server. Clients Clients are browser connections from heterogeneous devices that visit the master server’s url. Clients interact through a UI worker, called a boss, and can create slave workers to perform various tasks (see Workers). The boss is the main worker running in a client’s browser. It manages the slave and image download worker and functions as a bridge between the downloader and slaves. A simple wrapper handles UI interactions, and provides input/output to the boss. Client bosses use a data worker to download data from the data server using XHR. The data worker and server communicate using XHR and pass zip files in both directions. The boss handles unzipping and decoding data for slaves that request data. Clients therefore require no software installation other than its native browser. Clients can contribute to any project hosted by the master server. Clients can trigger several events through the UI worker. These include adjusting hyper-parameters, adding data, and adding slave workers, etc. (Fig. 3). Most tasks are run in a separate Web Worker thread (including the boss), ensuring a non-blocking and responsive client UI. Data downloading is a special task that, via the boss and the data worker, uses XHR to download from the data server. Workers In Fig. 3 the tasks implemented using Web Worker threads are shown. At the highest-level is the client UI, with which the user interacts with ML problems and controls their slave workers. From the client UI, a user can create a new project, load a project from file, upload data to a project, or add slave workers for a project. Slaves can perform several tasks; most important is the trainer, which connects to an event loop of a ML project and contributes to its computation (i.e. its map step). Each slave worker communicates directly to the master server using Web Sockets. For the latter three tasks, the communication is mainly for sending requests for models parameters and receiving them. The training slave has more complicated behavior because it must download data then perform computation as part of the main event loop. To train, the user sets the slave task to train and selects start/restart. This will trigger a join event at the master server; model parameters and data will be downloaded and the slave will begin computation upon completion of the data download. The user can remove a slave at any time. Other slave tasks are tracking, which requires receiving model parameters from the master, and allows users to monitor statistics of the model on a dataset (e.g. classification error) or to execute the model (e.g. classify an image on a mobile device). Each slave worker communicates directly to the master server using Web Sockets. 6/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript Figure 3. MLitB Client Workers. Each client connection to the master server initiates a UI worker, aka a boss. For uploading data from a client to the data server and for downloading data from the data server to a client, a separate Web Worker called the data worker is used. Users can add slaves through the UI worker; each slave performs a separate task using a Web Worker. 3.3 Events and Software Behavior The MLitB network is constructed as a master-slave relationship, with one server and multiple slave nodes (clients). The setup for computation is similar to a MapReduce network (Dean and Ghemawat, 2008); however, the master server performs many tasks during an iteration of the master event loop, including a reduce step, but also several other important tasks. The specific tasks will be dictated by events triggered by the client, such as requests for parameters, new client workers, removed/lost clients, etc. Our master event loop can be considered as a synchronized map-reduce algorithm with a user defined iteration duration T , where values of T may range from 1 to 30 seconds, depending on the size of the network and the problem. MLitB is not limited to a mapreduce paradigm and in fact we believe that our framework opens the door to peer-to-peer or gossip algorithms (Boyd et al., 2006). We are currently developing asynchronous algorithms to improve the scalability of MLitB. Master Event Loop The master event loop consists of five steps and is executed by the master server node as long there is at least one slave node connected. Each loop includes one map-reduce step, and runs for at least T seconds. The following steps are executed, in order: a) New data uploading and allocation. b) New client trainer initialization and data allocation. 7/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript c) Training workers reduce step. d) Latency monitoring and data allocation adjustment. e) Master broadcasts parameters. a) New data uploading and allocation When a client boss uploads data, it directly communicates with the data server using XHR. Once the data server has uploaded the zip file, it sends the data indices and classification labels to the boss. The boss then registers the indices with the master server. Each data index is managed: MLitB stores an allocated index (the worker that is allocated the id) and a cached index (the worker that has cached the id). The master ensures that the data allocation is balanced amongst its clients. Once a data set is allocated on the master server, the master allocates indices and sends the set of ids to workers. Workers can then request data from the boss, who in turn use its data downloader worker to download those worker specific ids from the data server. The data server sends a zipped file to the data downloader, which are then unzipped and processed by the boss (e.g. JPEG decoding for images). The zip file transfers are fast but the decoding can be slow. We therefore allow workers to begin computing before the entire dataset is downloaded and decoded, allowing projects to start training almost immediately while data gets cached in the background. b) New client trainer initialization and data allocation When a client boss adds a new slave, a request to join the project is sent to the master. If there is unallocated data, a balanced fraction of the data is allocated to the new worker. If there is no unallocated data, a pie-cutter algorithm is used to remove allocated data from other clients and assign it to the new client. This prevents unnecessary data transfers. The new worker is sent a set of data ids it will need to download from the client’s data worker. Once the data has been downloaded and put into the new worker’s cache, the master will then add the new worker to the computation performed at each iteration. The master server is immediately informed when a client or one of its workers is removed from the network.5 Because of this, it can manage the newly unallocated data (that were allocated to the lost client). c) Training workers’ reduce step The reduce step is completely problem specific. In our prototype, workers compute gradients with respect to model parameters over their allocated data vectors, and the reduce step sums over the gradients and updates the model parameters. d) Latency monitoring and data allocation adjustment The interval T represents both the time of computation and the latency between the client and the master node. The synchronization is stochastic and adaptive. At each reduce step, the master node estimates the latency between the client and the master and informs the client worker how long it should run for. A client does not need to have a batch size because it just clocks its own computation and returns results at the end of its scheduled work time. Under this setting, it is possible to have mobile devices that compute only a few gradients per second and a powerful desktop machine that performs hundreds or thousands. This simple approach also allows the master to account for unexpected user activity: if the user’s device slows or has increased latency, the master will decrease the load on the device for the next iteration. Generally, devices with a cellular network connection communicate with longer delays than hardwired machines. In practice, this means the reduction step in the master node receives delayed responses from slave nodes, forcing it to run the reduction function after the slowest slave node (with largest latency) has returned. This is called asynchronous reduction callback delay. e) Master broadcasts parameters An array of model parameters is broadcast to each clients’ boss worker using XHR; when the boss receives new parameters, they are given to each of its workers who then start another computation iteration. 3.4 ML use-case: Deep Neural Networks The current version of the MLitB software is built around a pervasive ML use-case: deep neural networks (DNNs). DNNs are the current state-of-the-art prediction models for many tasks, including computer 5 If a user closes a client tab, the master will know immediately and take action. In the current implementation, if a user closes the master tab, all current connections are lost. 8/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript vision (Krizhevsky et al., 2012; Lin et al., 2013), speech recognition (Hinton et al., 2012), and natural language processing and machine translation (Liu et al., 2014; Bahdanau et al., 2014; Sutskever et al., 2014). Our implementation only required superficial modifications to an existing JavaScript implementation (Karpathy, 2014) to fit into our network design. Figure 4. Effects of scaling on power and latency. Power—measured as the number of data vectors processed per second—scales linearly until 64 nodes, when the increase in latency jumps. The ideal linear scaling is shown in grey. 3.5 Scaling Behavior of MLitB We performed an experiment to study the scaling behavior of MLitB prototype. Using up to 32 4-core workstation machines connected on a local area network using a single router, we trained a simple convolutional NN on the MNIST dataset for 100 iterations (with 4 seconds per iteration/synchronization event).6 The number of slave nodes doubled from one experiment to the next (i.e. 1, 2, 4, . . . , 96). We are interested in the scaling behavior of two performance indicators: 1) power, measured in data vectors processed per second, and 2) latency in milliseconds between slaves and master node. Of secondary interest is the generalization performance on the MNIST test set. As a feasibility study of a distributed ML framework, we are most interested scaling power while minimizing latency effects during training, but we also want to ensure the correctness of the training algorithm. Since optimization using compiled JS and/or GPUs of the ML JavaScript library possible, but not our focus, we are less concerned with the power performance of a single slave node. Results for power and latency are shown in Fig. 4. Power increases linearly up to 64 slave nodes, at which point a large increase in latency limits additional power gains for new nodes. This is due to a single server reaching the limit of its capacity to process incoming gradients synchronously. Solutions include using multiple server processes, asynchronous updates, and partial gradient communication. Test error, as a function of the number of nodes is shown in Fig. 5 after 50 iterations (200 seconds) and 100 iterations (400 seconds); i.e. each point represents the same wall-clock computation time. This demonstrates the correctness of MLitB for a given model architecture and learning hyperparameters. Due to the data allocation policy that limits the data vector capacity of each node to 3000 vectors, experiments with more nodes process more of the training set during the training procedure. For example, using only 1 slave node trains on 3/60 of the full training set. With 20 nodes, the network is training on the full dataset. This policy could easily be modified to include data refreshment when running with unallocated data. 6 Slave node specifications (32 units): Intel Core i3-2120 3.3GHz (dual-core); 4GB RAM; Windows 7 Enterprise x64; Google Chrome 35. Master node specifications (1 unit): Intel Xeon E5620 2.4GHz (quad-core); 24 GB RAM; Ubuntu 10.04 LTS. NodeJS version: v0.10.28. The NN has a 28 × 28 input layer connected to 16 convolution filters (with pooling), followed by a fully connected output layer. 9/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript Figure 5. Effects of scaling on optimization. Convergence of the NN is measured in terms of test error after 50 and 100 iterations. Each point represents approximately the same wall-clock time (200/400 seconds for 50 and 100 iterations, respectively). The primary latency issue is due to all clients simultaneously sending gradients to the server at the end of each iteration. Three simple scaling solutions are 1) increasing the number of master node processes that receive gradients 2) using asynchronous update rules (each slave computes for a random amount of time, then sends updates), reducing the load of any one master node process, and 3) partial communication of gradients (decreasing bandwidth). Figure 6. CIFAR-10 project loaded in MLitB. 3.6 Walk-through of MLitB Prototype We briefly describe how MLitB works from a researcher’s point of view. Specification of Neural Network and Training Parameters Using a minimalist UI (not shown), the researcher can specify their neural network, for example they can add/remove layers of different types, and adjust regularization parameters (L1/L2/dropout) and learning rates. Alternatively, the researcher can load a previously saved neural network in JSON format (that may or may not have already been trained). Once a NN is specified (or loaded), it appears in the display, along 10/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript with other neural networks also managed by the master node. By selecting a specific neural network, the researcher can then add workers and data (e.g. project cifar10 in Fig. 6). Specification of Training Data Image classification data is simple to upload using named directory structures for image labels. For example, for CIFAR10 all files in the ”apple” subdirectory will be given label ”apple” once loaded (e.g. the image file /cifar10/apple/apple_apple_s_000022.png). The entire ”cifar10” directory can be zipped and uploaded. MLitB processes JPEG and PNG formats. A test set can be uploaded in tracker mode. Training Mode In the training mode, a training worker performs as many gradient computations as possible within the iteration duration T (i.e. during the map step of the main event loop). The total gradient and the number of gradients is sent to the master, which then in the reduce step computes a weighted average of gradients from all workers and takes a gradient step using AdaGrad (Duchi et al., 2011). At the end of the main event loop, new neural network weights are sent via Web Sockets to both trainer workers (for the next gradient computations) and to tracker workers (for computing statistics and executing the latest model). Figure 7. Tracking model (model execution): The label of a test image is predicted using the latest NN parameters. Users can execute a NN prediction using an image stored on their device or using their device’s camera. In this example, an image of a horse is correctly predicted with probability 0.687 (the class-conditional predictive probability). Tracking Mode There are two possible functions in tracking mode: 1) executing the neural network on test data, and 2) monitoring classification error on an independent data set. For 1, users can predict class labels for images taken with a device’s camera or locally stored images. Users can also learn a new classification problem on the fly by taking a picture and giving it a new label; this is treated as a new data vector and a new output neuron is added dynamically to the neural network if the label is also new. Fig. 7 shows a test image being classified by the cifar10 trained neural network. For 2, users create a statistics worker and can upload test images and track their error over time; after each complete evaluation of the test images, the latest neural network received from the master is used. Fig. 8 shows the error for cifar10 using a small test set for the first 600 parameter updates. Archiving Trained Neural Network Model The prototype does not include a research closure specification. However, it does provide easy archiving functionality. At any moment, users can download the entire model specification and current parameter values in JSON format. Users can then share or initialize a new training session with the JSON object 11/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript Figure 8. Tracking mode (classification error). A test dataset can be loaded and its classification error rate tracked over iterations; here using a NN trained on CIFAR-10. by uploading it during the model specification phase, which represents a high-level of reproducibility. Although the JSON object fully specifies the model, it does not include training or testing code. Despite this shortcoming, using a standard protocol is simple way of providing a lightweight archiving system. 3.7 Limitations of MLitB Prototype In this section we briefly discuss the limitations of the current prototype; later in Section 5 we will discuss the challenges we face in scaling MLitB to a massive level. Our scaling experiment demonstrates that the MLitB prototype can accommodate up to 64 clients before latency significantly degrades its performance. Latency, however, is primarily affected by the length of an iteration and by size of the neural network. For longer iterations, latency will become a smaller portion of the main event loop. For very large neural networks, latency will increase due to bandwidth pressure. As discussed previously, the main computational efficiency loss is due to the synchronization requirement of the master event loop. This requirement causes the master server to be idle while the clients are computing and the clients to wait while the master processes all the gradients. As the size of the full gradients can be large (at least > 1MB for small neural networks), the network bandwidth is quickly saturated at the end of a computation iteration and during the parameter broadcast. By changing to an asynchronous model, the master can continuously process gradients and the bandwidth can be maximally utilized. By communicating partial gradients, further efficiency can be attained. We leave this for future work. There is a theoretical limit of 500MB data storage per client (the viable memory of a web-browser). In our experience, the practical limit is closer to 100MB at which point performance is lost due to memory management issues. We found that 1MB/sec bandwidth was achievable on a local network, which meant that it could handle images on MNIST and CIFAR-10 easily, but would stall for larger images. With respect to Deep Neural Networks, the data processing ability of a single node was limited (especially is one compared to sophisticated GPU enables libraries (Bastien et al., 2012)). Although we were most interested in the scaling performance, we note that naive convolution implementations significantly slow performance. We found that reasonable sized images, up to 100 × 100 × 3 pixels, can be processed on mobile devices in less than a second without convolutions, but can take several seconds with convolutions, limiting its usefulness. In the future, near native or better implementations will be required for the convolutional layers. 12/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript 4 RELATED WORK MLitB has been influenced by a several different technologies and ideas presented by previous authors and from work in different specialization areas. We briefly summarize this related work below. 4.1 Volunteer Computing BOINC (Anderson, 2004) is an open-source software library used to set up a grid computing network, allowing anyone with a desktop computer connected to the internet to participate in computation; this is called public resource computing. Public resource or volunteer computing was popularized by SETI@Home (Anderson et al., 2002), a research project that analyzes radio signals from space in the search of signs of extraterrestrial intelligence. More recently, protein folding has emerged as significant success story (Lane et al., 2013). Hadoop (Shvachko et al., 2010) is an open-source software system for storing very large datasets and executing user application tasks on large networks of computers. MapReduce (Dean and Ghemawat, 2008) is a general solution for performing computation on large datasets using computer clusters. 4.2 JavaScript Applications In (Cushing et al., 2013) a network of distributed web-browsers called WeevilScout is used for complex computation (regular expression matching and binary tree modifications) using a JavaScript engine. It uses similar technology (Web Workers and Web Sockets) as MLitB. ConvNetJS (Karpathy, 2014) is a JavaScript implementation of a convolutional neural-network, developed primarily for educational purposes, which is capable of building diverse neural networks to run in a single web browser and trained using stochastic gradient descent; it can be seen as the non-distributed predecessor of MLitB. 4.3 Distributed Machine Learning The most performant deep neural network models are trained with sophisticated scientific libraries written for GPUs (Bergstra et al., 2010; Jia et al., 2014; Collobert et al., 2011) that provide orders of magnitude computational speed-ups compared to CPUs. Each implements some form of stochastic gradient descent (SGD) (Bottou, 2010) as the training algorithm. Most implementations are limited to running on the cores of a single machine and by extension the memory limitations of the GPU. Exceptionally, there are distributed deep learning algorithms that use a farm of GPUs (e.g. Downpour SGD (Dean et al., 2012)) and farms of commodity servers (e.g. COTS-HPS (Coates et al., 2013)). Other distributed ML algorithm research includes the parameter server model (Li et al., 2014), parallelized SGD (Zinkevich et al., 2010), and distributed SGD (Ahn et al., 2014). MLitB could potentially push commodity computing to the extreme using pre-existing devices, some of which may be GPU capable, with and without an organization’s existing computing infrastructure. As we discuss below, there are still many open research questions and opportunities for distributed ML algorithm research. 5 OPPORTUNITIES AND CHALLENGES In tandem with our vision, there are several directions the next version of MLitB can take, both in terms of the library itself and the potential kinds of applications a ubiquitous ML framework like MLitB can offer. We first focus on the engineering and research challenges we have discovered during the development of our prototype, along with some we expect as the project grows. Second, we look at the opportunities MLitB provides, not only based on the research directions the challenges uncovered, but also novel application areas that are perfect fits for MLitB. In Section 6 we preview the next concrete steps in MLitB development. 5.1 Challenges We have identified three keys engineering and research challenges that must be overcome for MLitB to achieve its vision of learning models a global scale. Memory Limitations State-of-the-art Neural Network models have huge numbers of parameters. This prevents them from fitting onto mobile devices. There are two possible solutions to this problem. The first solution is to learn or use smaller neural networks. Smaller NN models have shown promise on image classification performance, in particular the Network in Network (Lin et al., 2013) model from the Caffe model zoo, 13/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript is 16MB, and outperforms AlexNet which is 256MB (Jia et al., 2014). It is also possible to first train a deep neural network then use it to train a much smaller, shallow neural network (Ba and Caruana, 2014). Another solution is to distribute the NN (during training and prediction) across clients. An example of this approach is Downpour SGD (Dean et al., 2012). Communication Overhead With large models, large of numbers of parameters are communicated regularly. This is a similar issue to memory limitation and could benefit from the same solutions. However, given a fixed bandwidth and asynchronous parameter updates, we can ask what parameter updates (from master to client) and which gradients (from client to master) should be communicated. An algorithm could transmit a random subset of the weight gradients, or send the most informative. In other words, given a fixed bandwidth budget, we want to maximize the information transferred per iteration. Performance Efficiency Perhaps the biggest argument against scientific computing with JavaScript is its computation performance. We disagree that this should prevent the widespread adoption of browser-based, scientific computing because the goal of several groups to achieve native performance in JavaScript (JavaScript V8, 2014; asm.js, 2014) and GPU kernels are becoming part of existing web engines (e.g. WebCL7 ) and they can be seamlessly incorporated into existing JavaScript libraries, though they have yet to be written for ML. 5.2 Opportunities Massively Distributed Learning Algorithms The challenges just presented are obvious areas of future distributed machine learning research (and are currently being developed for the next version of MLitB). Perhaps more interesting is, at a higher level, that the MLitB vision raises novel questions about what it means to train models on a global scale. For instance, what does it mean for a model to be trained across a global internet of heterogeneous and unreliable devices? Is there a single model or a continuum of models that are consistent locally, but different from one region to another? How should a model adapt over long periods of time? These are largely untapped research areas for ML. Field Research Moving data collection and predictive models onto mobile devices makes is easy to bring models into the field. Connecting users with mobile devices to powerful NN models can aid field research by bringing the predictive models to the field, e.g. for fast labeling and data gathering. For example, a pilot program of crop surveillance in Uganda currently uses bespoke computer vision models for detecting pestilence (insect eggs, leaf diseases, etc) (Quinn et al., 2011). Projects like these could leverage publicly available, state-of-the-art computer vision models to bootstrap their field research. Privacy Preserving Computing and Mobile Health Our MLitB framework provides a natural platform for the development of real privacy-preserving application (Dwork, 2008) by naturally protecting user information contained on mobile devices, yet allowing the data to be used for valuable model development. The current version of MLitB does not provide privacy preserving algorithms such as (Han et al., 2010), but these could be easily incorporated into MLitB. It would therefore be possible for a collection of personal devices to collaboratively train machine learning models using sensitive data stored locally and with modified training algorithms that guarantee privacy. One could imagine, for example, using privately stored images of a skin disease to build a classifier based on large collection of disease exemplars, yet with the data always kept on each patient’s mobile device, thus never shared, and trained using privacy preserving algorithms. Green Computing One of our main objectives was to provide simple, cheap, distributed computing capability with MLitB. Because MLitB runs with minimal software installation (in most cases requiring none), it is possible to use this framework for low-power consumption distributed computing. By using existing organizational resources running in low-energy states (dormant or near dormant) MLitB can wake the machines, perform some computing cycles, and return them to their low-energy states. This is in stark contrast to a data center approach which has near constant, heavy energy usage (nrd, 2014). 7 WebCL by Kronos: www.khronos.org/webcl. 14/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript 6 FUTURE MLITB DEVELOPMENT The next phases of development will focus on the following directions: a visual programming user interface for model configuration, development of a library of ML models and algorithms, development of performant scientific libraries in JavaScript with and without GPUs, and model archiving with the development of a research closure specification. 6.1 Visual Programming Many ML models are constructed as chains of processing modules. This lends itself to a visual programming paradigm, where the chains can be constructed by dragging and dropping modules together. This way models can be visualized and compared, dissected, etc. Algorithms are tightly coupled to the model and a visual representation of the model can allow interaction with the algorithm as it proceeds. For example, learning rates for each layer of a neural network can be adjusted while monitoring error rates (even turned off for certain layers), or training modules can be added to improve learning of hidden layers for very deep neural networks, as done in (Szegedy et al., 2014). With a visual UI it would be easy to pull in other existing, pre-trained models, remove parts, and train on new data. For example, a researcher could start with a pre-trained image classifier, remove the last layer, and easily train a new image classifier, taking advantage of an existing, generalized image representation model. 6.2 Machine Learning Library We currently have built a prototype around an existing JavaScript implementation of DNNs (Karpathy, 2014). In the near future we plan on implementing other models (e.g. latent Dirichlet allocation) and algorithms (e.g. distributed MCMC (Ahn et al., 2014)). MLitB is agnostic to learning algorithms and therefore is a great platform for researching novel distributed learning algorithms. To do this, however, MLitB will need to completely separate machine learning model components from the MLitB network. At the moment, the prototype is closely tied to its neural network use-case. Once separated, it will be possible for external modules to be added by the open-source community. 6.3 GPU implementations Implementation of GPU kernels can bring MLitB performance up to the level of current state-of-the-art scientific libraries such as Theano (Bergstra et al., 2010; Bastien et al., 2012) and Caffe (Jia et al., 2014), while retaining the advantages of using heterogeneous devices. For example, balancing computational loads during training is very simple in MLitB and any learning algorithm can be shared by GPU powered desktops and mobile devices. Smart phones could be part of the distributed computing process by permitting the training algorithms to use short bursts of GPU power for their calculations, and therefore limiting battery drain and user disruption. 6.4 Design of Research closures MLitB can save and load JSON model configurations and parameters, allowing researchers to share and build upon other researchers’ work. However, it does not quite achieve our goal of a research closure where all aspects—code, configuration, parameters, etc–are saved into a single object. In addition to research closures, we hope to develop a model zoo, akin to Caffe’s for posting and sharing research. Finally, some kind of system for verifying models, like recomputation.org, would further strengthen the case for MLitB being truly reproducible (and provide backwards compatibility). 7 CONCLUSION In this paper we have introduced MLitB: Machine Learning in the Browser, an alternative framework for ML research based entirely on using the browser as the computational engine. The MLitB vision is based upon the overarching objectives that provide ubiquitous ML capability to every computing device, cheap distributed computing, and reproducible research. The MLitB prototype is written entirely in JavaScript and makes extensive use of existing JavaScript libraries, including Node.js for servers, Web Workers for non-blocking computation, and Web Sockets for communication between clients and servers. We demonstrated the potential of MLitB on a ML use-case: Deep Neural Networks trained with distributed Stochastic Gradient Descent using heterogenous devices, including dedicated grid-computing resources and mobile devices, using the same interface and with no client-side software installation. Clients simply connect to the server and computing begins. This use-case has provided valuable information 15/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript for future versions of MLitB, exposing both existing challenges and interesting research and application opportunities. We have also advocated for a framework which supports reproducible research; MLitB naturally provides this by allowing models and parameters to be saved to a single object which can be reloaded and used by other researchers immediately. REFERENCES (2014). Data center efficiency assessment. http://www.nrdc.org/energy/files/datacenter-efficiency-assessment-IP.pdf. Issue Paper IP:14-08-A, August 2014. Ahn, S., Shahbaba, B., and Welling, M. (2014). Distributed Stochastic Gradient MCMC. ICML. Anderson, D. P. (2004). BOINC: A system for public-resource computing and storage. In Proceedings of the Workshop on Grid Computing, Nov. 2004, pages 4–10. IEEE. Anderson, D. P., Cobb, J., Korpela, E., Lebofsky, M., and Werthimer, D. (2002). SETI@Home: an experiment in public-resource computing. Communications of the ACM, 45(11):56–61. asm.js, M. (2014). http://asmjs.org. Accessed: 2014-11-26. Ba, J. and Caruana, R. (2014). Do deep nets really need to be deep? In Advances in Neural Information Processing Systems, pages 2654–2662. Bahdanau, D., Cho, K., and Bengio, Y. (2014). Neural machine translation by jointly learning to align and translate. arXiv preprint arXiv:1409.0473. Bastien, F., Lamblin, P., Pascanu, R., Bergstra, J., Goodfellow, I., Bergeron, A., Bouchard, N., WardeFarley, D., and Bengio, Y. (2012). Theano: new features and speed improvements. arXiv preprint arXiv:1211.5590. Begoli, E. and Horey, J. (2012). Design principles for effective knowledge discovery from big data. In Software Architecture (WICSA) and European Conference on Software Architecture (ECSA), 2012 Joint Working IEEE/IFIP Conference on, pages 215–218. Bergstra, J., Breuleux, O., Bastien, F., Lamblin, P., Pascanu, R., Desjardins, G., Turian, J., Warde-Farley, D., and Bengio, Y. (2010). Theano: a CPU and GPU math expression compiler. In Proceedings of the Python for Scientific Computing Conference (SciPy). Oral Presentation. Bottou, L. (2010). Large-scale machine learning with stochastic gradient descent. In Proceedings of COMPSTAT’2010, pages 177–186. Springer. Boyd, S., Ghosh, A., Prabhakar, B., and Shah, D. (2006). Randomized gossip algorithms. Information Theory, IEEE Transactions on, 52(6):2508–2530. Can I Use (2014). Javascript API support comparison. http://caniuse.com/#cats=JS_API. Accessed: 2014-11-26. Casadevall, A. and Fang, F. C. (2010). Reproducible science. Infection and immunity, 78(12):4972–4975. Coates, A., Huval, B., Wang, T., Wu, D., Catanzaro, B., and Andrew, N. (2013). Deep learning with cots hpc systems. In Proceedings of The 30th International Conference on Machine Learning, pages 1337–1345. Collobert, R., Kavukcuoglu, K., and Farabet, C. (2011). Torch7: A matlab-like environment for machine learning. In BigLearn, NIPS Workshop, number EPFL-CONF-192376. Cushing, R., Putra, G. H. H., Koulouzis, S., Belloum, A., Bubak, M., and De Laat, C. (2013). Distributed computing on an ensemble of browsers. Internet Computing, IEEE, 17(5):54–61. Dean, J., Corrado, G., Monga, R., Chen, K., Devin, M., Mao, M., Senior, A., Tucker, P., Yang, K., Le, Q. V., et al. (2012). Large scale distributed deep networks. In Advances in Neural Information Processing Systems, pages 1223–1231. Dean, J. and Ghemawat, S. (2008). MapReduce: simplified data processing on large clusters. Communications of the ACM, 51(1):107–113. Duchi, J., Hazan, E., and Singer, Y. (2011). Adaptive subgradient methods for online learning and stochastic optimization. The Journal of Machine Learning Research, 12:2121–2159. Dwork, C. (2008). Differential privacy: A survey of results. In Theory and Applications of Models of Computation, pages 1–19. Springer. Han, S., Ng, W. K., Wan, L., and Lee, V. (2010). Privacy-preserving gradient-descent methods. Knowledge and Data Engineering, IEEE Transactions on, 22(6):884–899. Hinton, G., Deng, L., Yu, D., Dahl, G. E., Mohamed, A.-r., Jaitly, N., Senior, A., Vanhoucke, V., Nguyen, P., Sainath, T. N., et al. (2012). Deep neural networks for acoustic modeling in speech recognition: The shared views of four research groups. Signal Processing Magazine, IEEE, 29(6):82–97. 16/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript IETF (2011). The websocket protocol. http://tools.ietf.org/html/rfc6455. Accessed: 2014-12-04. JavaScript V8, C. (2014). https://developers.google.com/v8. Accessed: 2014-11-26. Jia, Y., Shelhamer, E., Donahue, J., Karayev, S., Long, J., Girshick, R., Guadarrama, S., and Darrell, T. (2014). Caffe: Convolutional architecture for fast feature embedding. arXiv preprint arXiv:1408.5093. Karpathy, A. (2014). ConvNetJS Deep Learning in the browser. http://www.convnetjs.com. Accessed: 2014-07-09. Khan, F., Foley-Bourgon, V., Kathrotia, S., Lavoie, E., and Hendren, L. (2014). Using javascript and webcl for numerical computations: A comparative study of native and web technologies. In Proceedings of the 10th ACM Symposium on Dynamic Languages, pages 91–102. ACM. Krizhevsky, A., Sutskever, I., and Hinton, G. E. (2012). Imagenet classification with deep convolutional neural networks. In Advances in neural information processing systems, pages 1097–1105. Lane, T. J., Shukla, D., Beauchamp, K. A., and Pande, V. S. (2013). To milliseconds and beyond: challenges in the simulation of protein folding. Current opinion in structural biology, 23(1):58–65. Li, M., Andersen, D. G., Park, J. W., Smola, A. J., Ahmed, A., Josifovski, V., Long, J., Shekita, E. J., and Su, B.-Y. (2014). Scaling distributed machine learning with the parameter server. In Proc. OSDI, pages 583–598. Lin, M., Chen, Q., and Yan, S. (2013). Network in network. arXiv preprint arXiv:1312.4400. Liu, S., Yang, N., Li, M., and Zhou, M. (2014). A recursive recurrent neural network for statistical machine translation. In Proceedings of ACL, pages 1491–1500. McNutt, M. (2014). Reproducibility. Science, 343(6168). Quinn, J. A., Leyton-Brown, K., and Mwebaze, E. (2011). Modeling and monitoring crop disease in developing countries. In AAAI. Ray, B., Posnett, D., Filkov, V., and Devanbu, P. (2014). A large scale study of programming languages and code quality in github. In Proceedings of the 22Nd ACM SIGSOFT International Symposium on Foundations of Software Engineering, FSE 2014, pages 155–165. ACM. Shvachko, K., Kuang, H., Radia, S., and Chansler, R. (2010). The Hadoop distributed file system. In Mass Storage Systems and Technologies (MSST), 2010 IEEE 26th Symposium on, pages 1–10. IEEE. Stodden, V., Borwein, J., and Bailey, D. H. (2013a). “setting the default to reproducible” in computational science research. Computational Science Research, 46(5). Stodden, V., Guo, P., and Ma, Z. (2013b). Toward reproducible computational research: an empirical analysis of data and code policy adoption by journals. PloS one, 8(6):e67111. Sutskever, I., Vinyals, O., and Le, Q. V. (2014). Sequence to sequence learning with neural networks. arXiv preprint arXiv:1409.3215. Szegedy, C., Liu, W., Jia, Y., Sermanet, P., Reed, S., Anguelov, D., Erhan, D., Vanhoucke, V., and Rabinovich, A. (2014). Going deeper with convolutions. CoRR, abs/1409.4842. Tilkov, S. and Vinoski, S. (2010). Node.js: Using javascript to build high-performance network programs. IEEE Internet Computing, 14(6):80–83. W3C (2014). Web workers, editor’s draft 19 may 2014. http://dev.w3.org/html5/workers. Accessed: 2014-12-04. W3Techs (2014). Usage of JavaScript for websites. http://w3techs.com/technologies/ details/cp-javascript/all/all. Accessed: 2014-06-17. Zinkevich, M., Weimer, M., Li, L., and Smola, A. J. (2010). Parallelized Stochastic Gradient Descent. In Advances in Neural Information Processing Systems, pages 2595–2603. 17/17 PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript Figure 1(on next page) Overview of MLitB. {\bf (1)} A researcher sets up a learning problem in his/her browser. {\bf (2)} Through the internet, grid and desktop machines contribute computation to solve the problem. {\bf (3)} Heterogeneous devices, such as mobile phone and tablets, connect to the same problem and contribute computation. At any time, connected clients can download the model configuration and parameters, or use the model directly in their browsing environment. Icon made by Freepik from www.flaticon.com PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript Figure 2(on next page) MLitB architecture and technologies. {\bf (1)} Servers are {\em Node.js} applications. The {\em master server} is the main server controlling communication between clients and hosts ML projects. {\bf (2)} Communication between the master server and clients occurs over Web Sockets. {\bf (3)} When heterogeneous devices connect to the master server they use Web Workers to perform different tasks. Upon connection, a UI worker, or boss, is instantiated. Web Workers perform all the other tasks on a client and are controlled by the boss. See Fig.~\ref{fig:mlitb_workers} for details. {\bf (4)} A special data worker on the client communicates with the data server using XHR. {\bf (5)} The {\em data server}, also a Node.js application, manages uploading of data in {\em zip} format and serves data vectors to the client data workers. Icon made by Freepik from www.flaticon.com PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript Figure 3(on next page) MLitB Client Workers. Each client connection to the master server initiates a {\em UI worker}, aka a {\em boss}. For uploading data from a client to the data server and for downloading data from the data server to a client, a separate Web Worker called the {\em data worker} is used. Users can add slaves through the UI worker; each slave performs a separate task using a Web Worker. Icon made by Freepik from www.flaticon.com PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript 4 Effects of scaling on power and latency. Power---measured as the number of data vectors processed per second---scales linearly until 64 nodes, when the increase in latency jumps. The ideal linear scaling is shown in grey. PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript 5 Effects of scaling on optimization. Convergence of the NN is measured in terms of test error after 50 and 100 iterations. Each point represents approximately the same wall-clock time (200/400 seconds for 50 and 100 iterations, respectively). PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript 6 CIFAR-10 project loaded in MLitB. PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript 7 Tracking model (model execution). The label of a test image is predicted using the latest NN parameters. Users can execute a NN prediction using an image stored on their device or using their device's camera. In this example, an image of a horse is correctly predicted with probability $0.687$ (the classconditional predictive probability). PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015) Reviewing Manuscript 8 Tracking mode (classification error). A test dataset can be loaded and its classification error rate tracked over iterations; here using a NN trained on CIFAR-10. PeerJ Comput. Sci. reviewing PDF \| (CS-2015:02:4063:1:1:ACCEPTED 18 Jun 2015)
https://openalex.org/W3037891775	https://www.researchsquare.com/article/rs-19168/v2.pdf	English	null	Validation of depressive symptoms, social support, and minority stress scales among gay, bisexual, and other men who have with men (GBMSM) in Nigeria, Africa: a mixed methods approach	BMC public health	2,020	cc-by	9,274	Validation of depressive symptoms, social support, and minority stress scales among Gay, Bisexual, and other men who have with men (GBMSM) in Nigeria, Africa: A Mixed Methods Approach Validation of depressive symptoms, social support, and minority stress scales among Gay, Bisexual, and other men who have with men (GBMSM) in Nigeria, Africa: A Mixed Methods Approach Validation of depressive symptoms, social support, and minority stress scales among Gay, Bisexual, and other men who have with men (GBMSM) in Nigeria, Africa: A Mixed Methods Approach Adedotun Ogunbajo (  adedotun_ogunbajo@brown.edu ) Brown University School of Public Health https://orcid.org/0000-0002-3074-6431 Stella Iwuagwu Centre for the Right to Health Rashidi Williams Equality Triangle for Health and People Development Initiative Katie B Biello Brown University School of Public Health Christopher W Kahler Brown University School of Public Health Theodorus Sandfort New York State Psychiatric Institute Matthew J Mimiaga Brown University School of Public Health Research article Keywords: Minority Stress; GBMSM; Nigeria; Mental Health; Validity; Reliability; Gay and bisexual men; Social Support; Depression; LGBT Posted Date: June 11th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-19168/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on June 29th, 2020. See the published version at https://doi.org/10.1186/s12889-020-09127-0. Validation of depressive symptoms, social support, and minority stress scales among Gay, Bisexual, and other men who have with men (GBMSM) in Nigeria, Africa: A Mixed Methods Approach Adedotun Ogunbajo (  adedotun_ogunbajo@brown.edu ) Brown University School of Public Health https://orcid.org/0000-0002-3074-6431 Stella Iwuagwu Centre for the Right to Health Rashidi Williams Equality Triangle for Health and People Development Initiative Katie B Biello Brown University School of Public Health Christopher W Kahler Brown University School of Public Health Theodorus Sandfort New York State Psychiatric Institute Matthew J Mimiaga Brown University School of Public Health Research article Keywords: Minority Stress; GBMSM; Nigeria; Mental Health; Validity; Reliability; Gay and bisexual men; Social Support; Depression; LGBT Posted Date: June 11th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-19168/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on June 29th, 2020. See the published version at https://doi.org/10.1186/s12889-020-09127-0. Research article Keywords: Minority Stress; GBMSM; Nigeria; Mental Health; Validity; Reliability; Gay and bisexual men; Social Support; Depression; LGBT Posted Date: June 11th, 2020 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on June 29th, 2020. See the published version at https://doi.org/10.1186/s12889-020-09127-0. Page 1/23 Abstract Background: Gay, bisexual, and other men who have sex with men (GBMSM) in Nigeria experience social marginalization, discrimination and violence due to their sexual identity, which may negatively impact physical, mental, and sexual health outcomes. Studies on GBMSM in Africa utilize measurement scales developed largely for populations in the Global North. The validity and reliability of these instruments—to our knowledge—have never been thoroughly investigated among GBMSM in Nigeria. The aim of the current study was to determine the validity and reliability of the English versions of the Center for Epidemiologic Studies Depression Scale (CESD-R), Multidimensional Scale of Perceived Social Support (MSPSS), and LGBT Minority Stress Measure among a large multi-state sample of GBMSM Nigeria. Methods: Between January and June 2019, we conducted cognitive interviews (N=30) and quantitative assessments (N=406) with GBMSM in Nigeria. The cognitive interviews assessed comprehension of scale items and elicited suggestions for scale modifications. The quantitative assessment was used to gather psychosocial health data and to evaluate psychometric properties and construct validity of the modified scales. We utilized confirmatory factor analysis to assess factor structure, correlation coefficients, and Cronbach’s alpha to examine scale validity and internal consistency. Results: Based on participant feedback from the cognitive interviews, we made slight modifications (i.e., culturally appropriate word substitutions) to all three scales. Results of quantitative analyses indicated good psychometric properties including high factor loadings, internal consistency and construct validity among the CESD-R, MSPSS, and LGBT Minority Stress Measure among GBMSM in Nigeria. Conclusion: These results suggests that modifying research scales to be more culturally relevant likely do not jeopardize their validity and reliability. We found that modified scales measuring depressive symptoms, perceived social support, and minority stress among GBMSM in Nigeria remained valid. More research is needed to explore whether the psychometric properties remain if the scales are translated into broken English (Pidgin) and other traditional Nigerian languages (Yoruba, Igbo and Hausa). Introduction Nigerian gay, bisexual, and other men who have sex with men (GBMSM) experience marginalization, discrimination and violence due to their sexual orientation and same-sex sexual activity[1-3], which may negatively impact physical, mental, and sexual health outcomes[4, 5]. The minority stress model posits that the prejudice, discrimination, and stigma experienced by sexual and gender minority individuals—as a result of sexual or gender identity—contributes to higher levels of stress, which may lead to mental health problems[6] and sexual risk behaviors[7, 8].Prior studies have found high levels of mental health problems (i.e. depression, anxiety, suicide ideation, and post-traumatic stress disorder (PTSD)) among Nigerian GBMSM[9, 10]. Consequently, it is important to explore these topics, especially considering the vulnerable and hostile situations Nigerian GBMSM are constantly confronted with. Page 2/23 Page 2/23 According to the minority stress model, the pathway between sexual and gender minority stress and mental health problem may be moderated by social support and coping strategies[6]. Several studies have been conducted among GBMSM that provide evidence for the theoretical underpinnings and pathways asserted by this model [11-13]; additionally, similar findings have been observed among sexual minority women[13-15], and transgender populations[16-18]. Consequently, being able to accurately measure depressive symptoms, social support, and minority stress is important to appropriately intervening to improve the quality of life of Nigerian GBMSM. Most quantitative studies conducted among African GBMSM utilize research instruments and scales developed and validated in the Global North (especially in North America and Europe) and includes participants from those settings. Consequently, these scales may contain cultural references and colloquialism that may not be applicable, easily understood or culturally relevant to African GBMSM. Formative research on the psychometric properties of these scales is essential to accurately quantify depressive symptoms, social support, and minority stress and subsequently devise intervention strategies to effectively address these issues. The aim of the current study was to adapt—after cognitive testing—and subsequently assess the validity, reliability, and psychometric properties of three widely used psychosocial measures in a large multi-state sample of GBMSM in Nigeria. The Center for Epidemiologic Studies Depression Scale (CESD-R)[19] is a 20-item scale used to screen for clinically significant depressive symptoms. The Multidimensional Scale of Perceived Social Support (MSPSS)[20], is a 12-item validated scale to measure perceived social support from family, friends, and significant others. Mixed-Methods Approach We utilized a sequential exploratory mixed method design[28], which is a methodological approach that combines qualitative and quantitative data collection and analysis in phases. In the first phase, we collected qualitative data on the cultural relevancy of the unmodified research instruments (cognitive testing) and analyzed the data. Next, we modified the instruments, based on the feedback from participants, and carried out the quantitative phase, where we tested the psychometric properties of the modified instruments. Introduction The LGBT Minority Stress Measure[21], is a 50-item scale developed to measure stress-related components of the minority stress model: prejudice events, victimization events, anticipation of rejection, identity concealment, internalized anti-LGBT stigma, everyday discrimination, and community support. These scales have been widely utilized to measure depressive symptoms, social support, and minority stress among GBMSM[22-25]. While many studies have investigated the psychometric characteristics of these scales, a vast majority been conducted in the Global North (largely in the United States of America)[21, 26, 27]. The validity and reliability of these instruments—to our knowledge—have never been investigated among GBMSM in Nigeria. Cognitive Testing Page 3/23 Participants and Procedures In January 2019, we recruited 30 GBMSM from Delta (n=15) and Lagos (n=15), Nigeria through local community-based organizations (CBOs) to participate in one-on-one cognitive interviews to assess cultural relevance and comprehension of the CESD-R, the MSPSS, and the LGBT Minority Stress. Participants were asked to provide suggestions for modification of these to make it easily understandable by Nigerian GBMSM. Inclusion criteria for participation were: 1) 18 years of age or older; 2) currently residing in Delta or Lagos; 3) cis-gender male; and 4) history of sex with another male. Peer educators at the two CBOs shared information about the cognitive testing with the target population at various community-centered events (e.g., HIV testing and counseling, health education, advocacy events, etc.) and provided study contact information to individuals who were interested. Study activities took place in private offices within our partner CBOs. The theoretical groundings for our cognitive testing approach emanate from the question-answer model, which proposes that in order for participants to accurately answer a question, they must: 1) understand the question, 2) retrieve the necessary information from their long-term memory, 3) decide what information is necessary to respond to the question, and 4) answer the question[29]. First, we read out- loud the instructions for each scale to participants. Next, we read each item and probed whether participants understood what the question was asking. We had the participants repeat back what they believed the question was attempting to ask. Next, we asked how they would modify the question to be more easily understood by and relevant to GBMSM in Nigeria. This protocol was repeated for each item within a scale. Lastly, participants were asked what overall construct the scale aimed to measure. This protocol was repeated for each individual scale. All interviewers were digitally recorded. Based on feedback from the cognitive interviews and iterative feedback from senior authors, the scales were modified and subsequently administered to a large, multi-state sample of GBMSM in Nigeria. Perceived Social Support Perceived social support was assessed using the MSPSS[20], a 12-item self-report scale used to measure perceived social support from family, friends, and significant other. These instructions were given to participants prior to completing this scale: “We are interested in how you feel about the following statements. Read each statement carefully. Indicate how you feel about each statement.” The items were scored on a 7-point Likert scale ranging from 1 “very strongly disagree” to 7 “very strongly agree”. Scores were summed and higher scores indicated greater perceived social support. We investigated the psychometric properties of the three-factors structure of the MSPSS, which assesses three distinct sources of social support (family, friends, and significant other), which has been demonstrated to have adequate data fit characteristics[20, 27, 31] Depressive Symptoms Depressive Symptoms Depressive symptoms were assessed using the CESD-R scale[19], a 20-item self-report scale used to screen for clinically significant depressive symptoms. These instructions were given to participants prior to completing this scale: “Below is a list of the ways you might have felt or behaved. Please check the box to tell me how often you have felt this way in the past week.” The items were scored on a 4-point scale ranging from 0 “not at all or less than one day” to 3 “5-7 days or nearly every day for two weeks”, and summed, with a higher score indicating more severe depressive symptoms. We investigated the psychometric properties of the one-factor structure of the CESD-R to assess overall depressive symptoms, which has been demonstrated to have adequate data fit characteristics[26, 30]. Participants and Procedures Between March and June 2019, 406 GBMSM were recruited from Abuja (n=107), Delta (n=102), Lagos (n=112), and Plateau (n=85) recruited through community-based organizations (CBOs) and snowball sampling. Peer educators, outreach workers, and key opinion leaders from CBOs based in the four study sites provided potential participants with information about the study and a study contact number. Individuals who showed interest in the study were screened for eligibility. Eligibility criteria were: 1) 18 years of age or older; 2) currently residing in one of four Nigerian states (Abuja, Delta, Lagos or Plateau); 3) identify as cis-gender male (i.e., participants who were assigned male sex at birth and currently identify as men); and 4) any self-reported history of sex (oral or anal) with another male. Eligible participants were asked to provide information about the study to other members of their social network. Data collection was conducted in the private offices of each CBO. Each participant provided verbal informed consent and completed the quantitative survey with the help of a trained research assistant. The survey took1 hour to 1.5 hours to complete. Upon completion of the survey, participants were compensated with 4,000 Naira Page 4/23 Page 4/23 (equivalent to 10 US dollars). The study protocols were approved by the institutional review boards at Brown University and the Nigerian Institute of Medical Research. (equivalent to 10 US dollars). The study protocols were approved by the institutional review boards at Brown University and the Nigerian Institute of Medical Research. Measures Tested Measures to Assess Construct Validity To assesses the validity of CESD-R, MSPSS, and the LGBT Minority Stress Measure, we chose two measures we hypothesized would be significantly (convergent validity) and non-significantly (divergent validity) correlated (Pearson correlation coefficient) with our measures. Convergent Validity. The purpose of convergent validity is to assess whether the scales are significantly related as predicted [32]. The UCLA Loneliness Scale was used to assess convergent validity as past research has demonstrated significant positive associations between depressive symptoms, minority stress and loneliness[33, 34]; and a significant negative association between social support and loneliness[35]. Loneliness was assessed using the UCLA Loneliness Scale[36], an 8-item validated scale that measures various aspects of loneliness on a 4-point Likert scale ranging from 1 “Never” to 4 “Often”. Scores were summed and higher scores indicated greater perceived loneliness. Divergent Validity. The purpose of divergent validity is to examine whether the construct of interest is different from another separate concept[32]. Healthcare utilization was used to assess divergent validity as we hypothesize that it would have smaller association with experiences of minority stress, depressive symptoms, and perceived social support. Healthcare utilization was assessed by asking participants: “When was the last time you went to a doctor for a medical check-up? A routine checkup is a general physical exam, not for a specific injury, illness or condition” with possible options response “within the last year,” “within the last two years,” “within the last five years,” “five years or more ago,” or “never”. Minority Stress Scales Five distinct constructs within minority stress were assessed using the LGBT Minority Stress Measure[21] : community connectedness, internalized stigma, rejection anticipation, identity concealment, and victimization events. These instructions were given to participants prior to completing these scales: “The next few questions will ask you about the LGBT community. LGBT stands for Lesbian, Gay, Bisexual, and Transgender individuals. Please think about your own identity within the community and your relation with the LGBT community when answering these questions.” Community connectedness was assessed using five items and scored on a 5-point Likert scale ranging from 1 “Strongly Disagree” to 5 “Strongly Agree” with a higher score indicating higher levels of community connectedness. Internalized stigma was assessed using 3 items and scored on a 5-point Likert scale ranging from 1 “Strongly Disagree to 5 “Strongly Agree” with a higher score indicating higher levels of internalized stigma. Rejection anticipation was assessed using four items and scored on a 5-point Likert scale ranging from 1 “Never Happens” to 5 “Happens all the time” with a higher sore indicating higher levels of rejection anticipation. Identity Page 5/23 Page 5/23 concealment was assessed using four items and scored on a 5-point Likert scale ranging from 1 “Never Happens” to 5 “Happens all the time” with a higher sore indicating higher levels of identity concealment. Victimization events was assessed using three items and scored on a 5-point Likert scale ranging from 1 “Never Happens” to 5 “Happens all the time” with a higher sore indicating higher levels of self-reported experiences of victimization events. We investigated the psychometric properties of the five distinctive constructs of minority stress that we were interested in (community connectedness, internalized stigma, rejection anticipation, identity concealment, and victimization events). Measures to Assess Construct Validity Cognitive Testing All interviews were transcribed by a professional transcribing company based in Nigeria. As English is the official language of Nigeria, scales were administered in English and no translation services were necessary. We analyzed the cognitive interviews consistent with best practice recommendations[37, 38] and previous research[39, 40]. The transcripts were independently analyzed by one study team member. Analyses were structured around the constructs of the question-answer model explained above. Each question within each scale was analyzed independently. We complied a comprehensive list of all suggested changes for each individual question. Modifications were made to individual questions when two or more participants suggested changes to that question. Next, all suggested modifications were Page 6/23 considered and a list of three or less possible revised questions were noted. After consultation with a group of experts—consisting of GBMSM and researchers who work with this population in Nigeria—the final version of the revised questions were reached. The most parsimonious and easily understandable questions were selected. Confirmatory Factor Analysis We used MPlus to conduct confirmatory factor analysis (CFA). CFA is a psychometric assessment that allows for testing of an a priori factor structure of a specific measurement instrument and estimation of latent constructs while correcting for measurement errors[32]. We conducted CFA, rather than exploratory factor analysis, because the scales we were validating have clearly defined subscales and constructs and have been widely utilized within the field of behavioral and public health research. Participants with any missing responses were excluded from the CFA. To assess fit of the model[41, 42], we examined the root mean square error of approximation (RSMEA) values (<0.06 considered excellent and <0.08 considered good); comparative fit index (CFI) and tucker-lewis index (TLI) values (<0.95 considered excellent and <0.90 considered good); and the akaike information criterion (AIC) assessed model parsimony, with a significant decrease in AIC suggesting a better fitting model. le 1: Sociodemographic characteristics of Cognitive Interview participants (N=30 Cognitive Interviews Demographics (N=30) Cognitive Interviews Demographics (N=30) As seen in Table 1, participants ranged in age from 20 to 40 years (mean=29.1, standard deviation [SD]= 5.3), and half (n=15, 50.0%) of participants identified as gay/homosexual. Most participants (n=19, 63.3%) reported their relationship status as single and more than three-fourths (n=23, 76.7%) had a university education or higher. Table 1: Sociodemographic characteristics of Cognitive Interview participants ( Page 7/23 Page 7/23 Demographics Mean (SD); N (%) Age (in years) 29.1 (5.3) Current Sexual Orientation Gay/Homosexual 15 (50.0%) Bisexual 15 (50.0%) Current Relationship Status Single, never been married 19 (63.3%) Long-term relationship with a man 7 (23.3%) Long-term relationship with a man 3 (10.0%) Separated 1 (3.3%) Current Housing Status Stable Housing 29 (96.7%) Unstable Housing 1 (3.3%) Educational Attainment Senior Secondary School (SSS) or lower 7 (23.3%) Some University/Vocation Education 15 (50.0%) University Degree 8 (26.7%) Current Employment Status Employed 16 (53.3%) Unemployed 14 (46.7%) Cognitive Interview Findings Cognitive Interview Findings Of the 20 statements contained in the CESD-R scale, 8 were modified (Table 3). A majority of the modifications constituted changing a few words to make the phrase more understandable (for example, we changed “I had trouble keeping my mind of what I was doing” to “I had trouble concentrating on what I was doing”). Only one of the statements was completely modified (“I could not get going” to “I lacked motivation”). Of the 12 statements contained in the MSPSS, 8 were modified (Table 3). In the significant other subscale, “special person” was replaced with “significant other”. The rest of the changes were minor word substitutions such as changing “I can talk about my problems with my family” to “I can share my problems with my family” Of the combined 16 statements contained in the 5 subscales of the LGBT Minority Stress Measure, 11 were modified (Table 3). A majority of the modifications constituted changing a few words to make the phrase more understandable (for example, we changed “I feel like I am a part of the LGBT community” to “I feel like I am a member of the LGBT community”). Cognitive Interviews Demographics (N=30) A few statements were completely changed (for Page 8/23 Page 8/23 example, we changed “If I was offered the chance to be someone who is not LGBT I would accept the opportunity” to “If I could change from being LGBT to be straight, I would.” example, we changed “If I was offered the chance to be someone who is not LGBT I would accept the opportunity” to “If I could change from being LGBT to be straight, I would.” Quantitative Sample Demographics (N=406) As seen in Table 2, participants ranged in age from 18 to 60 years (mean=29.2, SD=5.8), the majority (n=238, 58.6%) identified as bisexual, and 61.6% were single. We had an ethnically diverse sample (20.3% were Igbo, 17.8% were Hausa, 17.7% were Yoruba, 15.7% were Urhobo, and many more ethnic groups were represented) Most (n=238, 61.8%) participants reported experiencing high financial hardship and 22.3% reported a history of incarceration (n=86). One-fourth (n=99, 24.8%) of participants reported living with HIV and one third (n=124, 32.3%) reported a sexually transmitted infection diagnosis in the previous year. Quantitative Sample Demographics (N=406) phic characteristics of Quantitative Assessment participants (N= Demographics Mean (SD); N (%) Age (in years) 29.2 (5.8) Current Sexual Orientation Gay/Homosexual 160 (39.4%) Bisexual 238 (58.6%) Current Relationship Status Single 250 (61.6%) Not Single 150 (36.9%) Religious Affiliation Christian 253 (62.3%) Muslim 116 (28.6%) Other 30 (7.4%) Monthly income (in Naira) 0-10,000 105 (25.9%) 10,000-30,000 106 (26.1%) 30,000-50,000 81 (20.0%) 50,000-100,000 54 (13.3%) 100,000+ 49 (12.1%) Employment Status Employed 319 (78.6%) Unemployed 81 (20.0%) Table 2: Sociodemographic characteristics of Quantitative Assessment participants (N=406) Demographics Mean (SD); N (%) Age (in years) 29.2 (5.8) Current Sexual Orientation Gay/Homosexual 160 (39.4%) Bisexual 238 (58.6%) Current Relationship Status Single 250 (61.6%) Not Single 150 (36.9%) Religious Affiliation Christian 253 (62.3%) Muslim 116 (28.6%) Other 30 (7.4%) Monthly income (in Naira) 0-10,000 105 (25.9%) 10,000-30,000 106 (26.1%) 30,000-50,000 81 (20.0%) 50,000-100,000 54 (13.3%) 100,000+ 49 (12.1%) Employment Status Employed 319 (78.6%) Unemployed 81 (20.0%) ciodemographic characteristics of Quantitative Assessment participants (N=406) Table 2: Sociodemographic characteristics of Quantitative Assessment participa Page 9/23 Table 3: Original and modified measurement scale items Table 3: Original and modified measurement scale items Table 3: Original and modified measurement scale items Page 9/23 Scale Original Item Modified Item CESD-R My appetite was poor I didn’t have an appetite I could not shake off the blues I could not change my bad mood (final item) I could not think straight (other suggested item) I could not think properly (other suggested item) I had trouble keeping my mind of what I was doing I had trouble concentrating on what I was doing I could not get going I lacked motivation I lost interest in my usual activities I lost interest in my daily activities I felt fidgety I felt nervous I wanted to hurt myself I wanted to harm myself I had a lot of trouble getting to sleep I had trouble sleeping MSPSS There is a special person who is around when I am in need There is a significant other I can lean on There is a special person with whom I can share my joys and sorrows There is a significant other who I can share my joys and sorrows with I get the emotional help and support I need from my family I get the love and support I need from my family I have a special person who is a real source of comfort to me I have a significant other who is a real source of comfort to me My friends really try to help me My friends are there for me I can talk about my problems with my family I can share my problems with my family I have friends with whom I can share my joys and sorrows I have friends who I can share my joys and sorrows with There is a special person in my life who cares about my feelings There is a significant other in my life who cares about my feelings The LGBT Minority Stress Measure Community Connectedness I feel like I am a part of the GBMSM community I feel like I am a member of the LGBT community I feel that I could find information and pamphlets on GBMSM issues I feel that I could find information, books, flyers on LGBT issues I feel that I could find professional services for GBMSM issues if I needed to I feel that I could find friendly services for LGBT issues if I needed to I feel that I could find a public space that is supportive of GBMSM activities I feel like there is a safe space where LGBT social activities can take place Internalized Stigma If I was offered the chance to be someone who is not GBMSM, I would accept the opportunity If I could change from being LGBT to straight, I would I envy people who are not GBMSM. Quantitative Sample Demographics (N=406) I am jealous of people who are not LGBT Rejection Anticipation I brace myself to be treated disrespectfully because I am GBMSM I prepare myself to be treated disrespectfully because I am LGBT Identity Concealment I avoid telling people about certain things in my life that might imply I am GBMSM I avoid telling people about certain things in my life that might make them think I am LGBT because I do not want others to know I am GBMSM not want others to know I am LGBT I do not bring a date to social events because I do not want others to know I am GBMSM I do not bring a date to social gathering/ parties because I do not want others to know I am LGBT Victimization Events I have been verbally harassed or called names because I am GBMSM I have been called names or insulted because I am LGBT Confirmatory Factor Analysis Results (N=406) CESD-R All items significantly loaded onto the one-factor depression construct except item #9 (I slept much more than usual), (β=0.25) (Table 4). The fit indices for the one-factor model were acceptable (RMSEA=0.10; CFI=0.82; TLI=0.80). This provides evidence that the CESD-R is a reasonable instrument to ascertain depressive symptoms among Nigerian GBMSM. Table 4: Standardized factor loadings from confirmatory factor analysis for the Center for Epidemiologic Studies Depression Scale (CESD-R). because I do not want others to know I am GBMSM not want others to know I am LGBT I do not bring a date to social events because I do not want others to know I am GBMSM I do not bring a date to social gathering/ parties because I do not want others to know I am LGBT Victimization Events I have been verbally harassed or called names because I am GBMSM I have been called names or insulted because I am LGBT Confirmatory Factor Analysis Results (N=406) CESD-R CESD-R All items significantly loaded onto the one-factor depression construct except item #9 (I slept much more than usual), (β=0.25) (Table 4). The fit indices for the one-factor model were acceptable (RMSEA=0.10; CFI=0.82; TLI=0.80). This provides evidence that the CESD-R is a reasonable instrument to ascertain depressive symptoms among Nigerian GBMSM. Table 4: Standardized factor loadings from confirmatory factor analysis for the Center for Epidemiologic Studies Depression Scale (CESD-R). Table 4: Standardized factor loadings from confirmatory factor analysis for the Center for Epidemiologic Studies Depression Scale (CESD-R). Page 11/23 Item 1 Factor Depression (α=0.93) 1. I could not change my bad mood* 0.665 1. I felt depressed 0.710 1. I felt sad 0.656 1. Nothing made me happy 0.709 1. I lost interest in my daily activities* 0.710 1. I didn’t have an appetite* 0.452 1. I lost a lot of weight without trying to 0.617 1. My sleep was restless 0.679 1. I slept much more than usual 0.246 1. I had trouble sleeping 0.646 1. I had trouble concentrating on what I was doing 0.680 1. I could not focus on the important things 0.762 1. I felt like a bad person 0.668 1. I did not like myself 0.651 1. I lacked motivation* 0.649 1. I was tired all the time 0.684 1. I felt like I was moving too slowly 0.659 1. I felt nervous* 0.743 0 595 All items significantly loaded onto their respective factors (Table 5). The three-factor model measures three distinct sources of perceived social support (family, friends, and significant other). The fit indices for the one-factor model were acceptable (RMSEA=0.09; CFI=0.92; TLI=0.90). The better fit statistics and multidimensional nature of social support leads us to conclude that the three-factor model is parsimonious. Table 5: Standardized factor loadings from confirmatory factor analysis for Multidimensional Scale of Perceived Social Support (MSPSS). CESD-R Item 3 Factor Significant Other (α=0.81) Family (α=0.80) Friends (α=0.82) here is a significant other I can lean on* 0.672 here is a significant other who I can share my joys and rrows with* 0.675 have a significant other who is a real source of comfort me* 0.790 here is a significant other in my life who cares about my elings* 0.708 y family really tries to help me 0.820 get the love and support I need from my family* 0.835 an share my problems with my family* 0.581 y family is willing to help me make decisions 0.579 y friends are there for me* 0.755 an count on my friends when things go wrong 0.821 have friends who I can share my joys and sorrows with* 0.674 an talk about my problems with my friends 0.673 modified from original question ings from confirmatory factor analysis for Multidimensional Scale of Perceived Table 5: Standardized factor loadings from confirmatory factor analysis for Multidimensional Scale of Perceived Social Support (MSPSS). able 5: Standardized factor loadings from confirmatory factor analysis for Multid ocial Support (MSPSS). All items significantly loaded onto their respective factors (Table 6). The five-factor model measures five distinct experiences of minority stress (community connectedness, internalized stigma, rejection anticipation, identity concealment, and victimization events). The good fit statistics (RMSEA=0.08; CFI=0.91; TIL=0.90) and multidimensional nature of minority stress leads us to conclude that these measures accurately assessed various dimensions of experiences of minority stress among Nigerian GBMSM. CESD-R Page 14/23 Item 5 Factor Community Connectedness (α=0.86) Internalized Stigma (α=0.80) Rejection Anticipation (α=0.72) Identity Concealment (α=0.86) Victimization Events (α=0.92) eel connected to other LGBT ople 0.697 eel like I am a member of the GBT community 0.673 eel that I could find information, oks, flyers on LGBT issues* 0.849 eel that I could find friendly rvices for LGBT issues if I needed * 0.801 eel like there is a safe space here LGBT social activities can ke place* 0.664 I could change from being LGBT be straight, I would* 0.841 wish I wasn’t LGBT 0.904 am jealous of people who are not GBT* 0.554 hen I meet someone new, I worry at they secretly do not like me cause I am LGBT 0.645 prepare myself to be treated srespectfully because I am LGBT* 0.550 expect that others will not accept e because I am LGBT 0.764 worry about what will happen if ople find out I am LGBT 0.593 avoid telling people about certain ings in my life that might make em think I am LGBT* 0.865 avoid talking about my love life cause I do not want others to 0.888 do not bring a date to social thering/ parties because I do not ant others to know I am LGBT* 0.693 imit what I share on social media, who can see it, because I do not ant others to know I am LGBT 0.591 have been called names or insulted cause I am LGBT* 0.864 thers have threatened to harm me cause I am LGBT 0.905 have been bullied by others cause I am LGBT 0.898 modified from original question Scale Properties (N=406) Scores on the CESD-R (20 items) ranged from 0 to 55 (M=11.4, SD=12.2). Internal consistency was high (Cronbach’s α=0.93). Scores on the MSPSS (12 items) ranged from 12 to 84 (M=58.4, SD=12.6). Internal consistency was high (Cronbach’s α=0.86). Scores on the community connectedness subscale (5 items) ranged from 5 to 25 (M=19.8, SD=4.5). Internal consistency was high (Cronbach’s α=0.86). Scores on the internalized stigma subscale (3 items) ranged from 3 to 15 (M=8.0, SD=3.5). Internal consistency was high (Cronbach’s α=0.80). Scores on the rejection anticipation subscale (4 items) ranged from 4 to 20 (M=9.8, SD=4.0). Internal consistency was acceptable (Cronbach’s α=0.72). Scores on the identity concealment subscale (4 items) ranged from 4 to 20 (M=13.1, SD=4.8). Internal consistency was high (Cronbach’s α=0.86). CESD-R Scores on the victimization events subscale (3 items) ranged from 3 to 15 (M=5.4, SD=3.2). Internal consistency was very high (Cronbach’s α=0.92). Scores on the CESD-R (20 items) ranged from 0 to 55 (M=11.4, SD=12.2). Internal consistency was high (Cronbach’s α=0.93). Scores on the MSPSS (12 items) ranged from 12 to 84 (M=58.4, SD=12.6). Internal consistency was high (Cronbach’s α=0.86). Scores on the community connectedness subscale (5 items) ranged from 5 to 25 (M=19.8, SD=4.5). Internal consistency was high (Cronbach’s α=0.86). Scores on the internalized stigma subscale (3 items) ranged from 3 to 15 (M=8.0, SD=3.5). Internal consistency was high (Cronbach’s α=0.80). Scores on the rejection anticipation subscale (4 items) ranged from 4 to 20 (M=9.8, SD=4.0). Internal consistency was acceptable (Cronbach’s α=0.72). Scores on the identity concealment subscale (4 items) ranged from 4 to 20 (M=13.1, SD=4.8). Internal consistency was high (Cronbach’s α=0.86). Scores on the victimization events subscale (3 items) ranged from 3 to 15 (M=5.4, SD=3.2). Internal consistency was very high (Cronbach’s α=0.92). Scores on the CESD-R (20 items) ranged from 0 to 55 (M=11.4, SD=12.2). Internal consistency was high (Cronbach’s α=0.93). Scores on the MSPSS (12 items) ranged from 12 to 84 (M=58.4, SD=12.6). Internal consistency was high (Cronbach’s α=0.86). Scores on the community connectedness subscale (5 items) ranged from 5 to 25 (M=19.8, SD=4.5). Internal consistency was high (Cronbach’s α=0.86). Scores on the internalized stigma subscale (3 items) ranged from 3 to 15 (M=8.0, SD=3.5). Internal consistency was high (Cronbach’s α=0.80). Scores on the rejection anticipation subscale (4 items) ranged from 4 to 20 (M=9.8, SD=4.0). Internal consistency was acceptable (Cronbach’s α=0.72). Scores on the identity concealment subscale (4 items) ranged from 4 to 20 (M=13.1, SD=4.8). Internal consistency was high (Cronbach’s α=0.86). Scores on the victimization events subscale (3 items) ranged from 3 to 15 (M=5.4, SD=3.2). Internal consistency was very high (Cronbach’s α=0.92). Construct Validity Analysis To evaluate the convergent validity (Table 7), correlations (Pearson’s coefficients) were conducted between the CESD-R, the MSPSS, the LGBT Minority Stress Measure, and the UCLA Loneliness Scale. We hypothesized that there will be a positive significant relationship between depressive symptoms, minority stress, and loneliness. We also hypothesized a significant inverse relationship between perceived social support and loneliness. Upon calculation of Pearson’s coefficient, the UCLA Loneliness Scale was found to be correlated, but not strongly, in the expected direction with CESD-R (r=0.38, p<0.01), perceived social support (family [r=-0.23, p<.01], friends [r=-0.26, p<0.01], and significant other [r=-0.20, p<0.01]) and all but one of the minority stress scales (community connectedness [r=-0.09, not significant], internalized stigma Page 16/23 Page 16/23 [r=0.10, p<0.05], rejection anticipation [r=0.23, p<0.01], identity concealment [r=0.14, p<0.01], and victimization events [r=0.19, p<0.01]), thereby demonstrating evidence for convergent validity. Additionally, the social support and minority stress subscales were highly correlated with each other (\|r\|=0.23-0.48), p<0.01), providing evidence for concurrent validity. To evaluate discriminant validity (Table 7), correlations (Pearson’s coefficients) were conducted between CESD-R, MSPSS, LGBT Minority Stress Measure, and healthcare utilization. We hypothesized that there will be no statistically significant relationship between depressive symptoms, perceived social support, minority stress and healthcare utilization. Upon calculation of Pearson’s coefficient, healthcare utilization was found to be not strongly correlated with the CESD-R (r=0.02, not significant), perceived social support (family [r=-0.01, not significant], friends [r=-0.07, not significant], and significant other [r=-0.05, not significant]) and all the minority stress scales (community connectedness [r=0.09, not significant], internalized stigma [r=-0.03, not significant], rejection anticipation [r=0.06, not significant], identity concealment [r=-0.02, not significant], and victimization events [r=0.05, not significant]), thereby demonstrating strong evidence for discriminant validity. Discussion This is the first study, as far as we are aware, to investigate the psychometric properties of key psychosocial research instruments among Nigerian GBMSM. Confirmatory factor analysis, internal consistency, and construct validity all suggest that the CESD-R, the MSPSS, and the LGBT Minority Stress Measure have strong validity and reliability in this sample, especially after the modifications (21 out of 48 total question items were modified). These findings are especially strong given the geographical and ethnic diversity represented in our sample. Results suggest that modified versions of psychosocial scales can accurately measure the same constructs as the original scales even after modification to be more culturally relevant. The structural validity of these scales has major implications for use in future behavioral research and intervention studies among Nigerian and generally among African GBMSM. We found that the CESD-R had high factor loadings, internal consistency and construct validity. However, an item related to sleep quality (‘I slept much more than usual’) had poor factor loading on both the overall depression scale and sleep construct within the overall scale. This might be attributable to a differing cultural conceptualization of sleep, where quality of sleep may vary vastly on basis of age, geographical location, ethnic group membership, amongst other factors. Lastly, it is important to understand that sleep disturbance, as a result of depressive symptoms, can manifest as either hypersomnia or insomnia. The sleep-related question in the CESD-R only assesses hypersomnia, and not insomnia, which may partially explain the observed low factor loading. Additionally, we found the one- factor measurement of depressive symptoms to be parsimonious, providing more evidence that the CESD-R might be a reliable scale to measure depressive symptoms among Nigerian GBMSM. This is especially relevant since previous studies have found high prevalence of depressive symptoms among Nigerian GBMSM[9, 10]. Similar to the CESD-R, the MSPSS had sound psychometric properties, which suggests its’ potential to accurately measure perceived social support from three distinct sources—family, friends, and significant other—among Nigerian GBMSM. This is of particular importance as social support has been hypothesized as a potential moderator of the association between experiences of minority stress and mental health problems in sexual minority communities[17, 43]. Perceived social support might reduce or diminish the effects of minority stress on mental health problems among individuals with high levels of perceived social support compared to individuals with low levels of perceived social support. Construct Validity Analysis Table 7: Correlation demonstrating convergent and divergent validity between scales and validity measures Measure 1 2 3 4 5 6 7 8 9 10 11 ESD-R 1 -.26* -.04 .15 .37 .17 .24 .23 .27 0.38 0.02 SPSS (family) 1 .48 0.53 .23 -0.18 -0.17 -0.48 -0.28 0.23 -0.01 SPSS (friends) 1 0.56 .25 -0.20 -0.23 -0.17 -0.29 0.26 -0.07 SPSS (significant her) 1 0.19 0.10 0.14 -0.19 -0.24 0.20 -0.05 ommunity onnectedness 1 -0.29 -0.27 -0.25 -0.38 0.09 0.09 ternalized Stigma 1 0.29 0.25 0.33** 0.10* -0.03 ejection Anticipation 1 0.27 0.24 0.23 0.06 entity Concealment 1 0.30 0.14* -0.02 ctimization Events 1 0.19** 0.05 oneliness 1 0.04 ealthcare Utilization 1 P<.05, P<.01 Table 7: Correlation demonstrating convergent and divergent validity between ting convergent and divergent validity between scales and validity measures ation demonstrating convergent and divergent validity between scales and valid P<.05, **P<.01 Page 17/23 Conclusion The current study provides further evidence that cultural adaptation of research instruments does not jeopardize the validity and reliability of the original scales. If the goal of public health research is to prevent disease on a population level, it is incumbent upon population health researchers to ensure that the measurement scales that are being utilized are culturally relevant and have sound psychometric properties. Our study provides evidence that both goals can be successfully accomplished. Discussion Consequently, effectively measuring levels of perceived social support might help aid the design of interventions to help Nigerian GBMSM buffer the stress associated with their sexual orientation by identifying possible sources of social support and coping mechanisms. We found that the LGBT Minority Stress Measure provided an accurate measurement of the various aspects of minority stress (community connectedness, internalized stigma, rejection anticipation, identity concealment, and victimization events). There was further evidence that each subscale independently measured a specific construct of minority stress. This finding enables researchers who are interested in Page 18/23 Page 18/23 specific constructs within minority stress to administer that specific subscale independent of the longer, comprehensive scale. Our findings should be interpreted in the context of some limitations. While the sample was geographically and demographically diverse, the scales were evaluated among a sample mainly recruited through GBMSM community-based organizations and GBMSM social networks. This sampling frame limits our ability to generalize our findings to GBMSM who do not seek services at these organizations or who are outside of the social networks sampled. Further, social desirability bias may have influenced participants’ responses because the assessment was completed together with trained research assistants. Additionally, while we conducted cognitive interviews prior to administering the amended scales, we did not assess test-retest reliability, which would have provided stronger evidence of the validity of the scales after modification. Future studies should investigate whether these psychometric properties hold for scales that have been translated into Nigerian pidgin English or native languages (Yoruba, Igbo, & Hausa). Translating these scales into the major local languages will broaden the reach of public health research by allowing individuals who feel comfortable communicating in these local languages to participate. It is also important to investigate the temporality of minority stress and its’ effects on depressive symptoms, and social support, which is best accomplished by conducting longitudinal studies. Abbreviations Page 19/23 Abbreviations GBMSM: Gay, bisexual, and other men who have sex with men PTSD: Post-traumatic stress disorder CESD-R: Center for Epidemiologic Studies Depression Scale MSPSS: Multidimensional Scale of Perceived Social Support CBOs: Community-based organizations CFA: Confirmatory factor analysis Declarations GBMSM: Gay, bisexual, and other men who have sex with men PTSD: Post-traumatic stress disorder Authors' contributions AO, KBB, and MJM conceptualized the research study. AO conducted the research study, analyzed the data and drafted the article. SI, RW, KBB, CWK, TGMS, and MJM provided feedback on multiple drafts and approved the final manuscript draft. All authors read and approved the final manuscript. Consent for publication Not Applicable Availability of data and materials Data cannot be shared publicly because of the sensitive nature of the research subjects and the context in which the research was conducted. However, a codebook of data analysis procedures which allows replication of the reported results has been deposited for public access at the Brown University depository and is available here: https://doi.org/10.26300/bmq7-9k58. Funding This work was supported by grant R36 DA047216 from the National Institute on Drug Abuse (PI: Adedotun Ogunbajo), and the Robert Wood Johnson Health Policy Research Scholars Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests Coauthor Katie B. Biello is an Associate Editor of this journal . Ethics approval and consent to participate The study protocols were approved by the institutional review boards at Brown University and the Nigerian Institute of Medical Research. Each participant provided verbal informed consent, due to the sensitive nature of the research project. Verbal informed consent was suggested by both review boards during the review process and thereby adopted. Acknowledgments We will like to thank all the participants of the study for their time and efforts. We would also like to thank the staff at Centre for Right to Health (Abuja) Equality Triangle Initiative (Delta), Improved Sexual Health and Rights Advocacy Initiative (ISHRAI, Lagos) and Hope Alive Health Awareness Initiative (Plateau). We also extend our gratitude to Olubiyi Oludipe (ISHRAI), Bala Mohammed Salisu (Hope Alive Health Awareness Initiative), Chucks Onuoha, Prince Bethel, Eke Chukwudi, Tochukwu Okereke, Josiah Djagvidi, and Odi Iorfa Agev, for providing logistical support to the project. Page 20/23 References 1. Sekoni AO, Ayoola OO, Somefun EO: Experiences of social oppression among men who have sex with men in a cosmopolitan city in Nigeria. HIV/AIDS (Auckland, NZ) 2015, 7:21. 2. Allman D, Adebajo S, Myers T, Odumuye O, Ogunsola S: Challenges for the sexual health and social acceptance of men who have sex with men in Nigeria. Culture, health & sexuality 2007, 9(2):153-168. 3. Melhado L: In Nigeria, anti-gay law associated with increased stigma and discrimination. International Perspectives on Sexual and Reproductive Health 2015, 41(3):167. 4. Makanjuola O, Folayan MO, Oginni OA: On being gay in Nigeria: Discrimination, mental health distress, and coping. Journal of Gay & Lesbian Mental Health 2018, 22(4):372-384. 5. Oginni OA, Mosaku KS, Mapayi BM, Akinsulore A, Afolabi TO: Depression and associated factors among gay and heterosexual male university students in Nigeria. Archives of sexual behavior 2018, 47(4):1119-1132. 5. Oginni OA, Mosaku KS, Mapayi BM, Akinsulore A, Afolabi TO: Depression and associated factors among gay and heterosexual male university students in Nigeria. Archives of sexual behavior 2018, 47(4):1119-1132. 6. Meyer IH, Frost D: Minority stress and the health of sexual minorities. Handbook of psychology and sexual orientation 2013:252-266. 6. Meyer IH, Frost D: Minority stress and the health of sexual minorities. Handbook of psychology and sexual orientation 2013:252-266. 7. Fields EL, Bogart LM, Galvan FH, Wagner GJ, Klein DJ, Schuster MA: Association of discrimination- related trauma with sexual risk among HIV-positive African American men who have sex with men. American journal of public health 2013, 103(5):875-880. 8. Hidalgo MA, Cotten C, Johnson AK, Kuhns LM, Garofalo R: ‘Yes, I am more than just that’: Gay/bisexual young men residing in the United States discuss the influence of minority stress on their sexual risk behavior prior to HIV infection. International Journal of Sexual Health 2013, 25(4):291-304. 9. Ogunbajo A, Oke T, Jin H, Rashidi W, Iwuagwu S, Harper GW, Biello KB, Mimiaga MJ: A syndemic of psychosocial health problems is associated with increased HIV sexual risk among Nigerian gay, bisexual, and other men who have sex with men (GBMSM). AIDS care 2019:1-6. 10. Rodriguez-Hart C, Bradley C, German D, Musci R, Orazulike I, Baral S, Liu H, Crowell TA, Charurat M, Nowak RG: The Synergistic Impact of Sexual Stigma and Psychosocial Well-Being on HIV Testing: A Mixed-Methods Study Among Nigerian Men who have Sex with Men. AIDS and Behavior 2018, 22(12):3905-3915. 11. 19. Eaton WW, Smith C, Ybarra M, Muntaner C, Tien A: Center for Epidemiologic Studies Depression Scale: review and revision (CESD and CESD-R). 2004. 19. Eaton WW, Smith C, Ybarra M, Muntaner C, Tien A: Center for Epidemiologic Studies Depression Scale: review and revision (CESD and CESD-R). 2004. 20. Zimet GD, Dahlem NW, Zimet SG, Farley GK: The multidimensional scale of perceived social support. Journal of personality assessment 1988, 52(1):30-41. 20. Zimet GD, Dahlem NW, Zimet SG, Farley GK: The multidimensional scale of perceived social support. Journal of personality assessment 1988, 52(1):30-41. 21. Outland PL: Developing the LGBT minority stress measure. Colorado Sta 21. Outland PL: Developing the LGBT minority stress measure. Colorado State University. Libraries; 2016. 21. Outland PL: Developing the LGBT minority stress measure. Colorado State University. Libraries; 2016. 21. Outland PL: Developing the LGBT minority stress measure. Colorado State University. Libraries; 2016. 22. Hussen SA, Easley KA, Smith JC, Shenvi N, Harper GW, Camacho-Gonzalez AF, Stephenson R, del Rio 22. Hussen SA, Easley KA, Smith JC, Shenvi N, Harper GW, Camacho-Gonzalez AF, Stephenson R, del Rio C: Social capital, depressive symptoms, and HIV viral suppression among young black, gay, bisexual and other men who have sex with men living with HIV. AIDS and Behavior 2018, 22(9):3024-3032. 22. Hussen SA, Easley KA, Smith JC, Shenvi N, Harper GW, Camacho-Gonzalez AF, Stephenson R, del Rio C: Social capital, depressive symptoms, and HIV viral suppression among young black, gay, bisexual and other men who have sex with men living with HIV. AIDS and Behavior 2018, 22(9):3024-3032. 23. Mimiaga MJ, Reisner SL, Cranston K, Isenberg D, Bright D, Daffin G, Bland S, Driscoll MA, VanDerwarker R, Vega B: Sexual mixing patterns and partner characteristics of black MSM in Massachusetts at increased risk for HIV infection and transmission. Journal of Urban Health 2009, 86(4):602-623. 24. Ogunbajo A, Iwuagwu S, Williams R, Biello K, Mimiaga MJ: Awareness, willingness to use, and history of HIV PrEP use among gay, bisexual, and other men who have sex with men in Nigeria. PloS one 2019, 14(12). 24. Ogunbajo A, Iwuagwu S, Williams R, Biello K, Mimiaga MJ: Awareness, willingness to use, and history of HIV PrEP use among gay, bisexual, and other men who have sex with men in Nigeria. PloS one 2019, 14(12). 25. Fisher CM, Woodford MR, Gartner RE, Sterzing PR, Victor BG: Advancing research on LGBTQ microaggressions: A psychometric scoping review of measures. Journal of homosexuality 2019, 66(10):1345-1379. 25. References Meyer IH: Minority stress and mental health in gay men. Journal of health and social behavior 1995:38-56. 11. Meyer IH: Minority stress and mental health in gay men. Journal of health and social behavior 1995:38-56. 12. Hatzenbuehler ML, Nolen-Hoeksema S, Erickson SJ: Minority stress predictors of HIV risk behavior, substance use, and depressive symptoms: results from a prospective study of bereaved gay men. Health Psychology 2008, 27(4):455. 12. Hatzenbuehler ML, Nolen-Hoeksema S, Erickson SJ: Minority stress predictors of HIV risk behavior, substance use, and depressive symptoms: results from a prospective study of bereaved gay men. Health Psychology 2008, 27(4):455. 13. Feinstein BA, Goldfried MR, Davila J: The relationship between experiences of discrimination and mental health among lesbians and gay men: An examination of internalized homonegativity and rejection sensitivity as potential mechanisms. Journal of consulting and clinical psychology 2012, 80(5):917. 13. Feinstein BA, Goldfried MR, Davila J: The relationship between experiences of discrimination and mental health among lesbians and gay men: An examination of internalized homonegativity and rejection sensitivity as potential mechanisms. Journal of consulting and clinical psychology 2012, 80(5):917. Page 21/23 Page 21/23 14. Lehavot K, Simoni JM: The impact of minority stress on mental health and substance use among sexual minority women. Journal of consulting and clinical psychology 2011, 79(2):159. 15. Kuyper L, Fokkema T: Loneliness among older lesbian, gay, and bisexual adults: The role of minority stress. Archives of sexual behavior 2010, 39(5):1171-1180. 16. Testa RJ, Michaels MS, Bliss W, Rogers ML, Balsam KF, Joiner T: Suicidal ideation in transgender people: Gender minority stress and interpersonal theory factors. Journal of abnormal psychology 2017, 126(1):125. 17. Scandurra C, Amodeo AL, Valerio P, Bochicchio V, Frost DM: Minority stress, resilience, and mental health: A study of Italian transgender people. Journal of Social Issues 2017, 73(3):563-585. 18. Rood BA, Reisner SL, Surace FI, Puckett JA, Maroney MR, Pantalone DW: Expecting rejection: Understanding the minority stress experiences of transgender and gender-nonconforming individuals. Transgender Health 2016, 1(1):151-164. 19. Eaton WW, Smith C, Ybarra M, Muntaner C, Tien A: Center for Epidemiologic Studies Depression Scale: review and revision (CESD and CESD-R). 2004. Fisher CM, Woodford MR, Gartner RE, Sterzing PR, Victor BG: Advancing research on LGBTQ microaggressions: A psychometric scoping review of measures. Journal of homosexuality 2019, 66(10):1345-1379. 26. Van Dam NT, Earleywine M: Validation of the Center for Epidemiologic Studies Depression Scale— Revised (CESD-R): Pragmatic depression assessment in the general population. Psychiatry research 2011, 186(1):128-132. 26. Van Dam NT, Earleywine M: Validation of the Center for Epidemiologic Studies Depression Scale— Revised (CESD-R): Pragmatic depression assessment in the general population. Psychiatry research 2011, 186(1):128-132. 27. Canty-Mitchell J, Zimet GD: Psychometric properties of the Multidimensional Scale of Perceived Social Support in urban adolescents. American journal of community psychology 2000, 28(3):391- 400. 27. Canty-Mitchell J, Zimet GD: Psychometric properties of the Multidimensional Scale of Perceived Social Support in urban adolescents. American journal of community psychology 2000, 28(3):391- 400. Page 22/23 28. Creswell JW, Clark VLP: Designing and conducting mixed methods research: Sage publications; 2017. Page 22/23 28. Creswell JW, Clark VLP: Designing and conducting mixed methods research: Sage publications; 2017. Page 22/23 Page 22/23 29. Tourangeau R: Cognitive sciences and survey methods. Cognitive aspects of survey methodology: Building a bridge between disciplines 1984:73-100. 30. Edwards MC, Cheavens JS, Heiy JE, Cukrowicz KC: A reexamination of the factor structure of the Center for Epidemiologic Studies Depression Scale: Is a one-factor model plausible? Psychological assessment 2010, 22(3):711. 31. Kazarian SS, McCabe SB: Dimensions of social support in the MSPSS: Factorial structure, reliability, and theoretical implications. Journal of Community Psychology 1991, 19(2):150-160. 32. Boateng GO, Neilands TB, Frongillo EA, Melgar-Quiñonez HR, Young SL: Best practices for developing and validating scales for health, social, and behavioral research: a primer. Frontiers in public health 2018, 6. 33. Su X, Zhou AN, Li J, Shi L-e, Huan X, Yan H, Wei C: Depression, loneliness, and sexual risk-taking among HIV-negative/unknown men who have sex with men in China. Archives of sexual behavior 2018, 47(7):1959-1968. 34. Grov C, Golub SA, Parsons JT, Brennan M, Karpiak SE: Loneliness and HIV-related stigma explain depression among older HIV-positive adults. AIDS care 2010, 22(5):630-639. 35. Kao JC, Chuong A, Reddy MK, Gobin RL, Zlotnick C, Johnson JE: Associations between past trauma, current social support, and loneliness in incarcerated populations. Health & justice 2014, 2(1):7. 36. Hays RD, DiMatteo MR: A short-form measure of loneliness. Journal of personality assessment 1987, 51(1):69-81. 37. Beatty PC, Willis GB: Research synthesis: The practice of cognitive interviewing. 19. Eaton WW, Smith C, Ybarra M, Muntaner C, Tien A: Center for Epidemiologic Studies Depression Scale: review and revision (CESD and CESD-R). 2004. Public opinion quarterly 2007, 71(2):287-311. 38. Fowler Jr FJ, Lloyd SJ, Cosenza CA, Wilson IB: Coding cognitive interviews: an approach to enhancing the value of cognitive testing for survey question evaluation. Field Methods 2016, 28(1):3- 20. 39. Reisner SL, Conron KJ, Tardiff LA, Jarvi S, Gordon AR, Austin SB: Monitoring the health of transgender and other gender minority populations: validity of natal sex and gender identity survey items in a US national cohort of young adults. BMC public health 2014, 14(1):1224. 40. Wilson IB, Fowler FJ, Cosenza CA, Michaud J, Bentkover J, Rana A, Kogelman L, Rogers WH: Cognitive and field testing of a new set of medication adherence self-report items for HIV care. AIDS and Behavior 2014, 18(12):2349-2358. 40. Wilson IB, Fowler FJ, Cosenza CA, Michaud J, Bentkover J, Rana A, Kogelman L, Rogers WH: Cognitive and field testing of a new set of medication adherence self-report items for HIV care. AIDS and Behavior 2014, 18(12):2349-2358. 41. Marsh HW, Hau K-T, Wen Z: In search of golden rules: Comment on hypothesis-testing approaches to setting cutoff values for fit indexes and dangers in overgeneralizing Hu and Bentler's (1999) findings. Structural equation modeling 2004, 11(3):320-341. 41. Marsh HW, Hau K-T, Wen Z: In search of golden rules: Comment on hypothesis-testing approaches to setting cutoff values for fit indexes and dangers in overgeneralizing Hu and Bentler's (1999) findings. Structural equation modeling 2004, 11(3):320-341. 42. Brown TA: Confirmatory factor analysis for applied research: Guilford Publications; 2014. 42. Brown TA: Confirmatory factor analysis for applied research: Guilford Publications; 2014. 43. Wong CF, Schrager SM, Holloway IW, Meyer IH, Kipke MD: Minority stress experiences and psychological well-being: The impact of support from and connection to social networks within the Los Angeles house and ball communities. Prevention Science 2014, 15(1):44-55. 43. Wong CF, Schrager SM, Holloway IW, Meyer IH, Kipke MD: Minority stress experiences and psychological well-being: The impact of support from and connection to social networks within the Los Angeles house and ball communities. Prevention Science 2014, 15(1):44-55. Page 23/23 Page 23/23
https://openalex.org/W4290095242	https://hal.inrae.fr/hal-02663922/file/12953_20091028104822848_1.pdf	English	null	MATER protein expression and intracellular localization throughout folliculogenesis and preimplantion embryo development in the bovine	HAL (Le Centre pour la Communication Scientifique Directe)	2,006	cc-by	7,834	To cite this version: Sophie Pennetier, Christine Perreau, Svetlana Uzbekova, Aurore Thelie, Bernadette Delaleu, et al.. MATER protein expression and intracellular localization throughout folliculogenesis and preim- plantion embryo development in the bovine. BMC Developmental Biology, 2006, 6 (26), 9 p. 10.1186/1471-213X-6-26. hal-02663922 This article is available from: http://www.biomedcentral.com/1471-213X/6/26 This article is available from: http://www.biomedcentral.com/1471-213X/6/26 © 2006 Pennetier et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Centra BMC Developmental Biology Open Acces Research article MATER protein expression and intracellular localization throughout folliculogenesis and preimplantation embryo development in the bovine Sophie Pennetier, Christine Perreau, Svetlana Uzbekova, Aurore Thélie, Bernadette Delaleu, Pascal Mermillod and Rozenn Dalbiès-Tran* Address: Physiologie de la Reproduction et des Comportements, UMR 6175 Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique/Université François Rabelais de Tours/Haras Nationaux, F-37380 Nouzilly, France Email: Sophie Pennetier - pennetie@ifrance.com; Christine Perreau - Christine.Perreau@tours.inra.fr; Svetlana Uzbekova - Svetlana.Uzbekova@tours.inra.fr; Aurore Thélie - Aurore.Thelie@tours.inra.fr; Bernadette Delaleu - Bernadette.Delaleu@tours.inra.fr; Pascal Mermillod - mermillo@tours.inra.fr; Rozenn Dalbiès- Tran* - dalbies@tours.inra.fr * Corresponding author Abstract Published: 06 June 2006 BMC Developmental Biology 2006, 6:26 doi:10.1186/1471-213X-6-26 Received: 25 January 2006 Accepted: 06 June 2006 This article is available from: http://www.biomedcentral.com/1471-213X/6/26 © 2006 Pennetier et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central Abstract Background: Mater (Maternal Antigen that Embryos Require), also known as Nalp5 (NACHT, leucine rich repeat and PYD containing 5), is an oocyte-specific maternal effect gene required for early embryonic development beyond the two-cell stage in mouse. We previously characterized the bovine orthologue MATER as an oocyte marker gene in cattle, and this gene was recently assigned to a QTL region for reproductive traits. Results: Here we have analyzed gene expression during folliculogenesis and preimplantation embryo development. In situ hybridization and immunohistochemistry on bovine ovarian section revealed that both the transcript and protein are restricted to the oocyte from primary follicles onwards, and accumulate in the oocyte cytoplasm during follicle growth. In immature oocytes, cytoplasmic, and more precisely cytosolic localization of MATER was confirmed by immunohistochemistry coupled with confocal microscopy and immunogold electron microscopy. By real-time PCR, MATER messenger RNA was observed to decrease strongly during maturation, and progressively during the embryo cleavage stages; it was hardly detected in morulae and blastocysts. The protein persisted after fertilization up until the blastocyst stage, and was mostly degraded after hatching. A similar predominantly cytoplasmic localization was observed in blastomeres from embryos up to 8-cells, with an apparent concentration near the nuclear membrane. Conclusion: Altogether, these expression patterns are consistent with bovine MATER protein being an oocyte specific maternal effect factor as in mouse. ent on maternal transcripts and proteins synthesized dur- ing oogenesis. Maternal factors are able to support the first ent on maternal transcripts and proteins synthesized dur- ing oogenesis. Maternal factors are able to support the first BMC Developmental Biology Open Access BMC Developmental Biology 2006, 6:26 doi:10.1186/1471-213X-6-26 BMC Developmental Biology 2006, 6:26 doi:10.1186/1471-213X-6-26 esea c a t c e MATER protein expression and intracellular localization throughout folliculogenesis and preimplantation embryo development in the bovine Address: Physiologie de la Reproduction et des Comportements, UMR 6175 Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique/Université François Rabelais de Tours/Haras Nationaux, F-37380 Nouzilly, France Email: Sophie Pennetier - pennetie@ifrance.com; Christine Perreau - Christine.Perreau@tours.inra.fr; Svetlana Uzbekova - Svetlana.Uzbekova@tours.inra.fr; Aurore Thélie - Aurore.Thelie@tours.inra.fr; Bernadette Delaleu - Bernadette.Delaleu@tours.inra.fr; Pascal Mermillod - mermillo@tours.inra.fr; Rozenn Dalbiès- Tran* - dalbies@tours.inra.fr * Corresponding author Received: 25 January 2006 Accepted: 06 June 2006 Published: 06 June 2006 Published: 06 June 2006 HAL Id: hal-02663922 https://hal.inrae.fr/hal-02663922v1 Submitted on 31 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. http://www.biomedcentral.com/1471-213X/6/26 http://www.biomedcentral.com/1471-213X/6/26 http://www.biomedcentral.com/1471-213X/6/26 plantation embryos until the blastocyst stage and is mostly degraded after hatching. cleavages, while blastocyst formation involves both maternal and embryonic factors. Over the last years, oocyte-restricted maternal effect genes have been the focus of much attention due to their specific expression profile and crucial function in early embryo development. They are predominantly expressed in oocyte, remain present in early embryos and then are degraded at the time of maternal-to-embryo transition (MET), without compensation by embryonic transcription. Functional studies based on knock-out mouse models have demon- strated their essential role in preimplantation embryo development, whereas functions in the oocyte itself have not been elucidated until this day. Antibody characterization First, we checked our antipeptide serum and purified anti- body for sensitivity and specificity by western blotting (Fig. 1). Under normal exposure, a single intense band was detected at the MATER protein predicted molecular weight (121 kDa) with as few as 10 oocytes. No such band was detected in a protein extract from cumulus cells in huge excess (see the amount of TUBULIN), although faint bands were observed at various molecular weights. Specif- icity was further confirmed by the absence of MATER sig- nal in oocytes using preimmune serum. Mater (Maternal Antigen that Embryos Require) is one such oocyte-specific maternal effect genes and was first identified in mouse [1,2]. The transcript and protein expression profiles have been investigated during oogene- sis and preimplantation embryo development. Mater transcript and protein are first detected in oocyte from pri- mary follicles and accumulate during oocyte growth. The transcript level decreases after fertilization as shown by ribonuclease protection assay or DNA array [3,4]). The protein remains abundant until the morula stage and is still present in blastocysts [3]. Mater-null females present a normal phenotype regarding folliculogenesis, ovulation and fertilization, but their embryos do not develop beyond the 2-cell stage coincident with the maternal-to- embryo transition. Mater precise function remains to be elucidated, although the global transcription decrease described in two-cell embryos lacking MATER may sug- gest a role in embryonic genome activation [2]. In our pre- vious work, we identified the bovine orthologue of Mater. The longest open reading frame encodes a putative pro- tein of 1098 amino acids (121 kDa). As its human coun- terpart, bovine MATER includes the 3 domains characteristics of the Nacht, Leucine rich repeat and Pyrin domain containing (NALP) family: a N-terminal Pyrin domain, followed by a NACHT domain and twelve C-ter- minal leucine rich repeats of the ribonuclease inhibitor subtype (LRR-RI). It is localized within a QTL region for reproductive traits [5]. MATER transcript pattern, i.e. its tissue distribution and its disappearance in blastocyst, appeared in agreement with bovine MATER also being an oocyte-specific maternal effect gene and suggested a con- served function as in mouse. Background i l i g Preimplantation embryo development is largely depend- Page 1 of 9 (page number not for citation purposes) Page 1 of 9 (page number not for citation purposes) BMC Developmental Biology 2006, 6:26 development Evolution of MATER messenger RNAs during during oocyte maturation, fertilization and preimplantation embryo development was followed by real time PCR. Triplicate reactions were run onto four independent sam- ples at each stage. The level relative to immature oocytes is shown (Fig. 4A). Detection level decreased by 72% dur- ing maturation, then remained stable during fertilization. It decreased progressively during the cleavage stages, up to 10% of its initial level in 5 to 8 cell-embryos. In morulae and blastocysts, amplicons were produced only in one or two samples; the level of polyadenylated transcript was less than 0.5% of its initial value. Western blotting was then performed to characterize MATER protein expression (Fig. 4B). MATER was abundant during oocyte matura- tion, and after fertilization until the expanded blastocyst stage. Most protein was then degraded after hatching. This pattern was reproducibly observed with two independent oocyte and embryo collections. By contrast, our positive control β-TUBULIN increased in hatched blastocysts (Fig. 4B). To further characterize MATER gene expression during fol- liculogenesis, we studied its expression pattern at the pro- tein level. Using anti-MATER serum, the protein was exclusively detected in oocyte and no staining was observed in granulosa or theca cells. MATER was detected in oocyte of primary follicles at a low level, with a very dis- tinctive pattern: the staining localized to a single dot (Fig. 3A) which was specifically and reproductively observed. As folliculogenesis proceeds, staining intensity increases in oocyte from preantral to small antral and antral folli- cles (Fig. 3B–D), suggesting an accumulation of the pro- tein. Interestingly, on sections that went through the nucleus in pre-antral and early antral follicles, the protein appeared excluded from the germinal vesicle (Fig. 3B, C). Primary follicule A Ovarian localization of bovine MATER transcript by in situ hybridization Figure 2 Ovarian localization of bovine MATER transcript by in situ hybridization. Bright field (A, D) and dark field (B, C, E, F) photomicrographs of adjacent ovarian sections showing primary follicles (A-C, arrows), and an antral follicle (D-F) hybridized with either antisense (A, B, D, E) or sense (C, F) MATER-[35S] riboprobes. Scale bar: 100 μm. Primary follicule Antral follicle A D B E C F D D A MATER expression throughout folliculogenesis The expression of MATER transcript during folliculogene- sis was analysed using in situ hybridization onto ovarian sections. As previously described [6], MATER expression was restricted to the oocyte within bovine ovary. In this experiment, a signal could be detected in oocyte as early as the primary follicle stage and at a higher level in oocyte IO Cc IO Cc IO Cc pre-immune anti-sera purified antibody - 116kD MATER IO Cc IO Cc IO Cc pre-immune anti-sera purified antibody - 116kD MATER Characterization of anti-MATER serum and purified antibody by Western blot Figure 1 Characterization of anti-MATER serum and purified antibody by Western blot. Detection of MATER in imma- ture oocytes (IO) but not in cumulus cells (Cc) with anti- peptide serum or purified antibody, nor with the preimmune serum. Molecular weight is indicated on the left. IO Cc IO Cc IO Cc pre-immune anti-sera purified antibody - 116kD αααα-TUBULIN MATER IO Cc IO Cc IO Cc pre-immune anti-sera purified antibody - 116kD αααα-TUBULIN MATER pre-immune anti-sera purified antibody pre-immune anti-sera purified antibody αααα-TUBULIN αααα-TUBULIN To reinforce this hypothesis, we needed to refine tran- script expression during folliculogenesis, and mostly to characterize the expression and localization of the pro- tein. In this study, we show that bovine MATER transcript and protein are expressed in the oocyte as early as the pri- mary follicle stage and accumulate during folliculogene- sis. The protein localizes in the cytosol of immature oocytes. It remains abundant in the cytoplasm of preim- Characterization of anti-MATER serum and purified antibody by Western blot Figure 1 Characterization of anti-MATER serum and purified antibody by Western blot. Detection of MATER in imma- ture oocytes (IO) but not in cumulus cells (Cc) with anti- peptide serum or purified antibody, nor with the preimmune serum. Molecular weight is indicated on the left. p y yg Characterization of anti-MATER serum and purified antibody by Western blot. Detection of MATER in imma- ture oocytes (IO) but not in cumulus cells (Cc) with anti- peptide serum or purified antibody, nor with the preimmune serum. Molecular weight is indicated on the left. Page 2 of 9 (page number not for citation purposes) Page 2 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/6/26 BMC Developmental Biology 2006, 6:26 http://www.biomedcentral.com/1471-213X/6/26 from antral follicle. Hybridization with the corresponding sense probe was used as negative control (Fig. 2). Page 3 of 9 (page number not for citation purposes) MATER protein localization in oocyte and preimplantation embryos B C p p y Immunohistochemistry onto ovary sections suggested that MATER could be predominantly cytoplasmic. To refine protein localization, we performed confocal micro- scopy analysis on oocytes from 3 to 8 mm follicles and preimplantation embryos, after immunofluorescent stain- ing using purified antipeptide (Fig. 5). Firstly, we observed oocytes at germinal vesicle (GV) and nuclear envelope break down (NEBD) stages, as discriminated by LAMIN A/C staining (Fig. 5A). In most GV oocytes, MATER protein was predominantly localized in the cyto- plasm (Fig. 5A, left panel); however, a few oocytes (less than 10%) presented a distinct pattern, with staining within both the cytoplasm and the nucleus (Fig. 5A, mid- dle panel). In oocytes characterized by degradation of LAMIN A/C (Fig. 5A, right panel), MATER was distributed throughout the oocyte, only excluded from the chromatin area as revealed by Hoechst staining. Then we analysed mature oocytes, zygotes and preimplantation embryos (Fig. 5B). MATER was predominantly detected in the cyto- plasm, with the protein apparently concentrating in the cortical region of mature oocytes, zygotes and embryos at least until the 8-cell stage. In zygotes to 8-cell embryos, increased staining around the chromatin area (as revealed by Hoechst staining, not shown) suggested that MATER also accumulated around the nuclear membrane. In expanded blastocysts, MATER was distributed evenly between inner cell mass and trophectoderm. A dramatic decrease was observed in hatched blastocysts, as previ- ously observed by Western blotting. As negative control, B C B E E F C Ovarian lo hybridizatio Figure 2 Ovarian localization of bovine MATER transcript by in situ hybridization. Bright field (A, D) and dark field (B, C, E, F) photomicrographs of adjacent ovarian sections showing primary follicles (A-C, arrows), and an antral follicle (D-F) hybridized with either antisense (A, B, D, E) or sense (C, F) MATER-[35S] riboprobes. Scale bar: 100 μm. Page 3 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/6/26 BMC Developmental Biology 2006, 6:26 Ovarian localization of bovine MATER protein by immunohistochemistry Figure 3 Ovarian localization of bovine MATER protein by immunohistochemistry. Adjacent ovarian sections showing pri- mary follicle (A, E), pre-antral follicle (B, F), early antral follicle (C, G) and over 1 mm antral follicle (D, H) incubated with either anti-MATER serum (A-D) or preimmune serum (E-H) followed by biotinylated horse anti-rabbit IgG antibody. Scale bar: 100 μm. Inset in A is the same oocyte shown at higher magnification. MATER protein localization in oocyte and preimplantation embryos primary follicle pre-antral follicle early antral follicle antral follicle A E C G D H B F A early antral follicle C G antral follicle D H early antral follicle pre-antral follicle primary follicle antral follicle D C G H p y y g Ovarian localization of bovine MATER protein by immunohistochemistry. Adjacent ovarian sections showing pri- mary follicle (A, E), pre-antral follicle (B, F), early antral follicle (C, G) and over 1 mm antral follicle (D, H) incubated with either anti-MATER serum (A-D) or preimmune serum (E-H) followed by biotinylated horse anti-rabbit IgG antibody. Scale bar: 100 μm. Inset in A is the same oocyte shown at higher magnification. no staining was revealed using rabbit IgG as primary anti- body (not shown). MATER gene expression throughout bovine folliculogenesis and preimplantation embryo development In a previous study, we demonstrated that MATER tran- script was restricted to the oocyte within bovine ovary [6]. Here, we have refined our expression analysis. By in situ hybridization and immunohistochemistry experiments, we detected MATER transcript and protein in oocytes as early as the primary follicle stage (Fig. 2 and 3), as previ- ously described in mouse [1,3]. This shows that at least some transcripts support immediate translation, but it does not exclude that others are stored within the oocyte cytoplasm. Such an early protein synthesis may appear premature since functional studies did not reveal a delete- rious effect of Mater absence at this early stage, but rather in the 2-cell embryo [2]. Indeed, ZAR1, another mouse oocyte-specific maternal effect protein, was also detected in mouse primary follicles [7]. Several scenarios may rec- oncile these apparent discrepancies. The first hypothesis is that MATER indeed plays a role in the oocyte, but that another gene may be redundant at this stage, at least in Mater-null females. Obvious candidates are genes of the NALP family, and especially oocyte restricted members such as NALP9 in bovine [8] or the Nalp9 or Nalp4 genes in mouse [9,10]. To our knowledge, there are no func- tional data on these genes from knock-out models or other targeted inhibition experiments. This early expres- sion may also be indicative of an upstream role of MATER in a cascade of events, as demonstrated for the mouse To refine MATER protein subcellular localization, trans- mission electron microscopy analysis was performed on ultrathin sections of immature oocytes using purified antipeptide. MATER protein localization in oocyte and preimplantation embryos Immunogold particules were diffusely present in the cytosol at a low density. MATER protein was not detected in the nucleus, nor in organelles including mitochondria (Fig. 6). As a negative control, no particles were observed when omitting the primary antibody (not shown). Page 4 of 9 (page number not for citation purposes) Discussion In this study, we characterized the bovine MATER gene and its expression throughout folliculogenesis and in vitro preimplantation embryo development. We demon- strated that both the transcript and protein were present in oocyte from primary follicle and accumulated thereafter. During preimplantation embryo development, MATER messengers were hardly detected after the morula stage, while the protein remained present until the blastocyst stage before rapid degradation after hatching. In imma- ture oocytes and in embryos, the protein was predomi- nantly cytoplasmic. Page 4 of 9 (page number not for citation purposes) Page 4 of 9 (page number not for citation purposes) BMC Developmental Biology 2006, 6:26 http://www.biomedcentral.com/1471-213X/6/26 Localization of MATER protein in bovine oocytes and preim- plantation embryos by confocal microscopy Figure 5 Localization of MATER protein in bovine oocytes and preimplantation embryos by confocal microscopy. Oocytes and embryos were incubated with anti-MATER pep tide purified antibody (1:250) and revealed by green staining. Scale bar: 50 μm. (A) Oocytes at GV or NEBD stage. Lamins A/C are stained in red (middle row). Chromatin is stained in blue with Hoechst 33258 (lower row). (B) immature oocytes (IO), in vitro matured oocytes (MO), in vitro cultured zygote (1C), 2-, 4-, 8- cell embryos, morula (mo), expanded blasto- t (EB) d h t h d bl t t (HB) GV (A) (B) MATER MATER + LAMIN A/C (merged) Hoechst Mo GV NEBD 1C MO IO 2C Mo 8C 4C EB HB 1C MO IO Expression of bovine MATER in oocytes and preimplantation embryos Figure 4 Expression of bovine MATER in oocytes and preim- plantation embryos. Developmental stages are immature oocytes (IO), in vitro matured oocytes (MO), in vitro cultured zygote (1C), 2-, 4-, 5- to 8- cell embryos, morula (mo), expanded blastocysts (EB) and hatched blastocysts (HB). (A) Quantification of the messenger RNA using real-time PCR. Relative level to immature oocytes is shown (mean ± SEM). In late stage embryos, transcript level is indicated but too low to be seen on the histogram. Different superscripts indi- cate a significant difference (P ≤ 0.05). (B) Detection of the protein by Western blot; α-TUBULIN is shown as a positive control. Discussion (A) 0,0000 0,0016 0,0003 0,0 0,2 0,4 0,6 0,8 1,0 1,2 IO MO 1C 2C 4C 5-8C mo EB HB relative mRNA level * ** a a a b c c c b (B) IO MO 1C 2C 4C 5-8C mo EB HB MATER D-TUBULIN GV (A) MATER MATER + LAMIN A/C (merged) Hoechst GV NEBD (A) (A) (A) 0,0000 0,0016 0,0003 0,0 0,2 0,4 0,6 0,8 1,0 1,2 IO MO 1C 2C 4C 5-8C mo EB HB relative mRNA level * ** a a a b c c c b (B) Mo 1C MO IO 2C Mo 8C 4C EB HB 1C MO IO (B) IO IO (B) IO MO 1C 2C 4C 5-8C mo EB HB MATER D-TUBULIN (B) 8C 2C 4C EB Mo p y p p y g Expression of bovine MATER in oocytes and preim- plantation embryos. Developmental stages are immature oocytes (IO), in vitro matured oocytes (MO), in vitro cultured zygote (1C), 2-, 4-, 5- to 8- cell embryos, morula (mo), expanded blastocysts (EB) and hatched blastocysts (HB). (A) Quantification of the messenger RNA using real-time PCR. Relative level to immature oocytes is shown (mean ± SEM). In late stage embryos, transcript level is indicated but too low to be seen on the histogram. Different superscripts indi- cate a significant difference (P ≤ 0.05). (B) Detection of the protein by Western blot; α-TUBULIN is shown as a positive control. p y p p y g Expression of bovine MATER in oocytes and preim- plantation embryos. Developmental stages are immature oocytes (IO), in vitro matured oocytes (MO), in vitro cultured zygote (1C), 2-, 4-, 5- to 8- cell embryos, morula (mo), expanded blastocysts (EB) and hatched blastocysts (HB). (A) Quantification of the messenger RNA using real-time PCR. Relative level to immature oocytes is shown (mean ± SEM). In late stage embryos, transcript level is indicated but too low to be seen on the histogram. Different superscripts indi- cate a significant difference (P ≤ 0.05). (B) Detection of the protein by Western blot; α-TUBULIN is shown as a positive control. Localization of MATER protein in bovine oocytes and preim- plantation embryos by confocal microscopy Figure 5 Localization of MATER protein in bovine oocytes and preimplantation embryos by confocal microscopy. Oocytes and embryos were incubated with anti-MATER pep- tide purified antibody (1:250) and revealed by green staining. Scale bar: 50 μm. http://www.biomedcentral.com/1471-213X/6/26 BMC Developmental Biology 2006, 6:26 Subcellular localization of MATER protein in bovine imma- ture oocytes by transmission electron microscopy Figure 6 Subcellular localization of MATER protein in bovine immature oocytes by transmission electron micros- copy. Immunogold particles are pointed at by arrows. (A) region including a mitochondria (B) a detail of the nuclear membrane, separating the nucleus (below) and the cytoplasm (above). 200nm A 200nm B nuclear membrane nucleus cytoplasm mitochondria early embryos is of maternal origin. Second, degradation of MATER protein after hatching appears temporally con- trolled and substrate specific, since most proteins are rather synthesized at this stage following the MET. The bovine protein in fact persists longer than its mouse coun- terpart, which was mostly degraded by the expanded blas- tocyst stage. This is consistent with active degradation of MATER involving factors synthesized from embryonic transcripts in both species. The ubiquitin-proteasome pathway is one possible mechanism, and ubiquitin tran- scripts were actually shown to increase in bovine blasto- cysts [12]. On the other hand, ubiquitin cross-reactive structures were detected preferentially in the trophoblast over the the inner cell mass [13] whereas here we observe a simultaneous degradation of MATER in both structures at hatching (Fig. 5). Overall, the pattern of expression of bovine MATER, i.e. non reactivation at MET and degrada- tion of the protein before implantation, supports the hypothesis that it is a maternal effect gene as its mouse orthologue. A B Discussion (A) Oocytes at GV or NEBD stage. Lamins A/C are stained in red (middle row). Chromatin is stained in blue with Hoechst 33258 (lower row). (B) immature oocytes (IO), in vitro matured oocytes (MO), in vitro cultured zygote (1C), 2-, 4-, 8- cell embryos, morula (mo), expanded blasto- cysts (EB) and hatched blastocysts (HB). oocyte-specific homeobox gene Nobox [11]. Alternatively, the protein may be synthesized despite not being required at this stage, simply because specifically repressing the gene would be more complex for the cell than support minimal expression. Overall, we observed that immunos- taining of MATER protein increased in parallel with folli- cle development, from primary to large antral. This must involve a growing level of translation; in addition, it is possible that once synthesized, the protein remains stable for a long period of time and persists in the oocyte during folliculogenesis. Neo-synthesized proteins would then add to those already present, and early synthesis would allow for the accumulation of a largest stockpile of MATER. Page 5 of 9 (page number not for citation purposes) Page 5 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/6/26 MATER protein intracellular localization in bovine oocytes and preimplantation embryos Data from immunohistochemistry on ovary sections sug- gested that MATER protein was stored in the cytoplasm of oocyte during folliculogenesis (Fig. 3B, C). Intracellular localization of MATER was further investigated in oocytes and early embryos by immunofluorescence coupled to confocal microscopy analysis (Fig. 5). In most immature oocytes, MATER was predominantly localized in the cyto- plasm with an accumulation around the nuclear mem- brane and was hardly detected in the germinal vesicle. A few oocytes displayed a different pattern of protein distri- bution, where MATER was detected both in the cytoplasm and the germinal vesicle (Fig. 5A). After NEBD, the pro- tein was uniformly distributed throughout the cytoplasm and was only excluded from the chromatin area (Fig. 5A). Such transient nuclear localization of MATER was not described in mouse oocytes. We propose two hypotheses consistent with our observations. It may be that MATER passively diffuses to the germinal vesicle as the nuclear membrane starts to disaggregate, even though lamins are still detected. Alternatively, we can speculate that MATER actively translocates to the nucleus just before NEBD. MATER sequence does not display a nuclear localization signal as analyzed by the PredictNLS server [14,15]. But neither was such signal identified in the mouse oocyte specific variant of DNA (cytosine-5)-methyl-transferase 1 (Dnmt1o), whose temporarily controlled nucleocytoplas- mic trafficking was nevertheless demonstrated: this pro- tein localizes to the cytoplasm of oocytes and preimplantation embryos with exclusion from all nuclei except those of 8-cell embryos [16]. MATER might be involved in the short burst of transcription that has been Subcellular ture oocyt Figure 6 p y y py g Subcellular localization of MATER protein in bovine immature oocytes by transmission electron micros- copy. Immunogold particles are pointed at by arrows. (A) region including a mitochondria (B) a detail of the nuclear membrane, separating the nucleus (below) and the cytoplasm (above). By real-time PCR, bovine MATER transcripts showed a massive deadenylation and/or degradation during matu- ration (Fig 4A). Following fertilization, the messengers slowly decreased in embryos until the 5- to 8-cell stage; it was hardly detected in morulae and blastocysts, confirm- ing our previous report [6]. We did not evidence neo-syn- thesis or polyadenylation of MATER transcripts neither transiently nor after the MET. By contrast, we have dem- onstrated by Western-blot and immunofluorescence cou- pled to confocal microscopy that the protein is present at all stages, and displays a sharp decrease only after hatch- ing (Fig. Page 6 of 9 (page number not for citation purposes) Oocyte collection and in vitro embryo production Oocyte collection and in vitro embryo production Oocyte collection and in vitro embryo production Ovaries from adult cows (unless otherwise specified) were collected at a slaughterhouse and the oocyte-cumulus complexes were aspirated from 3–6 mm follicles, selected based on morphological criteria and washed in saline solution (for details see [21]). Denuded immature and in vitro matured oocytes were obtained as previously described [22]. Briefly, oocyte-cumulus complexes were denuded by mechanical treatment either before or after in vitro maturation in TCM199 (Sigma, Saint Quentin Falla- vier, France) supplemented with 10 ng/ml epidermal growth factor and 10% fetal calf serum for 24 hr at 39°C in water-saturated air with 5% carbon dioxide. For in vitro fertilization, groups of 50 intact in vitro matured oocyte- cumulus complexes were transferred into 500 μl fertiliza- tion medium containing 106 motile spermatozoa. 24 hr later, presumptive zygotes were denuded. Groups of 25 zygotes were transferred into 25 μl droplets (under paraf- fin oil) of modified synthetic oviduct fluid supplemented with 5% fetal calf serum. Embryos were cultured at 39°C in a water-saturated atmosphere with 5% CO2/5% O2/ 90% N2. Groups of 10 embryos were collected over preim- plantation development period: zygote and 2-cell embryos (day 2), 4-cell, 5 to 8-cell embryos (day 3), morulae (day 5), expanded blastocysts (day 6) and hatched blastocysts (day 8). All oocyte and embryo sam- ples were stored frozen at -80°C. Two and four independ- ent sets of oocytes and embryos were collected for immunological and real-time PCR analyses respectively. In GV oocytes, MATER localization was refined by immu- nogold coupled to transmission electron microscopy analysis. The protein was detected in the cytosol but not in organelles (Fig. 6). This pattern differs from MATER dis- tribution in mouse, where the protein was detected in the nucleus close to nuclear pores and in mitochondria [3]. Based on the presence of a leucine-rich repreat domain homologous to a porcine ribonuclease inhibitor, mouse MATER has been suggested to play a role in mRNA stabil- ity. Altogether, our observations on bovine MATER distri- bution are consistent with this hypothesis. In oocytes, MATER would stabilize transcripts from the moment when they are exported from the nucleus and during their storage in the oocyte cytoplasm up until the time when there are recruited for translation in the oocyte or after fer- tilization. Conclusion We have demonstrated that MATER transcript and protein are specifically expressed in oocyte from the primary folli- cle onwards in bovine ovary. The protein is stored in the cytoplasm of growing oocytes and persists during matura- tion, fertilization and in embryos until the expanded blas- tocyst stage. It is mostly degraded after hatching. MATER protein intracellular localization in bovine oocytes and preimplantation embryos 4 and 5). These distinct patterns for the transcript and the protein provide valuable information. First, later persistence of the protein as compared to the messenger is consistent with MATER being a fairly stable protein. This, together with the accumulation of MATER during follicu- logenesis, suggests that at least some protein detected in By real-time PCR, bovine MATER transcripts showed a massive deadenylation and/or degradation during matu- ration (Fig 4A). Following fertilization, the messengers slowly decreased in embryos until the 5- to 8-cell stage; it was hardly detected in morulae and blastocysts, confirm- ing our previous report [6]. We did not evidence neo-syn- thesis or polyadenylation of MATER transcripts neither transiently nor after the MET. By contrast, we have dem- onstrated by Western-blot and immunofluorescence cou- pled to confocal microscopy that the protein is present at all stages, and displays a sharp decrease only after hatch- ing (Fig. 4 and 5). These distinct patterns for the transcript and the protein provide valuable information. First, later persistence of the protein as compared to the messenger is consistent with MATER being a fairly stable protein. This, together with the accumulation of MATER during follicu- logenesis, suggests that at least some protein detected in Page 6 of 9 (page number not for citation purposes) Page 6 of 9 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/6/26 http://www.biomedcentral.com/1471-213X/6/26 BMC Developmental Biology 2006, 6:26 suggested to occur in bovine oocyte-cumulus complexes just before chromatin condenses [17-19] Therefore MATER transcript and protein expression pat- terns are consistent with a maternal effect function con- served in bovine. Challenging functional approaches are now required to confirm this hypothesis. Then, we followed MATER localization after oocyte matu- ration, fertilization and during preimplantation embryo development (Fig. 5B). Localization was predominantly cytoplasmic in embryos at least until the 8 cell stage. We noticed that the protein appeared to concentrate in the cortical region of mature oocytes and embryos up to 8- cells, as previously observed in mouse preimplantation embryos [3]. A similar peripheral concentration had also been reported for the ovary-specific 2',5'-oligoadenylate synthetase-like protein (OAS1D) in metaphase II oocytes [20], and for DNMT1o in mature oocytes, 2- and 4-cell embryos [16]. The authors suggested that DNMT1o may be sequestered near the plasma membrane through inter- action with annexin V, a calcium-sensitive phospholipid binding protein [16]. MATER includes protein-protein interacting domains and identifying potential partners may help elucidating this intriguing distribution. Oocyte collection and in vitro embryo production Through a similar peri-nuclear accumulation in early embryos, MATER could protect the rare transcripts produced from the embryonic genome at these stages. Finally, MATER is degraded after hatching, once the embryo synthesizes large amount of messengers and pro- teins. This model is consistent with the phenotype of embryos from Mater null female mice: transcripts required for development beyond the 2-cell stage (e.g. in genome activation) would not be stabilized, resulting in developmental block. In situ hybridization Ovaries from 6 month old calves were embedded in Tis- sue-Tek medium (Sekura, Bayer Diagnostics, France), fro- zen in liquid nitrogen and then serially sectioned (10 μm) with cryostat. The sections were fixed in 4% paraformalde- hyde. A 391 bp MATER PCR fragment (corresponding to nt 2819–3209 of the coding sequence) was subcloned in Dual Promoter pCRII plasmid (Invitrogen, Cergy-Ponto- ise, France). Sense and antisense probes were generated using Riboprobe combined system SP6/T7 (Promega, Charbonnières, France) and labelled with [35S]-UTP. Hybridization and washed were performed as described elsewhere [23]. Sections were counterstained with hema- toxylin. Transmission electron microscopy (TEM) Immature oocytes were fixed in 4% paraformaldehyde for 20 min at 4°C. Subsequently, they were washed in phos- phate buffer 0.1 M, incubated in ammonium chloride 0.05 M in phosphate buffer for 30 min at room tempera- ture and washed in phosphate buffer. Then, oocytes were dehydrated in ethanol and embedded in LRWhite resin (London Resin Company, Theale, England). Ultra-thin sections (80 nm) were placed on 200 mesh formvar- coated nickel grids and blocked in 10% goat serum and 1% BSA. The grids were then incubated with purified rab- bit anti-MATER antibody (1:1000) overnight at 4°C, washed with PBS/0,1% BSA/1% goat serum and incu- bated with colloidal gold-labelled (18 nm) secondary goat anti-rabbit antibody (1:10, (Immunotech, Marseille, France) for 2 hr at room temperature. After several wash- ing in PBS and deionised water, sections were counter- stained with 4% uranyl acetate and lead citrate. Specificity of immunostaining was checked by incubation of sections with PBS/0,1% BSA/1% goat serum. Sections were Immunohistochemistry A keyhole limpet hemocyanin (KLH)-coupled MATER peptide (C terminus, aa 1084–1098) was used to immu- nize rabbits to produce the primary antibody (Eurogentec, Angers, France). Bovine ovaries were fixed in Bouin solu- tion (50% saturated picric acid, 3.7% formaldehyde, 5% acetic acid), embedded in paraffin and serially sectioned (10 μm). The ovarian sections were immersed in antigen unmasking solution (Abcys, Paris, France) and warmed for 6 min in a microwave. After removing endogenous peroxydase activity in 0,3% H2O2 and blocking with horse serum, ovarian sections were incubated either with pri- mary anti-MATER serum or rabbit preimmune serum (1:10000) overnight at 4°C, followed by incubation for 1 hr at room temperature with secondary biotinylated horse anti-mouse/rabbit/goat IgG antibody (1:200) (Abcys). Avidin, biotinylated horseradish peroxydase (HRP) and diamino-benzidene (DAB) were applied according to the manufacturer's instructions using Vectastain elite ABC kit (Abcys). Sections were counterstained with hematoxylin. Real-time PCR cDNA amount equivalent to 0.05 oocyte or embryo was used in triplicate reactions. The standard curve was deduced from five serial dilutions (100 fg to 0.01 fg) of a plasmid including the target sequence included in each run. In each sample, the median value of PCR triplicates was considered (0 when not detected), and MATER data was normalized with exogenous luciferase to correct for loss of RNA; the MATER/luciferase value was then com- pared to this same ratio in immature oocytes (mean of four oocyte collections). Data are presented as mean ± SEM. After analysis of variance, first between immature oocytes and other stages, then between stages starting from mature oocyte onwards, differences were considered statistically significant at the 95% confidence level (P ≤ 0,05) using Tukey comparison test. separated on 7.5 or 10% SDS-PAGE gels and transferred onto a nitrocellulose membrane. After blocking with 5% dry milk in tris buffered saline, the blot was incubated with anti-MATER serum (1:10000) or pre-immune serum (1:10000) or purified antibody (1:2000) overnight at 4°C under agitation, and subsequently with secondary HRP- conjugated goat anti-rabbit IgG antibody (1:5000) for 1 hr 30 min at room. The signal was detected using an enhanced chemiluminescence kit according to the manu- facturer's instructions (Amersham Biosciences, Orsay, France). The membranes were stripped and blotted with an anti-α-TUBULIN monoclonal antibody (Sigma). Experiment was performed on two independent oocyte and embryo collections. Confocal scanning laser microscopy Oocytes and embryos were fixed for 20 min in 4% para- formaldehyde. After treatment in phosphate buffered saline 1X/Triton 0.2% followed by blocking in 5% goat serum, they were subsequently incubated with either rab- bit anti-MATER antibody purified onto a peptide affinity column by the manufacturer (1:250) or rabbit IgG over- night at 4°C, and then for 1 hr at room temperature with secondary goat anti-rabbit IgG antibody conjugated to fluoprobe 488 (1:100) or fluorescein isothiocyanate (1:400) for immature oocytes (both from Interchim, Montluçon, France). For LAMIN staining, immature oocytes were incubated with mouse anti-LAMIN A/C anti- body (1:200; Ozyme, Saint Quentin Yvelines, France) for 2 hr at room temperature and with secondary Texas Red- conjugated goat anti-mouse antibody (1:400; Sigma), for 1 hr at room temperature. Chromatin was stained using Hoechst 33258 (Sigma). Mowiol was used as mounting medium. Real-time PCR After adding 10 pg of luciferase mRNA (Promega) as an exogenous standard, total DNAse-treated RNA was extracted from 4 independent pools of 10 oocytes or Page 7 of 9 (page number not for citation purposes) Page 7 of 9 (page number not for citation purposes) BMC Developmental Biology 2006, 6:26 http://www.biomedcentral.com/1471-213X/6/26 http://www.biomedcentral.com/1471-213X/6/26 embryos at each stage using the Picopure RNA isolation kit (Alphelys, Plaisir, France). Reverse transcription was performed using oligo(dT)15 primers (Promega) during 50 min at 37°C by mouse Moloney leukaemia virus reverse transcriptase (Invitrogen, cergy Pontoise, France). Target cDNA were then quantified by real-time PCR using iQ SYBR green supermix (Bio-Rad, Marnes la Coquette, France) in a MyCycler system (Bio-Rad) using specific primers for MATER (forward 5'-GCTGGAGGCGTGT- GGACTG; reverse 5'-GGTCTGTAGATTAGAGGT- GGGATGC) and luciferase (forward 5'- TCATTCTTCGCCAAAAGCACTCTG; reverse 5'- AGCCCATATCCTTGTCGTATCCC). A 3-step protocol was repeated for 40 cycles (95°C for 30 sec, 60°C for 30 sec, 72°C for 20 sec), followed by acquisition of the melting curve. cDNA amount equivalent to 0.05 oocyte or embryo was used in triplicate reactions. The standard curve was deduced from five serial dilutions (100 fg to 0.01 fg) of a plasmid including the target sequence included in each run. In each sample, the median value of PCR triplicates was considered (0 when not detected), and MATER data was normalized with exogenous luciferase to correct for loss of RNA; the MATER/luciferase value was then com- pared to this same ratio in immature oocytes (mean of four oocyte collections). Data are presented as mean ± SEM. After analysis of variance, first between immature oocytes and other stages, then between stages starting from mature oocyte onwards, differences were considered statistically significant at the 95% confidence level (P ≤ 0,05) using Tukey comparison test. embryos at each stage using the Picopure RNA isolation kit (Alphelys, Plaisir, France). Reverse transcription was performed using oligo(dT)15 primers (Promega) during 50 min at 37°C by mouse Moloney leukaemia virus reverse transcriptase (Invitrogen, cergy Pontoise, France). Target cDNA were then quantified by real-time PCR using iQ SYBR green supermix (Bio-Rad, Marnes la Coquette, France) in a MyCycler system (Bio-Rad) using specific primers for MATER (forward 5'-GCTGGAGGCGTGT- GGACTG; reverse 5'-GGTCTGTAGATTAGAGGT- GGGATGC) and luciferase (forward 5'- TCATTCTTCGCCAAAAGCACTCTG; reverse 5'- AGCCCATATCCTTGTCGTATCCC). A 3-step protocol was repeated for 40 cycles (95°C for 30 sec, 60°C for 30 sec, 72°C for 20 sec), followed by acquisition of the melting curve. Authors' contributions 12. 12. Robert C, McGraw S, Massicotte L, Pravetoni M, Gandolfi F, Sirard MA: Quantification of housekeeping transcript levels during the development of bovine preimplantation embryos. Biol Reprod 2002, 67:1465-1472. SP participated in experimental design and carried out most experimental work. CP collected biological material, carried out in vitro embryo production, and participated in immunohistochemistry. BD carried out the TEM exper- iments. SU was involved in biological sample collection, Western blot, and provided input in writing the manu- script. AT performed oocyte/embryo collection and real- time PCR. PM provided expertise in experimental design and analysis. RDT designed and supervised the study, and participated in experimental work (oocyte collection, Western blot). SP and RDT wrote the manuscript. 13. p 13. Sutovsky P, Motlik J, Neuber E, Pavlok A, Schatten G, Palecek J, Hyttel P, Adebayo OT, Adwan K, Alberio R, Bagis H, Bataineh Z, Bjerregaard B, Bodo S, Bryja V, Carrington M, Couf M, de la Fuente R, Diblik J, Esner M, Forejt J, Fulka J Jr, Geussova G, Gjorret JO, Libik M, Hampl A, Hassane MS, Houshmand M, Hozak P, Jezova M, Kania G, Kanka J, Kandil OM, Kishimoto T, Klima J, Kohoutek J, Kopska T, Kubelka M, Lapathitis G, Laurincik J, Lefevre B, Mihalik J, Novakova M, Oko R, Omelka R, Owiny D, Pachernik J, Pacholikova J, Peknicova J, Pesty A, Ponya Z, Preclikova H, Sloskova A, Svoboda P, Strejcek F, Toth S, Tepla O, Valdivia M, Vodicka P, Zudova D: Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts. Cloning Stem Cells 2001, 3:157-161. 14. [http://cubic.bioc.columbia.edu/cgi/var/nair/resonline.pl]. Western blot Pools of 10 oocytes or embryos, or cumulus cells, were frozen and thawed three times, and then the proteins were Page 8 of 9 (page number not for citation purposes) BMC Developmental Biology 2006, 6:26 http://www.biomedcentral.com/1471-213X/6/26 observed with a CM10 electron microscope (Philips, Eindhoven, Netherlands). gene expression patterns in mouse oocytes. Hum Mol Genet 2004, 13:2263-2278. gene expression patterns in mouse oocytes. Hum Mol Genet 2004, 13:2263-2278. 11. 11. Rajkovic A, Pangas SA, Ballow D, Suzumori N, Matzuk MM: NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression. Science 2004, 305:1157-1159. Acknowledgements 15. Nair R, Rost B: LOC3D: annotate sub-cellular localization for protein structures. Nucleic Acids Res 2003, 31:3337-3340. We thank Dr Florence Guignot, Pascal Papillier, and Gaël Ramé for their assistance in ovary, oocyte and embryo collection, Pr Marc-André Sirard and Dr Philippe Monget for helpful discussions. Pr Lawrence Nelson is acknowledged for the gift of an antibody against mouse MATER that was tested in preliminary experiments but failed to recognize the bovine pro- tein. SP and AT are supported by fellowships from the Conseil Régional Région Centre and Institut National de la Recherche Agronomique. This work is part of the "Ovogenae" program, an Agenae selected project spon- sored by grants from the french Ministry of Research (#03P409) and APIS- GENE. 16. Howell CY, Bestor TH, Ding F, Latham KE, Mertineit C, Trasler JM, Chaillet JR: Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene. Cell 2001, 104:829-838. f 17. Sirard MA, Florman HM, Leibfried-Rutledge ML, Barnes FL, Sims ML, First NL: Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes. Biol Reprod 1989, 40:1257-1263. p 18. Kastrop PM, Hulshof SC, Bevers MM, Destree OH, Kruip TA: The effects of alpha-amanitin and cycloheximide on nuclear pro- gression, protein synthesis, and phosphorylation during bovine oocyte maturation in vitro. Mol Reprod Dev 1991, 28:249-254. 19. Memili E, Dominko T, First NL: Onset of transcription in bovine oocytes and preimplantation embryos. Mol Reprod Dev 1998, 51:36-41. References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 9 of 9 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 9 of 9 Publish with BioMed Central and every scientist can read your work free of charge y p 7. Wu X, Viveiros MM, Eppig JJ, Bai Y, Fitzpatrick SL, Matzuk MM: Zygote arrest 1 (Zar1) is a novel maternal-effect gene criti- cal for the oocyte-to-embryo transition. Nat Genet 2003, 33:187-191. 8. Dalbies-Tran R, Papillier P, Pennetier S, Uzbekova S, Monget P: Bovine mater-like NALP9 is an oocyte marker gene. Mol Reprod Dev 2005, 71:414-421. 9. Dade S, Callebaut I, Paillisson A, Bontoux M, Dalbies-Tran R, Monget P: In silico identification and structural features of six new genes similar to MATER specifically expressed in the oocyte. Biochem Biophys Res Commun 2004, 324:547-553. p y 10. Hamatani T, Falco G, Carter MG, Akutsu H, Stagg CA, Sharov AA, Dudekula DB, VanBuren V, Ko MS: Age-associated alteration of References 1. Tong ZB, Nelson LM: A mouse gene encoding an oocyte anti- gen associated with autoimmune premature ovarian failure. Endocrinology 1999, 140:3720-3726. 20. 20. Yan W, Ma L, Stein P, Pangas SA, Burns KH, Bai Y, Schultz RM, Matzuk MM: Mice deficient in oocyte-specific oligoadenylate syn- thetase-like protein OAS1D display reduced fertility. Mol Cell Biol 2005, 25:4615-4624. gy 2. Tong ZB, Gold L, Pfeifer KE, Dorward H, Lee E, Bondy CA, Dean J, Nelson LM: Mater, a maternal effect gene required for early embryonic development in mice. Nat Genet 2000, 26:267-268. 21. Mermillod P, Tomanek M, Marchal R, Meijer L: High developmen- tal competence of cattle oocytes maintained at the germinal vesicle stage for 24 hours in culture by specific inhibition of MPF kinase activity. Mol Reprod Dev 2000, 55:89-95. 3. Tong ZB, Gold L, De Pol A, Vanevski K, Dorward H, Sena P, Palumbo C, Bondy CA, Nelson LM: Developmental expression and sub- cellular localization of mouse MATER, an oocyte-specific protein essential for early development. Endocrinology 2004, 145:1427-1434. y p 22. Dalbies-Tran R, Mermillod P: Use of heterologous complemen- tary DNA array screening to analyze bovine oocyte tran- scriptome and its evolution during in vitro maturation. Biol Reprod 2003, 68:252-261. 4. Hamatani T, Carter MG, Sharov AA, Ko MS: Dynamics of global gene expression changes during mouse preimplantation development. Dev Cell 2004, 6:117-131. 23. p 23. Besnard N, Pisselet C, Monniaux D, Locatelli A, Benne F, Gasser F, Hatey F, Monget P: Expression of messenger ribonucleic acids of insulin-like growth factor binding protein-2, -4, and -5 in the ovine ovary: localization and changes during growth and atresia of antral follicles. Biol Reprod 1996, 55:1356-1367. 5. Ponsuksili S, Brunner RM, Goldammer T, Kuhn C, Walz C, Chomdej S, Tesfaye D, Schellander K, Wimmers K, Schwerin M: Bovine NALP5, NALP8, and NALP9 Genes: Assignment to a QTL Region and the Expression in Adult Tissues, Oocytes, and Preimplantation Embryos. Biol Reprod 2005. p y p 6. Pennetier S, Uzbekova S, Perreau C, Papillier P, Mermillod P, Dalbies- Tran R: Spatio-Temporal Expression of the Germ Cell Marker Genes MATER, ZAR1, GDF9, BMP15, andVASA in Adult Bovine Tissues, Oocytes, and Preimplantation Embryos. Biol Reprod 2004, 71:1359-1366.
https://openalex.org/W2591677258	https://www.scielo.br/j/aem/a/kgDWdbxTy5rGBDN7rGrNjnt/?lang=en&format=pdf	English	null	Metabolic syndrome in children and teenagers: worth assessing it, but how?	Archives of Endocrinology and Metabolism	2,017	cc-by	2,723	Metabolic syndrome in children and teenagers: worth assessing it, but how? Gilberto J. Paz-Filho1 R eaven has defined metabolic syndrome (at that time, “syndrome X”) as a clustering of risk factors for coronary heart disease and type 2 diabetes that include elevated glucose, hypertension, central obesity, and dyslipidemia (1). Since then, significant breakthroughs have been achieved in the field, but there is still much controversy regarding its diagnosis, and whether it should be considered as a single disease at all. Several organizations have proposed different diagnostic criteria, which have in common many caveats, such as the fact that they treat each component in a dichotomous way (above or below a cutoff) rather than in a continuous manner. Furthermore, equal weight toward the diagnosis of the metabolic syndrome is given to each criterion. After a couple of decades, Reaven himself questioned the clinical utility of metabolic syndrome as a diagnostic category (2), and some suggested that the time of metabolic syndrome as a condition has already passed (3). Nevertheless, much research on the diagnosis of metabolic syndrome is being published: up to January 23, 2017, 24,173 abstracts could be found on PubMed, when searching for the MeSH term “metabolic syndrome X”. R Correspondence to: Gilberto J. Paz-Filho 131 Garran Rd Acton, ACT 2612 Australia gilbertjpf@hotmail.com Received on Jan/23/2017 Accepted on Jan/23/2017 DOI: 10.1590/2359-3997000000249 Since children are also affected by all components of metabolic syndrome, it is nothing but natural to expand current research to a younger population. In this issue of AE&M, two interesting cross-sectional studies propose to determine different tests to diagnose metabolic syndrome in children and teenagers. In particular, Madeira and cols. aim to determine a cutoff value for serum leptin levels in prepubertal children, as a predictor of metabolic syndrome (4). Conversely, Stroescu and cols. provide a carotid intima media thickness (CIMT) cutoff value in children and teenagers, which intends to predict an increased risk of metabolic syndrome (5). In the paper by Madeira and cols., cross-sectional data from 340 Brazilian prepubertal children between ages 5-11 years were analyzed. As widely replicated in previous studies, several biomarkers were altered in the obese/overweight groups, indicating increased risk for cardiovascular disease and type 2 diabetes (6). In other words, children in those groups were more likely to have metabolic syndrome. 1 John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia editorial editorial Correspondence to: Gilberto J. Paz-Filho 131 Garran Rd Acton, ACT 2612 Australia gilbertjpf@hotmail.com Received on Jan/23/2017 Accepted on Jan/23/2017 DOI: 10.1590/2359-3997000000249 Arch Endocrinol Metab. 2017;61/1 Correspondence to: Gilberto J. Paz-Filho 131 Garran Rd Acton, ACT 2612 Australia gilbertjpf@hotmail.com Metabolic syndrome in children and teenagers: worth assessing it, but how? In fact, the prevalence of metabolic syndrome (adapted IDF criteria) was higher in the group of obese children (n = 33, 20%), when compared to what was observed in overweight (n = 1, 2%) and in age-matched, normal weight children (0%). However, the most significant finding of that study is the determination of a serum leptin cutoff value of 13.4 ng/mL that indicates the presence or absence of metabolic syndrome. Leptin has been proposed as a biomarker of metabolic syndrome due to its effects that contribute to the development of type 2 diabetes mellitus and cardiovascular disease. It has atherogenic, thrombotic, angiogenic, and proinflammatory effects. Furthermore, it increases oxidative stress, and promotes vascular smooth muscle Diagnosis of metabolic syndrome hypertrophy (7,8). Therefore, leptin is associated with the components of metabolic syndrome, and can contribute to the development of cardiovascular disease and diabetes. However, the direct role of leptin in the pathogenesis of some components of metabolic syndrome is controversial. Although leptin participates in the activation of the sympathetic system, it is unclear whether it is a causal agent of hypertension in humans (9). In case of plasma glucose levels (and insulin sensitivity), leptin seems to be beneficial only in the absence of leptin resistance (10,11), which could deter its use as a widespread biomarker of metabolic syndrome and all its components. Furthermore, Madeira and cols. consider only absolute serum leptin levels in their paper. It has been observed that leptin levels adjusted to fat mass, but not absolute leptinemia, were correlated to the severity of metabolic syndrome in adults, suggesting a state of relative leptin deficiency in obesity associated with more advanced stages of metabolic syndrome (12). cutoff depends on the prevalence of the disease in a target population, and the consequences of false- positive and false-negative results. For the screening of metabolic syndrome in this Brazilian prepubertal population, a serum leptin cutoff of 13.4 ng/mL seems more adequate than 12.3 ng/mL; based on the aforementioned factors, that cutoff could, however, be adjusted to even higher levels. j g The study by Madeira and cols. has the merit of studying prepubertal young children, a population that is not widely assessed for metabolic syndrome and the components associated with it. Important descriptive data for that population are presented (which can be used as a reference in future studies), and a cutoff for leptin is proposed for the diagnosis of metabolic syndrome. Metabolic syndrome in children and teenagers: worth assessing it, but how? However, the title may be a bit misleading, since the proposed cutoff does not allow the prediction of the development of metabolic syndrome – the cross- sectional results are merely descriptive, and do not provide long-term information on the development of cardiovascular disease and diabetes – the outcomes that ultimately matter. Only long-term, prospective studies through late adulthood would really answer questions that are relevant to prepubertal children: 1) can their serum leptin levels predict the development of cardiovascular disease and diabetes? 2) if yes, what cutoff should be used, so early intervention can be adopted? At least in adults, the answer to the first question seems to be “probably not” (16,17). The authors correctly present two receiver operating characteristic (ROC) curves for the determination of optimal cutoff values, one based on a crude model (i.e., obtained by pooling together all available data), and another based on a sex- and age-adjusted model. When constructing a ROC curve where the accuracy of a diagnostic test (leptin) is affected by covariates (sex and age), failure to incorporate information furnished by them may lead to erroneous conclusions (13). Since it is well-known that leptin levels behave differently according to both age and gender (even in prepubertal individuals) (14), the optimal serum leptin cutoff seems to be indeed 13.4 ng/mL, based on the adjusted model. The study by Stroescu and cols. involves cross- sectional data on 122 obese and 42 nonobese Romanian children from 4 to 20 years old, who were further categorized as born small for gestational age (SGA) or appropriate for gestational age (AGA). As previously observed, CIMT was increased among obese children. When analyzing only that group, obese SGA children had higher CIMT values than those born AGA. Subsequently, the authors claimed direct correlation between CIMT and leptin, and between CIMT and high sensitivity C-reactive protein (hsCRP). Inverse weak correlation was described between CIMT and adiponectin. These correlations led the authors to try to determine the optimal CIMT cutoff value that is associated with metabolic syndrome in that population. In that case, a cutoff equal to 0.049 cm is proposed, above which obese children would have increased risk of developing metabolic syndrome. However, the construction of a ROC curve provides a fixed cutoff value where sensitivity and specificity are optimal (i.e., the point in the curve closest to the upper left corner, and farthest from the diagonal line). Metabolic syndrome in children and teenagers: worth assessing it, but how? When applying cutoff values to public health, one has to take into account the prevalence of the disorder being tested, and the purpose of the test. For example, a disorder that is very low prevalent and that leads to an unacceptable increase in costs in case of false-positive diagnoses may require the selection of a cutoff that maximizes specificity. On the other hand, if a disorder is highly prevalent, and if missing a diseased individual leads to serious consequences, a lower cutoff value should be selected, to maximize sensitivity. If a test is used for screening purposes, then a higher cutoff value with higher sensitivity and negative predictive values must be used (15). In other words, the optimal In this paper, instead of using leptin as a biomarker of metabolic syndrome, the authors chose to employ 2 Arch Endocrinol Metab. 2017;61/1 Diagnosis of metabolic syndrome CIMT – a marker of atherosclerosis that has been associated with the components of metabolic syndrome, and able to predict cardiovascular risk (18). In their statement, the Working Group on Cardiovascular Prevention of the Association for European Pediatric Cardiology “recommends use CIMT in screening patients with elevated cardiovascular risk, even if the long-term benefit of CIMT measurement on the single patient’s vascular health remains to be determined” (19). These observations strengthen the importance of measuring CIMT in obese children. SGA. Due to the limitations of the study, it is advisable that this recommendation is not followed until larger, prospective studies are conducted. The screening of diseases can certainly impose economic burden to individuals and governments. When proposing the use of serum leptin levels or CIMT for the screening of metabolic syndrome, it is necessary to discuss its cost-effectiveness in terms of their ability to predict cardiovascular disease and diabetes in that population. Is it viable to use those tests for the screening of metabolic syndrome in children, considering that the components of metabolic syndrome are invariably measured in the management of obesity (25)? Possibly not. Most importantly, before trying to define a new test for diagnosing metabolic syndrome, researchers should first try to solve the underlying controversy: is metabolic syndrome an entity per se carrying a single weight as a risk factor, or is it a constellation of different conditions carrying different risks? In the literature, there are heterogeneous results regarding leptin, adiponectin and hsCRP levels in children born SGA (20-22). Metabolic syndrome in children and teenagers: worth assessing it, but how? In the present cohort, SGA obese children had increased CIMT, leptin and hsCRP, and decreased adiponectin, when compared to their obese AGA counterparts. Although the sample size is very small, the results strengthen the hypothesis that SGA babies are at higher risk of developing metabolic syndrome and its consequences (23), mainly due to a low-grade inflammatory state and to altered adipokines levels, independent of adiposity. Disclosure: no potential conflict of interest relevant to this article was reported. Disclosure: no potential conflict of interest relevant to this article was reported. It is unclear which criteria the authors used for the diagnosis of metabolic syndrome in the Romanian cohort. As pointed out by Madeira and cols., there is a lack of consensus definition for metabolic syndrome (4). Therefore, not outlining the criteria used to diagnose metabolic syndrome can seriously compromise the validity of the results. Similar to the points made herein to the article by Madeira and cols., the study by Stroescu and cols. is not prospective. Therefore, it cannot propose CIMT as a predictor for future development of metabolic syndrome. Furthermore, the presented coefficients of correlation between CIMT and leptin, adiponectin and hsCRP must be interpreted with caution, since they suggest only weak to moderate correlation. The accuracy of the results presented in Table 2 may be questioned due to the fact that the mean age of the obese AGA group is different from the mean age of the same group that is presented in an article based on data from the same cohort (24). Finally, the authors cannot answer the question posed in the title, due to the small sample size; indeed, they had to combine SGA and AGA data to construct the ROC curve. In their conclusion, the authors do not answer to their own question posed in the title; they expand the most potentially significant result to all obese children, and recommend screening for metabolic syndrome in all of those with CIMT above 0.049 mm, not only Arch Endocrinol Metab. 2017;61/1 REFERENCES 1. Reaven GM. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes. 1988;37(12):1595-607. 1. Reaven GM. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes. 1988;37(12):1595-607. 2. Reaven GM. The metabolic syndrome: time to get off the merry- go-round? J Intern Med. 2011;269(2):127-36. 2. Reaven GM. The metabolic syndrome: time to get off the merry- go-round? J Intern Med. 2011;269(2):127-36. 3. Quintao EC. Metabolic syndrome: did the creator kill the creature? Arq Bras Endocrinol Metabol. 2011;55(5):355-6. 4. Madeira I, Bordallo MA, Rodrigues NC, Carvalho C, Gazolla F, Collett-Solberg P, et al. Leptin as a predictor of metabolic syndrome in prepubertal children. Arch Endocrinol Metab. 2017;61(1):7-13. 5. Stroescu R, Bizerea T, Doroş G, Marazan M, Lesovici M, Mãrginean O. Correlation between adipokines and carotid intima media thickness in a group of obese Romanian children: is small for gestational age status an independent factor for cardiovascular risk? Arch Endocrinol Metab. 2017;61(1):14-20. 6. Ayer J, Charakida M, Deanfield JE, Celermajer DS. Lifetime risk: childhood obesity and cardiovascular risk. Eur Heart J. 2015;36(22):1371-6. 7. Paz-Filho G, Mastronardi CA, Licinio J. Leptin treatment: facts and expectations. Metabolism. 2015;64(1):146-56. 8. Paz-Filho G, Mastronardi C, Franco CB, Wang KB, Wong ML, Licinio J. Leptin: molecular mechanisms, systemic pro-inflammatory effects, and clinical implications. Arq Bras Endocrinol Metabol. 2012;56(9):597-607. 9. Simonds SE, Pryor JT, Cowley MA. Does leptin cause an increase in blood pressure in animals and humans? Curr Opin Nephrol Hypertens. 2017;26(1):20-5. 10. Martin SS, Qasim A, Reilly MP. Leptin resistance: a possible interface of inflammation and metabolism in obesity-related cardiovascular disease. J Am Coll Cardiol. 2008;52(15):1201-10. Arch Endocrinol Metab. 2017;61/1 3 Diagnosis of metabolic syndrome thickness for cardiovascular risk assessment: systematic review and meta-analysis. Atherosclerosis. 2013;228(1):1-11. 11. Paz-Filho G, Mastronardi C, Wong ML, Licinio J. Leptin therapy, insulin sensitivity, and glucose homeostasis. Indian J Endocrinol Metab. 2012;16(Suppl 3):S549-55. 19. Dalla Pozza R, Ehringer-Schetitska D, Fritsch P, Jokinen E, Petropoulos A, Oberhoffer R, et al. Intima media thickness measurement in children: a statement from the Association for European Paediatric Cardiology (AEPC) Working Group on Cardiovascular Prevention endorsed by the Association for European Paediatric Cardiology. Atherosclerosis. 2015;238(2):380-7. 12. Paz-Filho GJ, Volaco A, Suplicy HL, Radominski RB, Boguszewski CL. Decrease in leptin production by the adipose tissue in obesity associated with severe metabolic syndrome. Arq Bras Endocrinol Metabol. 2009;53(9):1088-95. 13. Pardo-Fernández JC, Rodríguez-Álvarez MX, Van Keilegom I. Arch Endocrinol Metab. 2017;61/1 REFERENCES A review on ROC curves in the presence of covariates. Revstat Stat J. 2014;12:21-41. 20. Ibanez L, Lopez-Bermejo A, Suarez L, Marcos MV, Diaz M, de Zegher F. Visceral adiposity without overweight in children born small for gestational age. J Clin Endocrinol Metab. 2008;93(6):2079-83. 14. Garcia-Mayor RV, Andrade MA, Rios M, Lage M, Dieguez C, Casanueva FF. Serum leptin levels in normal children: relationship to age, gender, body mass index, pituitary-gonadal hormones, and pubertal stage. J Clin Endocrinol Metab. 1997;82(9):2849-55. 21. Ibanez L, Lopez-Bermejo A, Diaz M, de Zegher F. Catch-up growth in girls born small for gestational age precedes childhood progression to high adiposity. Fertil Steril. 2011;96(1):220-3. 15. Doi SAR. Using and Interpreting Diagnostic Tests with Quantitative Results. In: Williams GM, Doi SAR, editors. Methods of Clinical Epidemiology. Heidelberg: Springer; 2013. 22. Melo AS, Bettiol H, Silva AA, Rosa-e-Silva AC, Cardoso VC, Reis RM, et al. Small for gestational age babies are not related to changes in markers of adipose tissue dysfunction during reproductive age. Early Hum Dev. 2014;90(5):231-5. 16. Martin SS, Blaha MJ, Muse ED, Qasim AN, Reilly MP, Blumenthal RS, et al. Leptin and incident cardiovascular disease: the Multi- ethnic Study of Atherosclerosis (MESA). Atherosclerosis. 2015;239(1):67-72. 23. Hernandez MI, Mericq V. Metabolic syndrome in children born small-for-gestational age. Arq Bras Endocrinol Metabol. 2011;55(8):583-9. 17. Sattar N, Wannamethee G, Sarwar N, Chernova J, Lawlor DA, Kelly A, et al. Leptin and coronary heart disease: prospective study and systematic review. J Am Coll Cardiol. 2009;53(2):167-75. 24. Stroescu R, Micle I, Marginean O, Bizerea T, Marazan M, Puiu M, et al. Is small for gestational age status associated with an increased risk of atherogenesis? Maedica (Buchar). 2013;8(4):315-20. 18. van den Oord SC, Sijbrands EJ, ten Kate GL, van Klaveren D, van Domburg RT, van der Steen AF, et al. Carotid intima-media 25. Ryan DH. Guidelines for Obesity Management. Endocrinol Metab Clin North Am. 2016;45(3):501-10. Arch Endocrinol Metab. 2017;61/1 Arch Endocrinol Metab. 2017;61/1
https://openalex.org/W2806448551	https://europepmc.org/articles/pmc5928146?pdf=render	English	null	High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria-infected human macrophages	Scientific reports	2,018	cc-by	9,682	High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria- infected human macrophages Manabu Inoue1, Mamiko Niki1, Yuriko Ozeki2, Sachiyo Nagi3, Evans Asena Chadeka3, Takehiro Yamaguchi2, Mayuko Osada-Oka 4, Kenji Ono5, Tetsuya Oda5, Faith Mwende6, Yukihiro Kaneko1, Makoto Matsumoto5, Satoshi Kaneko7,8, Yoshio Ichinose8,9, Sammy M. Njenga6, Shinjiro Hamano3,8 & Sohkichi Matsumoto2 Received: 12 October 2017 Accepted: 23 March 2018 Published: xx xx xxxx Received: 12 October 2017 Accepted: 23 March 2018 Published: xx xx xxxx Manabu Inoue1, Mamiko Niki1, Yuriko Ozeki2, Sachiyo Nagi3, Evans Asena Chadeka3, Takehiro Yamaguchi2, Mayuko Osada-Oka 4, Kenji Ono5, Tetsuya Oda5, Faith Mwende6, Yukihiro Kaneko1, Makoto Matsumoto5, Satoshi Kaneko7,8, Yoshio Ichinose8,9, Sammy M. Njenga6, Shinjiro Hamano3,8 & Sohkichi Matsumoto2 Immune responses to parasitic pathogens are affected by the host physiological condition. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) are transporters of lipids between the liver and peripheral tissues, and modulate pro-inflammatory immune responses. Pathogenic mycobacteria are parasitic intracellular bacteria that can survive within macrophages for a long period. Macrophage function is thus key for host defense against mycobacteria. These basic facts suggest possible effects of HDL and LDL on mycobacterial diseases, which have not been elucidated so far. In this study, we found that HDL and not LDL enhanced mycobacterial infections in human macrophages. Nevertheless, we observed that HDL remarkably suppressed production of tumor necrosis factor alpha (TNF-α) upon mycobacterial infections. TNF-α is a critical host-protective cytokine against mycobacterial diseases. We proved that toll-like receptor (TLR)-2 is responsible for TNF-α production by human macrophages infected with mycobacteria. Subsequent analysis showed that HDL downregulates TLR2 expression and suppresses its intracellular signaling pathways. This report demonstrates for the first time the substantial action of HDL in mycobacterial infections to human macrophages. Low-density lipoprotein (LDL)-cholesterol (LDL-C) transfers cholesterol from the liver to peripheral tissues. However, a high level of LDL-C is a risk factor for cardiovascular diseases1,2 because it initiates atherosclerosis, leading to peripheral inflammations including the production of inflammatory cytokines and accumulation of macrophages and activated T cells. High-density lipoprotein (HDL), on the other hand, inversely transfers cho- lesterol from peripheral tissues to the liver. Indeed, high levels of serum HDL-cholesterol is correlated with a reduced risk of atherosclerosis and cardiovascular diseases3–6. When macrophages are filled with cholesterol, they become foam cells, which trigger massive inflammation during atherosclerosis. HDL removes cholesterol from macrophages through lipid transporter proteins, such as, ABCA17,8, ABCG18,9 and SR-B110. This is considered as a part of the mechanism of anti-inflammatory effects by HDL. However, accumulated evidence also suggests a direct role of HDL in the suppression of inflammation11–15. It is thus likely that LDL and HDL have impacts on human health status concomitant with inflammation, such as, in infectious diseases. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 12 October 2017 Accepted: 23 March 2018 Published: xx xx xxxx Results M b Mycobacteria-infected human macrophages produce a large amount of TNF-α, which is sup- pressed by HDL. We differentiated the THP1 human acute monocytic cell line to macrophages by using phorbol 12-myristate 13-acetate (PMA) (THP1 macrophages). Macrophages were then cultured with or without adding varying doses of HDL or LDL (5–50 µg/ml) for 24 hours. We confirmed no significant differences in the viability rates of cells by addition of 5 to 50 µg/ml HDL or LDL, based on the results of the trypan blue-exclusion assays or assessment of cytoplasmic lactate dehydrogenase (LDH) enzyme activity in the culture medium.f y y y g y y To assess the effects of human plasma-derived HDL and LDL on mycobacteria infection of human mac- rophages, THP1 macrophages were infected with M. tuberculosis complexes, such as Mycobacterium bovis BCG (BCG) or M. tuberculosis H37Rv. Twenty-four hours after infection, we assessed inflammatory responses of the macrophages by measuring the levels of cytokines, including granulocyte/macrophage colony-stimulating factor (GM-CSF), IFN-γ, interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10 and TNF-α, secreted in the culture supernatant. Upon infection with BCG, we found a robust production of TNF-α (14820.02 ± 165.82 pg/ml), whose level was remarkably reduced with HDL treatment (Supplementary Fig. S1d). We found insignificant levels of IL-6 (76.64 ± 15.797 pg/ml) and IL-10 (40.03 ± 5.404 pg/ml) production (Supplementary Fig. S1a and b) and moderate levels of IFN-γ production (287.266 ± 4.738 pg/ml) (Supplementary Fig. S1c), all of whose expression levels were suppressed with HDL treatment. However, LDL suppressed the production of TNF-α and IFN-γ, but its effect was lower than that of HDL treatment, and it did not suppress the production of IL-6 or IL-10 (Supplementary Fig. S1a and b). The levels of other tested cytokines, such as IL-4, GM-CSF, IL-2 or IL-8, were below the detection limits in all samples. Similar to infection with BCG, we found a high level of TNF-α production upon M. tuberculosis infec- tion (9057.91 ± 219.07 pg/ml, Fig. 1e). We also found moderate levels of IL-6 (498.01 ± 4.78 pg/ml), IL-10 (1203.29 ± 168 pg/ml) and IFN-γ (1046.18 ± 16.83 pg/ml) production, and an insignificant level of IL-4 pro- duction (79.16 ± 2.6 pg/ml) (Fig. 1a–d). Notably, treatment with HDL significantly reduced the production of all of these cytokines in a dose-dependent manner, except for IL-10. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Mycobacterial infections are still a serious threat to human health, especially Mycobacterium tuberculosis com- plex, an etiologic agent of tuberculosis (TB), which is responsible for the highest mortality among all single path- ogens. The World Health Organization (WHO) estimated that 10.4 million people newly developed TB and 1.7 million people died from this disease in 2015, as indicated in the most recent report (WHO; Global Tuberculosis Report, 2017). p , ) M. tuberculosis is an intracellular pathogen that is well adapted to ensure its survival in macrophages. Therefore, the function of macrophages and the pro-inflammatory cytokines that activate them are critical for the host defense16. A key pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α), activates macrophages and is essential for granuloma formation. A granuloma is the hallmark of mycobacterial infections17. It is a roundish immunopathological structure made up of activated macrophages, which prevent the dissemination of mycobacteria. The significant role of TNF-α in granuloma formation and hence TB control in humans was proven with the administration of TNF-α-neutralizing therapy, which disrupted TB granuloma and increased TB reactivation18.h Thus, activated macrophages participate in the prevention of TB progression. However, M. tuberculosis can persist without complete sterilization, accounting for the huge TB reservoir19–21. During persistent infections, M. tuberculosis uses cholesterol as a carbon source22–26. Host cholesterol is also essential for phagocytosis of myco- bacteria by macrophages27. However, the immunomodulatory effects of cholesterol transporters, LDL and HDL, on mycobacterial diseases remain to be elucidated. In this study, we assessed the action of LDL and HDL on mycobacteria-infected human macrophages. High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria- infected human macrophages Manabu Inoue1, Mamiko Niki1, Yuriko Ozeki2, Sachiyo Nagi3, Evans Asena Chadeka3, Takehiro Yamaguchi2, Mayuko Osada-Oka 4, Kenji Ono5, Tetsuya Oda5, Faith Mwende6, Yukihiro Kaneko1, Makoto Matsumoto5, Satoshi Kaneko7,8, Yoshio Ichinose8,9, Sammy M. Njenga6, Shinjiro Hamano3,8 & Sohkichi Matsumoto2 1Department of Bacteriology and Virology, Osaka-City University Graduate School of Medicine, Osaka, Japan. 2Department of Bacteriology, Niigata University School of Medicine, Niigata, Japan. 3Department of Parasitology, Institute of Tropical Medicine (NEKKEN) and the Graduate School of Biomedical Science, Nagasaki University, Nagasaki, Japan. 4Food Hygiene and Environmental Health, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan. 5Microbiological Research Institute, Otsuka Pharmaceutical, Tokushima, Japan. 6Eastern and Southern Africa Centre of International Parasite Control (ESACIPAC), Kenya Medical Research Institute, Nairobi, Kenya. 7Department of Eco-Epidemiology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan. 8Nagasaki University Institute of Tropical Medicine(NUITM)-Kenya Medical Research Institute (KEMRI), Nairobi, Kenya. 9Department of Bacteriology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan. Correspondence and requests for materials should be addressed to Y.O. (email: yuriozeki@med.niigata-u.ac.jp) or S.M. (email: sohkichi@med.niigata-u.ac.jp) SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 1 Results M b LDL treatment reduced the levels of IL-4, IL-6, IFN-γ and TNF-α production, but to a lesser extent than HDL treatment, and these changes did not show dose-responsiveness. The levels of other tested cytokines, such as GM-CSF, IL-2 and IL-8, were below the detec- tion limits in all samples. These data suggest that among the tested cytokines, TNF-α is the most abundantly produced upon infection of THP1 macrophages with M. tuberculosis complex and HDL suppresses its production in a dose-dependent manner.ff p We also assessed the effects of HDL on M. tuberculosis-infected human macrophages that were differentiated from CD14 positive monocytes derived from peripheral blood mononuclear cells (PBMC). As observed in THP1 macrophages, HDL but not LDL treatment of the M. tuberculosis-infected macrophages significantly suppressed TNF-α production by PBMC-derived human macrophages (Supplementary Fig. S2). This shows the common responses to HDL by both THP1 macrophages and PBMC-derived ones. HDL but not LDL promotes entry of both BCG and M. tuberculosis into THP1 macrophages. HDL functions as a remover of cellular cholesterol in the periphery. In addition, previous reports showed that the removal of cholesterol suppressed BCG-infection of mouse macrophages27. This suggests the possibility that HDL inhibits phagocytosis of mycobacteria and in turn reduces infection-dependent TNF-α production. To inves- tigate this possibility, we determined the infecting number of mycobacteria by counting colony forming units (CFUs) 24 hours after infection of THP1 macrophages. The data revealed that CFUs of BCG and M. tuberculosis in LDL-treated macrophages were similar to those in control macrophages. In contrast, HDL-treated macrophages harbored a significantly higher number of both BCG and M. tuberculosis (Supplementary Fig. S3). These data suggest that HDL enhances mycobacterial infections of macrophages contrary to prior expectations. HDL inhibits the transcription of TNF-α and TNF receptors. Despite elevated mycobacterial entry into THP1 macrophage, the production of pro-inflammatory cytokines is inhibited by addition of HDL, suggest- ing some unknown systematic mechanism of HDL to this effect. We next analyzed whether the regulation occurs SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 2 www.nature.com/scientificreports/ ntificreports/ Figure 1. Effects of HDL and LDL on the expression of pro-inflammatory cytokines by M. tuberculosis-infected THP1macrophages. THP1-macrophages were cultured with or without adding HDL or LDL (5–50 µg/ml) for 24 hours. The treated macrophages were then infected with M. tuberculosis (multiplicity of infection = 10) for 24 hours (n = 4). Results M b The amounts of IL-4 (a), IL-6 (b), IL-10 (c), IFN-gamma (d), and TNF-α (e) from macrophages were measured using the Bio-Plex Multiplex System. ANOVA was used for test the differences in means. Figure 1. Effects of HDL and LDL on the expression of pro-inflammatory cytokines by M. tuberculosis-infected THP1macrophages. THP1-macrophages were cultured with or without adding HDL or LDL (5–50 µg/ml) for 24 hours. The treated macrophages were then infected with M. tuberculosis (multiplicity of infection = 10) for 24 hours (n = 4). The amounts of IL-4 (a), IL-6 (b), IL-10 (c), IFN-gamma (d), and TNF-α (e) from macrophages were measured using the Bio-Plex Multiplex System. ANOVA was used for test the differences in means. Figure 1. Effects of HDL and LDL on the expression of pro-inflammatory cytokines by M. tuberculosis-infected THP1macrophages. THP1-macrophages were cultured with or without adding HDL or LDL (5–50 µg/ml) for 24 hours. The treated macrophages were then infected with M. tuberculosis (multiplicity of infection = 10) for 24 hours (n = 4). The amounts of IL-4 (a), IL-6 (b), IL-10 (c), IFN-gamma (d), and TNF-α (e) from macrophages were measured using the Bio-Plex Multiplex System. ANOVA was used for test the differences in means. at the transcriptional level. In this experiment, the amount of TNF-α from BCG-infected THP1 macrophages in the control group, the group treated with 50 µg/mL HDL or LDL was 2067.2 pg/ml, 388.2 pg/ml, and 1161.3 pg/ ml, respectively (Fig. 2a). The mRNA levels of TNF-α in BCG-infected THP1 macrophages pretreated with HDL were suppressed by 64% (Fig. 2b), suggesting the presence of transcriptional regulation. Prior work indicated that TNF-α transcription is regulated by autocrine systems through the activation of the NF-κB pathway by the bonding of TNF-α with TNF receptors (TNFR-1 and TNFR-2)28. We evaluated the effects of HDL on TNFR-1 and TNFR-2 mRNA expression levels. We found that these were both significantly suppressed by pretreatment with HDL (Fig. 2c and d), but to a lesser extent compared with LDL-treatment. This suggested the presence of a HDL-specific mechanism other than down-regulation of TNFRs. at the transcriptional level. In this experiment, the amount of TNF-α from BCG-infected THP1 macrophages in the control group, the group treated with 50 µg/mL HDL or LDL was 2067.2 pg/ml, 388.2 pg/ml, and 1161.3 pg/ ml, respectively (Fig. 2a). The mRNA levels of TNF-α in BCG-infected THP1 macrophages pretreated with HDL were suppressed by 64% (Fig. Results M b (b–d) THP1 macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 24 hours and were then infected with BCG (multiplicity of infection = 10) for 6 hours. TNF-α (b), TNFR-1 (c), and TNFR-2 (d) mRNA expression levels were quantified using real-time PCR (n = 3) and normalized to GAPDH expression. ANOVA was used to test for the differences of in means. gf p p p g ( ) THP1 macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 24 hours. The treated macrophages were then infected with BCG (multiplicity of infection = 10) for 24 hours. The amounts of TNF-α in the cell culture supernatants were measured by ELISA. This assay was repeated three times. (b–d) THP1 macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 24 hours and were then infected with BCG (multiplicity of infection = 10) for 6 hours. TNF-α (b), TNFR-1 (c), and TNFR-2 (d) mRNA expression levels were quantified using real-time PCR (n = 3) and normalized to GAPDH expression. ANOVA was used to test for the differences of in means. HDL suppresses TLR2-dependent production of TNF-α by THP1 macrophages. A recent study showed that, in mouse bone marrow-derived macrophages, HDL induces transcription factor 3 (ATF3), which in turn suppresses the transcription of the genes encoding pro-inflammatory cytokines12. We, therefore, assessed the upregulation of ATF3 in BCG-infected THP1 macrophages. However, we could not find any significant increase in ATF3 with HDL-treatment compared with the untreated control and with LDL treatment (Supplementary Fig. S4). Stimulation of pattern recognition receptors, such as TLRs, is the predominant trigger of TNF-α transcription in mycobacteria-infected macrophages. TLR1/2, 2/6, 4 and 9 are involved in the recognition of mycobacterial components, and TLR2 is considered to be the most responsible receptor for induction of TNF-α29–32. We ana- lyzed the effect of HDL on the mRNA levels of TLR2. In uninfected macrophages, HDL reduced the TLR2 mRNA level by around 80% compared with that in untreated or LDL-treated ones (Fig. 3a). Importantly, HDL treat- ment reduced the TLR2 mRNA level to around 40% in mycobacteria-infected macrophages compared with the untreated control (Fig. 3b). Flow cytometry data analysis showed that the level of surface TLR2 of BCG-infected THP1 macrophages was reduced from 55.8% to 22.4% by the treatment with HDL as shown in Fig. 3c. Results M b 2b), suggesting the presence of transcriptional regulation. Prior work indicated that TNF-α transcription is regulated by autocrine systems through the activation of the NF-κB pathway by the bonding of TNF-α with TNF receptors (TNFR-1 and TNFR-2)28. We evaluated the effects of HDL on TNFR-1 and TNFR-2 mRNA expression levels. We found that these were both significantly suppressed by pretreatment with HDL (Fig. 2c and d), but to a lesser extent compared with LDL-treatment. This suggested the presence of a HDL-specific mechanism other than down-regulation of TNFRs. Prior work indicated that TNF-α transcription is regulated by autocrine systems through the activation of the NF-κB pathway by the bonding of TNF-α with TNF receptors (TNFR-1 and TNFR-2)28. We evaluated the effects of HDL on TNFR-1 and TNFR-2 mRNA expression levels. We found that these were both significantly suppressed by pretreatment with HDL (Fig. 2c and d), but to a lesser extent compared with LDL-treatment. This suggested the presence of a HDL-specific mechanism other than down-regulation of TNFRs. SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 3 www.nature.com/scientificreports/ Figure 2. Effect of HDL on the expression of TNF-α and its receptors in BCG-infected macrophages. (a) THP1 macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 24 hours. The treated macrophages were then infected with BCG (multiplicity of infection = 10) for 24 hours. The amounts of TNF-α in the cell culture supernatants were measured by ELISA. This assay was repeated three times. (b–d) THP1 macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 24 hours and were then infected with BCG (multiplicity of infection = 10) for 6 hours. TNF-α (b), TNFR-1 (c), and TNFR-2 (d) mRNA expression levels were quantified using real-time PCR (n = 3) and normalized to GAPDH expression. ANOVA was used to test for the differences of in means. f Figure 2. Effect of HDL on the expression of TNF-α and its receptors in BCG-infected macrophages. (a) h Figure 2. Effect of HDL on the expression of TNF-α and its receptors in BCG-infected macrophages. (a) THP1 macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 24 hours. The treated macrophages were then infected with BCG (multiplicity of infection = 10) for 24 hours. The amounts of TNF-α in the cell culture supernatants were measured by ELISA. This assay was repeated three times. Results M b p g y g We next assessed whether TLR2-dependent TNF-α production was impaired by HDL. We found that, indeed, HDL reduced TNF-α production by macrophages stimulated with 50 ng/ml of PAM3CSK4, a ligand for the TLR2/TLR1 homodimer (Fig. 3d).i To confirm that TLR2-mediated signaling pathways were impaired by HDL, we analyzed the activation (phos- phorylation) of NF-κB (p65) and p38, ERK and JNK mitogen-activated protein kinases (MAPKs) by immu- noblot of the cell lysates derived from HDL-treated BCG-infected macrophages. The data indicated 29%, 38% and 45% reduced phosphorylation of P65, P38 and JNK, respectively. This showed that HDL indeed suppressed activation of both major cellular signaling pathways for the transcription of the TNF-α gene30,33–35 (Fig. 4 and Supplementary Fig. S5). By contrast, HDL did not influence the phosphorylation of ERK. TLR2 is responsible for TNF-α production by THP1 macrophages upon infection with mycobacteria. TLR2 is a receptor responsible for TNF-α production upon mycobacterial infection of mouse macrophages36,37. While its similar role in human macrophages has been implied38, its exact responsibility has not been SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 4 www.nature.com/scientificreports/ Figure 3. HDL suppresses TLR2 expression of THP1 macrophages. (a) Macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 6 hours. The TLR2 mRNA expression levels were then quantified using real-time PCR (n = 3) and normalized to GAPDH expression. (b) Macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 24 hours and then infected with BCG (multiplicity of infection = 10) for 6 hours. The TLR2 mRNA expression level was quantified by real-time PCR (n = 3). ANOVA was used to test for the differences of in means. These assays were repeated twice. (c) THP1macrophages were cultured with (merged with pink) or without (filled with purple) addition of HDL (50 µg/ml) for 6 hours. Surface TLR2 was detected with PE-labeled anti-TLR2 antibody and analyzed by FACScan. The border of M1 and M2 was set at the peak of untreated THP1 cells stained by TLR2 antibody. Calculated % of cells in the M2 area of control cells and HDL-treated cells were 55.8% and 22.4%, respectively. (d) THP1 macrophages were cultured with or without addition of HDL (50 µg/ml) for 24 hours and were then stimulated with the TLR2 ligand Pam3CSK4 (50 ng/ml) for 24 hours. Results M b The amounts of TNF-α in the cell culture supernatants (n = 2) were measured by ELISA. ANOVA was used to test for the differences of in means. This assay was repeated twice. Figure 3. HDL suppresses TLR2 expression of THP1 macrophages. (a) Macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 6 hours. The TLR2 mRNA expression levels were then quantified using real-time PCR (n = 3) and normalized to GAPDH expression. (b) Macrophages were cultured with or without adding HDL or LDL (50 µg/ml) for 24 hours and then infected with BCG (multiplicity of infection = 10) for 6 hours. The TLR2 mRNA expression level was quantified by real-time PCR (n = 3). ANOVA was used to test for the differences of in means. These assays were repeated twice. (c) THP1macrophages were cultured with (merged with pink) or without (filled with purple) addition of HDL (50 µg/ml) for 6 hours. Surface TLR2 was detected with PE-labeled anti-TLR2 antibody and analyzed by FACScan. The border of M1 and M2 was set at the peak of untreated THP1 cells stained by TLR2 antibody. Calculated % of cells in the M2 area of control cells and HDL-treated cells were 55.8% and 22.4%, respectively. (d) THP1 macrophages were cultured with or without addition of HDL (50 µg/ml) for 24 hours and were then stimulated with the TLR2 ligand Pam3CSK4 (50 ng/ml) for 24 hours. The amounts of TNF-α in the cell culture supernatants (n = 2) were measured by ELISA. ANOVA was used to test for the differences of in means. This assay was repeated twice. elucidated36–38. To establish its exact role, we generated TLR-2 knock-out (KO) THP1 cells using the CRISPR-Cas9 system (Supplementary Fig. S6). As expected, the TLR-2 KO THP1 macrophage could not produce TNF-α when stimulated with PAM3CSK4. In this experimental setting, we observed that TNF-α production was impeded because of TLR2-deficiency upon BCG (Fig. 5a) and M. tuberculosis (Fig. 5b) infection. This demonstrated the critical role of TLR2 in TNF-α production by mycobacteria-infected human macrophages. SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 Figure 4. HDL impairs activation of TLR2-mediated intracellular signalings. THP1 macrophages were cultured with or without (control) adding HDL (50 µg/ml) for 24 hours. The macrophages were then infected with BCG (multiplicity of infection = 10) for 15 minutes. Immunoblot of p65 phosphorylation (phospho-p65), total p65, p38 phosphorylation (phospho-p38), total p38, ERK phosphorylation (phospho-ERK), total ERK, JNK phosphorylation (phospho-JNK) and total JNK (relative to total β-actin) were detected. Each whole cell lysate was fractionated by SDS-PAGE and transferred on a membrane.Immunoblot experiments were then performed to validate the level of each target protein. The western blot bands of phospho-p65 (65 kDa) total p65 (65 kDa), phospho-p38 (43 kDa), total p38 (43 kDa), phospho-ERK (44 and 42 kDa), total ERK (44 and 42 kDa), phospho- JNK (46 and 54 kDa) and total JNK(46 and 54 kDa) and total beta-actin (43 kDa)are displayed in the figure. Wholemembranes were also displayed in Supplemental Fig. S5. The immunoblots are representative of three independent experiments. as well29,50. We investigated the expression of these receptors but could not find any upregulation in HDL-treated THP1 macrophages. It is thus considered that other uncharacterized receptors may contribute to this process. The other possibility relates to the dynamic quantitative change of the macrophage membrane fluidity as a result of a change in cholesterol levels that should affect phagocytosis51. f Recently ATF3 was proposed to be the master transcriptional regulator in HDL-dependent suppression of pro-inflammatory cytokines in mice bone marrow-derived macrophages12. However, we could not find any upregulation of ATF3 in THP1 macrophages treated with HDL (Supplementary Fig. S4). This discrepancy may be due to the difference in responses between mouse and human macrophages. f g We investigated other possible molecular mechanisms to explain the HDL-dependent suppression of TNF-α production. TLR2 is a host-protective molecule against mycobacterial infections in both mouse and human52–56 and is considered a predominant receptor for TNF-α production. We thus next focused on TLR238. We found that HDL suppressed TLR2 expression (Fig. 3), cellular response to the TLR2-specific ligand (Fig. 3d) and TLR2-related cellular signaling pathways (Fig. 4). We then proved that TLR2 was a key receptor for TNF-α production upon mycobacterial infection by creating a TLR2-KO THP1 cell (Fig. 5). Discussion This study demonstrates for the first time that HDL dose-dependently suppresses the production of pro-inflammatory cytokines, such as, IL-6, TNF-α and IFN-γ by THP1 macrophages infected with M. tubercu- losis (Fig. 1). The same was true when macrophages differentiated from CD14+ monocytes derived from human blood were used (Supplementary Fig. S2). LDL treatment also reduced the production of cytokines but its effect was lower than that of HDL and it did not show dose responsiveness (Fig. 1), suggesting its minor function or non-functional artifacts. TNF-α was the most abundantly produced cytokine among those tested. It plays a key role in the immune defense during both active and latent TB39–44. IL-6 and IFN-γ also contribute to protection against mycobacterial diseases40,45–48. Inversely, however, massive production of these pro-inflammatory cytokines is harmful to the body. Our data thus suggest that HDL has the potential to modulate immune responses against mycobacterial infections.l HDL-dependent suppression of pro-inflammatory cytokine production was not owing to inhibition of myco- bacterial infection of macrophages. Rather, HDL treatment enhanced these infections (Supplementary Fig. S3). In the presence of sera, mycobacteria enters macrophages mainly through the complement receptor49. The con- tribution of other receptors, such as mannose receptor and the scavenger receptor B1 (SR-B1) have been reported SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 5 www.nature.com/scientificreports/ Figure 4. HDL impairs activation of TLR2-mediated intracellular signalings. THP1 macrophages were cultured with or without (control) adding HDL (50 µg/ml) for 24 hours. The macrophages were then infected with BCG (multiplicity of infection = 10) for 15 minutes. Immunoblot of p65 phosphorylation (phospho-p65), total p65, p38 phosphorylation (phospho-p38), total p38, ERK phosphorylation (phospho-ERK), total ERK, JNK phosphorylation (phospho-JNK) and total JNK (relative to total β-actin) were detected. Each whole cell lysate was fractionated by SDS-PAGE and transferred on a membrane.Immunoblot experiments were then performed to validate the level of each target protein. The western blot bands of phospho-p65 (65 kDa) total p65 (65 kDa), phospho-p38 (43 kDa), total p38 (43 kDa), phospho-ERK (44 and 42 kDa), total ERK (44 and 42 kDa), phospho- JNK (46 and 54 kDa) and total JNK(46 and 54 kDa) and total beta-actin (43 kDa)are displayed in the figure. Wholemembranes were also displayed in Supplemental Fig. S5. The immunoblots are representative of three independent experiments. Taken together these data indicate the signifi- cant role of TLR2 in TNF-α production upon mycobacterial infection and the involvement of down-regulation of TLR2 by HDL in the suppression of TNF-α production by mycobacteria-infected THP1 macrophages. SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 6 www.nature.com/scientificreports/ Figure 5. TLR2 is critical for TNF-α production from THP1 macrophages upon mycobacterial infection. (a) THP1 macrophages and its TLR2-KO cells were stimulated with the TLR2 ligand, Pam3CSK4 (50 ng/ ml) or infected with BCG (multiplicity of infection = 10) for 24 hours, and the amount of TNF-α in the cell culture supernatant (n = 3) was measured by ELISA. (b) THP1 macrophages and TLR2-KO cells were infected M. tuberculosis (multiplicity of infection = 10) for 24 hours, and the amount of TNF-α in the cell culture supernatant (n = 3) was measured by ELISA. ANOVA was used to test for the differences of in means. n from THP1 macrophages upon mycobacterial infectio Figure 5. TLR2 is critical for TNF-α production from THP1 macrophages upon mycobacterial infection. Figure 5. TLR2 is critical for TNF-α production from THP1 macrophages upon mycobacterial infection. (a) THP1 macrophages and its TLR2-KO cells were stimulated with the TLR2 ligand, Pam3CSK4 (50 ng/ ml) or infected with BCG (multiplicity of infection = 10) for 24 hours, and the amount of TNF-α in the cell culture supernatant (n = 3) was measured by ELISA. (b) THP1 macrophages and TLR2-KO cells were infected M. tuberculosis (multiplicity of infection = 10) for 24 hours, and the amount of TNF-α in the cell culture supernatant (n = 3) was measured by ELISA. ANOVA was used to test for the differences of in means. Figure 5. TLR2 is critical for TNF-α production from THP1 macrophages upon mycobacterial infection. (a) THP1 macrophages and its TLR2-KO cells were stimulated with the TLR2 ligand, Pam3CSK4 (50 ng/ ml) or infected with BCG (multiplicity of infection = 10) for 24 hours, and the amount of TNF-α in the cell culture supernatant (n = 3) was measured by ELISA. (b) THP1 macrophages and TLR2-KO cells were infected M. tuberculosis (multiplicity of infection = 10) for 24 hours, and the amount of TNF-α in the cell culture supernatant (n = 3) was measured by ELISA. ANOVA was used to test for the differences of in means. Previously, Tang et al. showed that apolipoprotein A-1, a component of HDL, interacts with ABCA1 and acti- vates STAT3, which in turn suppresses the production of pro-inflammatory cytokines57. ABCA1 transcription was remained in HDL-treated THP1 macrophages, although it was significantly reduced upon LDL treatment (Fig. S7). This implies the possibility of interaction between down-regulation of TLR2 and activation of the JAK/ STAT3 signaling pathway, which should be clarified in future studies.h Previously, Tang et al. showed that apolipoprotein A-1, a component of HDL, interacts with ABCA1 and acti- vates STAT3, which in turn suppresses the production of pro-inflammatory cytokines57. ABCA1 transcription was remained in HDL-treated THP1 macrophages, although it was significantly reduced upon LDL treatment (Fig. S7). This implies the possibility of interaction between down-regulation of TLR2 and activation of the JAK/ STAT3 signaling pathway, which should be clarified in future studies.h i There are few epidemiological cohort studies that estimate the relationship between HDL levels and tuber- culosis. Two papers showed that decrease of HDL-cholesterol levels was correlated with severity of the disease in active TB patients58. Another study showed that the level of HDL-c, but not LDL-c or total cholesterol was significantly lower in 115 TB patients compared to 70 pneumonia patients and 30 healthy control59. In contrast, hypercholesterolemia impairs protective immunity to tuberculosis in mice model60. A recent adolescent cohort study with 6,363 enrolled participants identified the upregulation of apolipoprotein A-1 as a risk factor of TB progression from latent infection61. It is also known that M. tuberculosis uses a host cholesterol as a major carbon source during infection22–26, implying that higher cellular cholesterol levels may activate TB disease progression. Taken together, these suggest the important but controversial role of HDL in the determination of TB disease out- come. Further studies are in demand to clarify the effect of HDL and LDL in clinical situations of mycobacterial diseases including initial infection, asymptomatic states, and active diseases.h g y p HDL is a highly condensed lipoprotein fraction in plasma. The most abundant apolipoprotein is A-I and the second one is apo A-II, but it is composed of over different 80 kinds of proteins and their variants. To identify which components and sub-fractions of HDL are responsible for their activity are next important issues to be clarified.h i The downregulation of TLR2 by HDL interferes with the general recognition of pathogen-associated molec- ular patterns and is not limited to mycobacterial diseases. SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 It is thus likely that HDL modulates host protective responses against other pathogens. While HDL is related to lower risk of vascular disorders contributing to human longevity, this study suggests the importance of taking into account the risk of immune suppression posed by HDL. Methods The cells were subsequently incubated with PE-labeled anti-human CD282 (TLR2) (clone: TL2.1) (eBioscience) in PBS at room temperature in the dark for 20 min. Cells were then washed twice with ice-cold PBS, resuspended in ice-cold PBS, and analyzed on a flow cytometer (FACSCalibur HG, Becton Dickinson). An isotype-matched antibody, mouse IhG2aκ Iso Control PE (clone: eBM2a) (eBioscience), of irrelevant specificity was used as a control. The control cells were analyzed using FACScan (BD Bioscience, Franklin Lakes, USA). Gene editing of TLR2 by CRISPR-Cas9 system. To generate TLR2-KO THP1 cells, the CRISPR-Cas9 system was applied. Niigata University Recombinant DNA Advisory Committee approved CRISPR/Cas9 gene knock out of TLR2 gene in THP1 cells in the BSL2 laboratory of Department of Bacteriology, Niigata University School of Medicine. The sequence of guide RNA (gRNA) synthesis, which targets the human TLR2 gene, was GACTGTACCCTTAATGGAGT(TGG). It was inserted into pSpCas9 (BB)-2A-Puro (PX459) V2.0 (Addgene, Cambridge, MA, USA) as previously described63. After insertion of the gRNA sequence, the pX459 vector was transfected into THP1 cells by electroporation with Gene Pulser Xcell (Bio-Rad, Hercules, CA, USA). For elec- troporation, THP1 cells were collected by centrifugation from culture and were suspended by the serum-free RPMI-1640 medium at 1 × 107 cells/ml. In a 0.4-cm cuvette, 10 μg of plasmid vector and 0.4 ml of cell suspension were mixed and incubated for 10 min, and then pulsed once with 260 V and 1050 μF. After electroporation, cells were grown in RPMI-1640 medium supplemented with 10% of FBS. Forty-eight hours after electroporation, cells were selected by 1 μg of puromycin (Invivogen, San Diego, CA, USA) for 48 hours. To obtain a monoclonal cell population, cloning by limiting dilution was performed. Gene editing of TLR2 was confirmed by the PAGE-based genotyping protocol64 and by determining the DNA sequence of a targeted region of the TLR2 exon. Immunoblotting of p65, p38, ERK1/2 and JNK. More than 5 × 106 differentiated THP1 macrophages were lysed with 1 Xlysis buffer containing 1% NP-40, 20 mM Tris-HCl pH 7.5, 2 mM EDTA and 150 mM NaCl, supplemented with a protease inhibitor cocktail (Nacalai Tesque) and 100 mM NaF, on ice for 10 min. The samples were then centrifuged at 16,000 g for 5 min at 4 °C, and the supernatant was analyzed by immunoblotting of whole cell lysates. Methods When using the Cytotoxicity Detection Kitsplus (LDH), the cytoplasmic LDH enzyme activity in the culture medium was measured according to the manufacturer’s instructions. Measurement of mycobacterial infection by CFU assays. In vitro infection experiments using BCG and M. tuberculosis were conducted in BSL2 and BSL 3 level facilities in the Department of Bacteriology, Niigata University School of Medicine after approval by the Institutional Biosafety Committee of Niigata University. Differentiated macrophages were infected with M. tuberculosis strain H37Rv or BCG at a multiplicity of infec- tion of 10 for 3 hours, washed, and lysed with PBS containing 0.5% Triton X-100. CFU assays were performed by harvesting serially diluted suspensions on Middlebrook 7H11 agar plates supplemented with oleic, albumin, dextrose, and catalase (OADC) at 37 °C for 3 weeks to quantify the number of mycobacteria that had infected the macrophages. Quantitative measurement of TNF-α, TNFR-1, TNFR-2, TLR2, and ATF3 mRNA expression lev- els in THP1 macrophages. Differentiated macrophages were harvested, and total RNA was extracted using TRIzol® (Thermo Fisher Scientific) according to the manufacturer’s instructions. The resulting RNA was then treated with RNase-free DNase (Ambion, California, USA) to remove any potential DNA contamination. After being reverse-transcribed to cDNA using the Super Script First Strand Synthesis System for reverse-transcription PCR (Invitrogen, Massachusetts, USA), triplicate cDNA aliquots were amplified with sequence-specific primers and SYBR green (Applied Biosystems by Life Technologies, California, USA) using 7500 Fast Real-Time PCR Systems (Applied Biosystems by Life Technologies). All reactions were repeated independently at least three times to ensure the reproducibility of the results. All primers were purchased from Sigma (Supplementary Table S1). Measurement of secreted TNF-α level by enzyme-linked immunoabsorbent assay (ELISA). TNF-α levels in the cell-culture supernatant were measured using human TNF-α ELISA kits (R&D Systems) according to the manufacturer’s instructions. Measurement of secreted IL-4, IL-6, IL-10, IFN-γ and TNF-α level by the Bio-Plex Multiplex System. IL-4, IL-6, IL-10, IFN-γ and TNF-α levels in cell-culture supernatants were measured using the Bio-Plex Multiplex System (Bio-Rad, California, USA) according to the manufacturer’s instructions. Flow cytometry. Following treatment with HDL (50 µg/ml), cells were washed with ice-cold PBS and then fixed with 80% methanol for 5 min and washed again with ice-cold PBS. Cells were then incubated with unlabe- led anti-Fc receptor antibody (eBioscience, San Diego, USA) in PBS in the dark at 4 °C for 20 min to block the Fc receptor. Methods Isolation of HDL and LDL from healthy donors. HDL and LDL were isolated by a method previously developed62. Briefly, freshly drawn blood samples were collected (1 mM EDTA as anti-coagulant) and centrifuged at 2,000 × g for 15 min. Chylomicrons were then removed from plasma by additional centrifugation at 100,000 × g for 10 min. For each sample, four volumes of plasma were mixed with one volume of OptiPrep™ (Axis-Shield, Dundee, Scotland), and 2.8 ml of the resulting mixture was transferred to an OptiPrep™ tube. Solution B (0.85 g of NaCl dissolved in 50 ml water) was layered on top of this mixture, followed by a layer of 10 ml of 100-mM Hepes stock solution. After sealing the tubes, the samples were centrifuged at approximately 350,000 × g for 3 hours at 16 °C. The HDL and LDL fractions were collected by using disposable syringes with 23-gauge needles. Cells. THP1 cells, a human acute monocytic cell line that was purchased from the Human Science Foundation (Tokyo, Japan), were cultured in RPMI-1640 supplemented with 10% HI-FBS (Thermo Fisher Scientific, Waltham, MA, USA). Differentiation was achieved by re-suspending the cells at 8 × 105 cells/ml in Dulbecco’s modified Eagle’s medium (Wako) supplemented with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin with the addition of 100 nM phorbol 12-myristate 13-acetate (Sigma) for 48 hours. Non-adherent cells were removed by aspiration of the supernatant followed by replacement with fresh medium.h The human blood PBMC was obtained by using Optiprep (Axis-Shield, Dundee, Scotland) in accordance with the manufacturer’s instructions. CD14+ monocytes were then isolated from PBMC by the AutoMacs separation system using anti-CD14 magnetic microbeads (Miltenyi Biotec, Germany). The CD14+ human monocytes were differentiated to macrophages by commercially available macrophage differentiation kit (R&D systems, Minnesota). SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 7 www.nature.com/scientificreports/ Reagents. Salmonella-derived LPS and Pam3CSK4 were purchased from Sigma and R&D Systems (Minneapolis, USA), respectively. Cell viability. We assessed the cell viability by using two methods: 1) trypan blue-exclusion assay; and 2) Cytotoxicity Detection Kitplus, which measures the LDH release from dead cells. Differentiated macrophages were cultured with or without adding HDL or LDL (5–50 µg/ml) for 24 hours. In trypan blue-exclusion assays, cells were stained with trypan blue solution, loaded into a hemocytometer and immediately examined under a micro- scope. Methods A total of 5 µg of each lysate, of which the protein concentration was determined by Advanced Protein Assay (Cytoskeleton, Inc.), was run on 12.5% SDS-PAGE gel. After SDS-PAGE, fractionated proteins were trans- ferred onto PVDF membranes (Millipore) and blocked in Tris-buffered saline (pH 7.5) containing 1% (wt/vol) skim milk and 0.05% Tween 20 before overnight incubation with each specific primary antibody. The following SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 8 www.nature.com/scientificreports/ antibodies were purchased from Cell Signaling TECHNOLOGY and used in this study: anti-phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (1:1000 dilution), anti-NF-κB p65 (D14E12) XP Rabbit mAb (1:1000 dilu- tion), anti-phospho-p38 MAP Kinase (Thr180/Tyr182) antibody (1:1000 dilution), anti-p38 MAPK antibody (1:1000 dilution), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb (1:2000 dilution), anti-p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (1:1000 dilution), anti-phospho-SAPK/JNK (Thr183/ Tyr185) (81E11) Rabbit mAb (1:1000 dilution), anti-SAPK/JNK antibody (1:1000 dilution) and anti-β-Actin (C4): sc-47778 antibody (1:2500 dilution). The membranes were then washed and incubated with appropriate secondary antibodies, and the antibody reactions were detected with Immobilon™ western (Millipore). Data analysis. ANOVA was used to analyze in vitro assay data. All statistical analyses were carried out using SPSS (ver. 21, IBM, NY, USA), and p-values less than 0.05 were considered significant65–69. References Elevation of plasma high-density lipoprotein concentration reduce E-selectin in an in vivo model of acute inflammation. Circulation 103, 108–112 (2001).l l 2. De Nardo, D. et al. High-density lipoprotein mediates anti-inflammatory reprogramming of macrophages via the transcriptiona regulator ATF3. Nature Immunology 15, 152–160, https://doi.org/10.1038/Ni.2784 (2014). 12. De Nardo, D. et al. High-density lipoprotein mediates anti-inflammatory reprogramming of macrophages via the transcriptional regulator ATF3. Nature Immunology 15, 152–160, https://doi.org/10.1038/Ni.2784 (2014). 13. Libby, P., Ridker, P. M. & Hansson, G. K. Progress and challenges in translating the biology of atherosclerosis. Nature 473, 317–325, https://doi.org/10.1038/nature10146 (2011).f g gy p g 13. Libby, P., Ridker, P. M. & Hansson, G. K. Progress and challenges in translating the biology of atherosclerosis. Nature 473, 317–325, https://doi.org/10.1038/nature10146 (2011).f p g 14. Murphy, A. J., Westerterp, M., Yvan-Charvet, L. & Tall, A. R. Anti-atherogenic mechanisms of high density lipoprotein: effec myeloid cells. Biochimica et biophysica acta 1821, 513–521, https://doi.org/10.1016/j.bbalip.2011.08.003 (2012). y p y p g j p ( ) 15. Norata, G. D., Pirillo, A., Ammirati, E. & Catapano, A. L. Emerging role of high density lipoproteins as a player in the imm system. Atherosclerosis 220, 11–21, https://doi.org/10.1016/j.atherosclerosis.2011.06.045 (2012). 16. Flynn, J. L. & Chan, J. Immunology of tuberculosis. Annual review of immunology 19, 93–129, https://doi.org/10.1146/annurev. immunol.19.1.93 (2001). 7. Flynn, J. L. et al. Tumor necrosis factor-alpha is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2, 561–572 (1995).l 8. Keane, J. et al. Tuberculosis associated with infliximab, a tumor necrosis factor (alpha)-neutralizing agent. New England Journal o Medicine 345, 1098–1104, https://doi.org/10.1056/Nejmoa011110 (2001). 19. Manabe, Y. C. & Bishai, W. R. Latent Mycobacterium tuberculosis-persistence, patience, and winning by waiting. Nature medicine 6, 1327–1329, https://doi.org/10.1038/82139 (2000). p g 20. Getahun, H. et al. Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries. The European respiratory journal 46, 1563–1576, https://doi.org/10.1183/13993003.01245-2015 (2015). h p p y j g 1. Getahun, H., Matteelli, A., Chaisson, R. E. & Raviglione, M. Latent Mycobacterium tuberculosis Infection. New England Journal o Medicine 372, 2127–2135, https://doi.org/10.1056/Nejmra1405427 (2015). 22. Pandey, A. K. & Sassetti, C. M. Mycobacterial persistence requires the utilization of host cholesterol. Proceedings of the National Academy of Sciences of the United States of America 105, 4376–4380, https://doi.org/10.1073/pnas.0711159105 (2008). y f f f p g p 23. Brzostek, A., Pawelczyk, J., Rumijowska-Galewicz, A., Dziadek, B. & Dziadek, J. References 1. Kannel, W. B., Dawber, T. R., Kagan, A., Revotskie, N. & Stokes, J. 3rd. Factors of risk in the development of coronary heart disease–six year follow-up experience. The Framingham Study. Annals of internal medicine 55, 33–50 (1961). y p ph g y f , ( ) 2. Wilson, P. W. et al. Prediction of coronary heart disease using risk factor categories. Circulation 97, 1837–1847 (1998). 3. Gordon, T., Castelli, W. P., Hjortland, M. C., Kannel, W. B. & Dawber, T. R. High density lipoprotein as a protective factor against coronary heart disease. The Framingham Study. The American journal of medicine 62, 707–714 (1977).h hh 4. Gordon, D. J. & Rifkind, B. M. High-density lipoprotein–the clinical implications of recent studies. The New England journal o medicine 321, 1311–1316, https://doi.org/10.1056/nejm198911093211907 (1989). 321, 1311–1316, https://doi.org/10.1056/nejm198911093211907 (1 p g j 5. Assmann, G., Schulte, H., von Eckardstein, A. & Huang, Y. High-density lipoprotein cholesterol as a predictor of coronary heart disease risk. The PROCAM experience and pathophysiological implications for reverse cholesterol transport. Atherosclerosis 124(Suppl), S11–20 (1996). ( pp ) ( ) 6. Tanne, D., Yaari, S. & Goldbourt, U. High-density lipoprotein cholesterol and risk of ischemic stroke mortality. A 21-year follow-up of 8586 men from the Israeli Ischemic Heart Disease Study. Stroke 28, 83–87 (1997). 7. Oram, J. F., Lawn, R. M., Garvin, M. R. & Wade, D. P. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages. The Journal of biological chemistry 275, 34508–34511, https://doi.org/10.1074/jbc. M006738200 (2000). 8. Wang, X. et al. Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo. Journal of Clinical Investigation 117, 2216–2224, https://doi.org/10.1172/Jc32057 (2007). 9. Wang, N., Lan, D., Chen, W., Matsuura, F. & Tall, A. R. ATP-binding cassette transporters G1 and G4 mediate cellular cholestero efflux to high-density lipoproteins. Proceedings of the National Academy of Sciences of the United States of America 101, 9774–9779 https://doi.org/10.1073/pnas.0403506101 (2004). p g p ( ) 10. Zhang, Y. et al. Hepatic expression of scavenger receptor class B type I (SR-BI) is a positive regulator of macrophage reverse cholesterol transport in vivo. The Journal of clinical investigation 115, 2870–2874, https://doi.org/10.1172/jci25327 (2005). h 11. Cockerill, G. W. et al. Elevation of plasma high-density lipoprotein concentration reduces interleukin-1-induced expression of E-selectin in an in vivo model of acute inflammation. Circulation 103, 108–112 (2001).l h 11. Cockerill, G. W. et al. www.nature.com/scientificreports/ p g j 4. Stenger, S. Immunological control of tuberculosis: Role of tumour necrosis factor and more. Annals of the Rheumatic Diseases 64 24–28, https://doi.org/10.1136/Ard.2005.042531 (2005).h p g 5. Cooper, A. M. et al. Disseminated tuberculosis in interferon gamma gene-disrupted mice. The Journal of experimental medicine 178 2243–2247 (1993). 46. Flesch, I. E. & Kaufmann, S. H. Role of cytokines in tuberculosis. Immunobiology 189, 316–339, https://doi.org/10.1016/s0171- 2985(11)80364-5 (1993).ih 47. Jouanguy, E. et al. Interferon-gamma-receptor deficiency in an infant with fatal bacille Calmette-Guerin infection. The New Eng journal of medicine 335, 1956–1961, https://doi.org/10.1056/nejm199612263352604 (1996).i 48. Ladel, C. H. et al. Lethal tuberculosis in interleukin-6-deficient mutant mice. Infection and immunity 65, 4843–4849 (1997). , ki f y , ( 49. Ernst, J. D. Macrophage receptors for Mycobacterium tuberculosis. Infection and immunity 66, 1277–1281 (1998). 0. Aderem, A. & Underhill, D. M. Mechanisms of phagocytosis in macrophages. Annual review of immunology 17, 593–623, https:/ doi.org/10.1146/annurev.immunol.17.1.593 (1999).f g 1. Goluszko, P. & Nowicki, B. Membrane cholesterol: a crucial molecule affecting interactions of microbial pathogens with mammalian cells. Infection and immunity 73, 7791–7796, https://doi.org/10.1128/iai.73.12.7791-7796.2005 (2005). f y p g 52. Bafica, A. et al. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. The Journal of experimental medicine 202, 1715–1724, https://doi.org/10.1084/jem.20051782 (2005).i h f p g j 3. Drennan, M. B. et al. Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection. Am J Pathol 164, 49–57 (2004). 4. Heldwein, K. A. et al. TLR2 and TLR4 serve distinct roles in the host immune response against Mycobacterium bovis BCG. J Leukoc Biol 74, 277–286 (2003).hh 5. Ogus, A. C. et al. The Arg753GLn polymorphism of the human toll-like receptor 2 gene in tuberculosis disease. The European respiratory journal 23, 219–223 (2004). p y j 6. Schroder, N. W. et al. Heterozygous Arg753Gln polymorphism of human TLR-2 impairs immune activation by Borrelia burgdorfer and protects from late stage Lyme disease. Journal of immunology (Baltimore, Md.: 1950) 175, 2534–2540 (2005).h g y f gy 57. Tang, C., Liu, Y., Kessler, P. S., Vaughan, A. M. & Oram, J. F. The macrophage cholesterol exporter ABCA1 functions as an inflammatory receptor. The Journal of biological chemistry 284, 32336–32343, https://doi.org/10.1074/jbc.M109.047472 (2009 O l S l h l l C d C fi l l h h d l l lh 58. Deniz, O. et al. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 1. Carmona, J. et al. Mycobacterium tuberculosis Strains Are Differentially Recognized by TLRs with an Impact on the Immune Response. PloS one 8, e67277, https://doi.org/10.1371/journal.pone.0067277 (2013). p p g j p 32. Stamm, C. E., Collins, A. C. & Shiloh, M. U. Sensing of Mycobacterium tuberculosis and consequences to both host and bac Immunological reviews 264, 204–219, https://doi.org/10.1111/imr.12263 (2015). g p g ( ) 33. Akira, S. & Takeda, K. Toll-like receptor signalling. Nat Rev Immunol 4, 499–511 (2004). p g g 34. Kleinnijenhuis, J., Oosting, M., Joosten, L. A., Netea, M. G. & Van Crevel, R. Innate immune recognition of Mycobacterium tuberculosis. Clinical & developmental immunology 2011, 405310, https://doi.org/10.1155/2011/405310 (2011). 35. Rajaram, M. V. et al. Mycobacterium tuberculosis activates human macrophage peroxisome proliferator-activated receptor gamma linking mannose receptor recognition to regulation of immune responses. Journal of immunology (Baltimore, Md.: 1950) 185, 929–942, https://doi.org/10.4049/jimmunol.1000866 (2010). p g j 6. Harding, C. V. & Boom, W. H. Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors. Na Rev Microbiol 8, 296–307, https://doi.org/10.1038/nrmicro2321 (2010). p g ( ) 7. Saraav, I., Singh, S. & Sharma, S. Outcome of Mycobacterium tuberculosis and Toll-like receptor interaction: immune response or immune evasion? Immunol Cell Biol 92, 741–746, https://doi.org/10.1038/icb.2014.52 (2014).h g 38. Thoma-Uszynski, S. et al. Induction of direct antimicrobial activity through mammalian toll-like receptors. Science (New York, N.Y.) 291, 1544–1547, https://doi.org/10.1126/science.291.5508.1544 (2001).i h 291, 1544–1547, https://doi.org/10.1126/science.291.5508.1544 p g 39. Bean, A. G. D. et al. Structural deficiencies in granuloma formation in TNF gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin. Journal of Immunology 162, 3504–3511 (1999).h 40. Flynn, J. L. et al. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. The Journal of experimental medicine 178, 2249–2254 (1993). p 1. Kaneko, S. et al. Health and Demographic Surveillance System in the Western and coastal areas of Kenya: an infrastructure for epidemiologic studies in Africa. J Epidemiol 22, 276–285 (2012).f p g p 42. Mohan, V. P. et al. Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: Possible role for limiting pathology. Infection and immunity 69, 1847–1855, https://doi.org/10.1128/Iai.69.3.1847-1855.2001 (2001). g p gy f y p g 3. Saunders, B. M. & Britton, W. J. Life and death in the granuloma: immunopathology of tuberculosis. Immunology and Cell Biology 85, 103–111, https://doi.org/10.1038/Sj.Icb.7100027 (2007). References Mycobacterium tuberculosis is able to accum and utilize cholesterol Journal of bacteriology 191 6584 6591 https://doi org/10 1128/jb 00488 09 (2009) 23. Brzostek, A., Pawelczyk, J., Rumijowska-Galewicz, A., Dziadek, B. & Dziadek, J. Mycobacterium tuberculosis is able to accumulate and utilize cholesterol. Journal of bacteriology 191, 6584–6591, https://doi.org/10.1128/jb.00488-09 (2009).fiii f gy g j 24. Griffin, J. E. et al. High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabo PLoS pathogens 7, e1002251, https://doi.org/10.1371/journal.ppat.1002251 (2011).h 25. Marques, M. A. et al. The Essential Role of Cholesterol Metabolism in the Intracellular Survival of Mycobacterium leprae Is Not Coupled to Central Carbon Metabolism and Energy Production. Journal of bacteriology 197, 3698–3707, https://doi.org/10.1128/ jb.00625-15 (2015). j ( ) 26. Soto-Ramirez, M. D. et al. Cholesterol plays a larger role during Mycobacterium tuberculosis in vitro dormancy and reactivation than previously suspected. Tuberculosis (Edinburgh, Scotland) 103, 1–9, https://doi.org/10.1016/j.tube.2016.12.004 (2017).i 27. Gatfield, J. & Pieters, J. Essential role for cholesterol in entry of mycobacteria into macrophages. Science (New York, N.Y.) 1647–1650 (2000). 8. Pekalski, J. et al. Spontaneous NF-kappaB activation by autocrine TNFalpha signaling: a computational analysis. PloS one 8, e78887 https://doi.org/10.1371/journal.pone.0078887 (2013).f p g j p ( ) 29. van Crevel, R., Ottenhoff, T. H. & van der Meer, J. W. Innate immunity to Mycobacterium tuberculosis. Clinical microbiology rev 15, 294–309, https://doi.org/10.1128/Cmr.15.2.294-309.2002 (2002). 30. Jo, E. K., Yang, C. S., Choi, C. H. & Harding, C. V. Intracellular signalling cascades regulating innate immune responses to Mycobacteria: branching out from Toll-like receptors. Cell Microbiol 9, 1087–1098, https://doi.org/10.1128/Cmr.15.2.294-309.2002 (2007). SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 9 www.nature.com/scientificreports/ Serum total cholesterol, HDL-C and LDL-C concentrations significantly correlate with the radiological extent of disease and the degree of smear positivity in patients with pulmonary tuberculosis. Clin Biochem 40, 162–166, https://doi. org/10.1016/j.clinbiochem.2006.10.015 (2007). g j 9. Sahin, F. & Yildiz, P. Distinctive biochemical changes in pulmonary tuberculosis and pneumonia. Arch Med Sci 9, 656–661, https:/ doi.org/10.5114/aoms.2013.34403 (2013). g 0. Martens, G. W. et al. Hypercholesterolemia impairs immunity to tuberculosis. Infection and immunity 76, 3464–3472, https://doi org/10.1128/iai.00037-08 (2008). g 61. Zak, D. E. et al. A blood RNA signature for tuberculosis disease risk: a prospective cohort study. Lancet 387, 2312–2322, https://doi. org/10.1016/s0140-6736(15)01316-1 (2016). 2. Graham, J. M. et al. A novel method for the rapid separation of plasma lipoproteins using self-generating gradients of iodixanol Atherosclerosis 124, 125–135 (1996). 3. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281–2308, https://doi.org/10.1038 nprot.2013.143 (2013).fii p 64. Zhu, X. et al. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Sci Rep 4, 6420, https://doi.org/10.1038/srep06420 (2014). 65. Ma, M. J. et al. Toll-like receptors, tumor necrosis factor-alpha, and interleukin-10 gene polymorphisms in risk of pulmonary tuberculosis and disease severity. Hum Immunol 71, 1005–1010, https://doi.org/10.1016/j.humimm.2010.07.009 (2010).h y p g j 66. Jafari, M. et al. TheNRAMP1, VDR, TNF-alpha, ICAM1, TLR2 and TLR4 gene polymorphisms in Iranian patients wi tuberculosis: A case-control study. Infect Genet Evol 39, 92–98, https://doi.org/10.1016/j.meegid.2016.01.013 (2016). y p g j 66. Jafari, M. et al. TheNRAMP1, VDR, TNF-alpha, ICAM1, TLR2 and TLR4 gene polymorphisms in Iranian patients with pul y p g j fari, M. et al. TheNRAMP1, VDR, TNF-alpha, ICAM1, TLR2 and TLR4 gene polymorphisms in Iranian patients with pulmonary b l i A t l t d I f t G t E l 39 92 98 http //d i /10 1016/j id 2016 01 013 (2016) fari, M. et al. TheNRAMP1, VDR, TNF-alpha, ICAM1, TLR2 and TLR4 gene polymorphisms in Iranian patients with pulmonary berculosis: A case-control study. Infect Genet Evol 39, 92–98, https://doi.org/10.1016/j.meegid.2016.01.013 (2016).h h p g p y p p p tuberculosis: A case-control study. Infect Genet Evol 39, 92–98, https://doi.org/10.1016/j.meegid.2016.01.013 (2016).h 67. Thuong, N. T. et al. A polymorphism in human TLR2 is associated with increased susceptibility to tuberculous meningitis. Genes Immun 8, 422–428, https://doi.org/10.1038/sj.gene.6364405 (2007).l p g j g ( ) 68. Velez, D. R. et al. Author Contributions Conceptualization: Y.O., S.M.N., S.H., and S.M. Formal analysis: M.I., M.N., Y.O., S.N., E.C., M.O.O., T.Y., K.O., T.O., F.M., and Y.K. Funding acquisition: S.H. and S.M. Investigation: M.I., M.N., Y.O., S.N., E.C., M.O.O., T.Y., K.O., T.O., F.M., S.H., and S.M. Methodology: K.O., T.O., and M.M. Project administration: S.K., Y.I., S.M.N., and S.H. Supervision: S.K., Y.I., S.M.N., and S.H. Validation: M.I., M.N., Y.O., S.M.N., S.H., and S.M. Writing – original draft: M.I., Y.O., T.Y., S.H., and S.M. Writing – review and editing: M.I., Y.O., T.Y., S.H., S.K., Y.I., and S.M. ddi i l f i Conceptualization: Y.O., S.M.N., S.H., and S.M. Formal analysis: M.I., M.N., Y.O., S.N., E.C., M.O.O., T.Y., K.O., T.O., F.M., and Y.K. Funding acquisition: S.H. and S.M. Investigation: M.I., M.N., Y.O., S.N., E.C., M.O.O., T.Y., K.O., T.O., F.M., S.H., and S.M. Methodology: K.O., T.O., and M.M. Project administration: S.K., Y.I., S.M.N., and S.H. Supervision: S.K., Y.I., S.M.N., and S.H. Validation: M.I., M.N., Y.O., S.M.N., S.H., and S.M. Writing – original draft: M.I., Y.O., T.Y., S.H., and S.M. Writing – review and editing: M.I., Y.O., T.Y., S.H., S.K., Y.I., and S.M. Conceptualization: Y.O., S.M.N., S.H., and S.M. Formal analysis: M.I., M.N., Y.O., S.N., E.C., M.O.O., T.Y., K.O., T.O., F.M., and Y.K. Funding acquisition: S.H. and S.M. Investigation: M.I., M.N., Y.O., S.N., E.C., M.O.O., T.Y., K.O., T.O., F.M., S.H., and S.M. Methodology: K.O., T.O., and M.M. Project administration: S.K., Y.I., S.M.N., and S.H. Supervision: S.K., Y.I., S.M.N., and S.H. Validation: M.I., M.N., Y.O., S.M.N., S.H., and S.M. Writing – original draft: M.I., Y.O., T.Y., S.H., and S.M. Writing – review and editing: M.I., Y.O., T.Y., S.H., S.K., Y.I., and S.M. www.nature.com/scientificreports/ Variants in toll-like receptors 2 and 9 influence susceptibility to pulmonary tuberculosis in Caucasians, Afr Americans and West Africans Human genetics 127 65 73 https://doi org/10 1007/s00439 009 0741 7 (2010) 68. Velez, D. R. et al. Variants in toll-like receptors 2 and 9 influence susceptibility to pulmonary tuberculosis in Caucasians, African- Americans, and West Africans. Human genetics 127, 65–73, https://doi.org/10.1007/s00439-009-0741-7 (2010). g p g ( ) 9. Nagi, S. et al. Risk factors and spatial distribution of Schistosoma mansoni infection among primary school children in Mbita District Western Kenya PLoS neglected tropical diseases 8 e2991 https://doi org/10 1371/journal pntd 0002991 (2014) 9. Nagi, S. et al. Risk factors and spatial distribution of Schistosoma mansoni infection among primary school children in Mbita District, Western Kenya. PLoS neglected tropical diseases 8, e2991, https://doi.org/10.1371/journal.pntd.0002991 (2014). SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 10 www.nature.com/scientificreports/ Acknowledgements g We are grateful to Ms. Yuko Kobayashi, Sara Matsumoto, and Shaban Amina Kaboso for their assistance, English editing, and heartfelt encouragement. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, the Japanese Ministry of Health, Labour and Welfare (Research on Emerging and Re-emerging Infectious Diseases, Health Sciences Research Grants), the Japan Health Science Foundation, the United States-Japan Cooperative Medical Science Program against Tuberculosis and Leprosy, and the Collaborative Research Foundation of Otsuka. This paper is published with the permission of the Director of the Kenya Medical Research Institute. SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 Additional Information Additional Information upplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-24233-1. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCIENTIFIC REpOrTS \| (2018) 8:6736 \| DOI:10.1038/s41598-018-24233-1 11
https://openalex.org/W3008327606	https://newogneup.elpub.ru/jour/article/download/1145/1005	Russian	null	Aluminum oxide and alumina ceramics (Review). Part 1. Properties of Al<sub>2</sub>O<sub>3</sub> and industrial production of dispersed Al<sub>2</sub>O<sub>3</sub>	Novye ogneupory	2,019	cc-by	7,251	УДК 546.623-31:666.3 ОКСИД АЛЮМИНИЯ И АЛЮМООКСИДНАЯ КЕРАМИКА (Обзор). Часть 1. Свойства Al2O3 и промышленное производство дисперсного Al2O3 Рассмотрены свойства оксида алюминия, связанные с его применением, разновидности промышлен- ных продуктов, способы производства в дисперсном состоянии, в виде поликристаллической керами- ки и монокристаллических изделий, а также химические аспекты технологических процессов. Ключевые слова: оксид алюминия, алюмооксидная керамика, абразивы, композиты. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ ФГБОУ ВО «Санкт-Петербургский государственный технологический институт (технический университет)», Санкт-Петербург, Россия ОКСИД АЛЮМИНИЯ И ЕГО ПРОМЫШЛЕННОЕ ПРОИЗВОДСТВО [1‒5] О ксид алюминия1 обладает такими полезными свойствами, как высокие температура плавле- ния, твердость и прочность, износоустойчивость, химическая стойкость. Сапфир и рубин ― моно- кристаллический корунд с примесями (Ti, Fe и Cr), которые дают окрашивание (синий, красный цвет), известны в ювелирном деле. Синтетические бесцветные лейкосапфиры применяют для произ- водства высокопрочных оптических элементов в иллюминаторах, в качестве активных элементов в лазерах и т. п. Поликристаллический Al2O3 имеет относительно низкую стоимость, является одним из самых широко распространенных керамиче- ских материалов и используется как огнеупор, как изолятор в электротехнике, для износостойких деталей механизмов, как абразивный материал для механической обработки и т. п. В электронике применяют диэлектрические подложки как из по- ликристаллического Al2O3, так и из монокристал- лического. Большая часть производимого α-Al2O3 идет как промежуточный продукт на получение алюминия путем электролиза в расплаве. Белый цвет спеченного Al2O3 (в отличие от прозрачного сапфира) обусловлен рассеянием света на порах и включениях примесных фаз. Оксид алюминия используется в некоторых композиционных мате- риалах как наполнитель (волокна Al2O3) или как матрица (например, с волокнами SiC в качестве ар- мирующих). Помимо прочего, Al2O3 обладает био- совместимостью и используется в медицине для имплантатов, в том числе для синтетических хру- сталиков глаза. Благодаря высоким механическим показателям, малой плотности и доступности Al2O3 является самым распространенным материалом керамической броневой защиты. О к с Разнообразие применения обусловлено много- образием полезных характеристик Al2O3. Неко- торые его свойства приведены в табл. 1 наряду со свойствами других широко распространенных ок- сидов, которые могут присутствовать в алюмоок- сидных материалах как примеси или специально введенные добавки. Приведенные значения пока- зателей свойств относятся как к кристаллическим (и аморфным в случае SiO2) материалам, так и к плотной спеченной керамике; последнее справед- ливо в большей степени для базы данных [6]. Так, диапазон значений плотности γ подразумевает ряд технических продуктов. Характерно, что по твердости Н корунд пре- восходит любые другие оксиды и обладает также очень высоким пределом прочности при сжатии σc. По пределам прочности при изгибе σf и рас- тяжении σt, а также трещиностойкости К1с он уступает некоторым оксидам, например ZrO2. По износостойкости поликристаллический Al2O3 (плотная корундовая керамика) превосходит монокристаллический (сапфир). Корундовая ке- рамика, сапфир являются наилучшими изолято- рами среди доступных оксидов, исключая SiO2, который обладает значительно более высокой пробойной напряженностью электрического поля (электрической прочностью) Ebr и, по некоторым данным, более высоким удельным электрическим 1 Если упоминается просто оксид алюминия Al2O3, часто подразумевается корунд α-Al2O3 (хотя в строгом пони- мании Al2O3 включает все, в том числе метастабильные, модификации). Д. А. Провоторов E-mail: Provotorov@vulkantm.com А. М. ОКСИД АЛЮМИНИЯ И ЕГО ПРОМЫШЛЕННОЕ ПРОИЗВОДСТВО [1‒5] Абызов E-mail: andabyz@mail.ru Д. А. Провоторов ail: Provotorov@vulkantm.c А. М. Абызов E-mail: andabyz@mail.ru ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 1 2019 16 ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Таблица 1. Некоторые свойства оксидов алюминия, кремния, циркония, титана и магния Свойство α-Al2O3 SiO2 ZrO2 TiO2 MgO Источник Плотность γ, г/см3 Температура плавления Tm, °C Предел прочности, МПа: при изгибе σf при сжатии σс Модуль упругости E, ГПа Трещиностойкость K1с, МПа·м0,5 Твердость: по Виккерсу HV, ГПа по Моосу HM ТКЛР, ppm/К Теплопроводность λ, Вт/(м·К) Удельное объемное электросопротивление ρ, Ом·см Электрическая проч- ность Ebr, кВ/мм Относительная диэлек- трическая проницае- мость εr 3,99 3‒3,98 2054 2004‒2096 282 152‒800 2550‒3100 690‒5500 365‒393 215‒413 ‒ 3,3‒5,0 20,6‒29,4 5,5‒22,0 9 9 7,1‒8,3 4,5‒10,9 36‒39 12,0‒38,5 2·1017 1012‒1018 13 8‒43 9,38, 11,59 7,8‒11,1 2,20‒2,65 2,201, 2,652 17104 ‒ 310 110‒200 680‒1380 20701, (690‒1380)2 73 66‒75 ‒ 0,62‒0,67 8 4,5‒9,5 7 7 0,55 0,41, 12,32 1,4 1,41(6,26, 10,67)2 1014 (1012‒1016)1, (>1018)2 (470‒670)1 15‒40 4,48, 4,69 3,6‒4,2 (5,80‒6,05)3 5‒6,15 2710 2550‒2700 430‒7203 177‒1000 18503 1200‒5200 2003 100‒250 (7‒15)3 1‒8 14,4, 15,7 5,5‒15,8 6,55 8 10,13 2,3‒12,2 (1‒2)3 1,7‒2,7 ‒ 107‒1012 11 4‒6 12,5 10‒23 4,24 4 1855 ‒ 340 140 800‒940 680 248‒282 230 ‒ 3,2 ‒ 8,6 7,0‒7,5 6,5 7,1 ‒ 7,46, 10,47 11,7 1013 1012 ‒ 4 868, 1709 85 3,58 3,54‒3,58 2852 2807‒2862 441 100‒200 1300‒1380 830‒1670 303 270‒330 ‒ 2,7‒2,8 7,4 5‒7 5,5‒6,0 – 11,5 9‒12 50‒75 30‒60 109 1014‒1015 ‒ 6‒10 9,6 6,8‒9,6 [1] [6] [1] [6] [1] [6] [1] [6] [1] [6] [1] [6] [1] [6] [1] [7] [1] [6] [1] [6] [1] [6] [8] [6] [8] [6] 1 Кварцевое стекло. 2 Кварц (α-SiO2). 3 Кубический, частично стабилизированный MgO (~3 мас. %). 4 Кристобалит, в который переходит кварц при нагреве. 5 Бадделеит. 6 Перпендикулярно кристаллографической оси с (┴ с). 7 Параллельно кристаллографической оси с (\| \| с). 8 Компоненты тензора по главным осям кристалла (ε11= ε22). 9 Компонента тензора по главным осям кристалла (ε33). конденсаторах, СВЧ-резонаторах. Тем не менее известны типы конденсаторов с диэлектрическим слоем Al2O3 (тонкопленочные, электролитиче- ские). Кроме того, Al2O3 представляет интерес в СВЧ-технике, поскольку сапфир обладает мини- мальными значениями тангенса угла диэлектри- ческих потерь tgδ среди известных веществ [11]. Это позволяет использовать непористый мелко- зернистый Al2O3 высокой чистоты для резонаторов высокой добротности в низкошумных осциллято- рах. Некоторые добавки (например, TiO2 в количе- стве 0,25 мас. %) улучшают диэлектрические свой- ства Al2O3 ― уменьшают диэлектрические потери. Т λ Al O й конденсаторах, СВЧ-резонаторах. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Каждое из этих соединений существу- ет в виде двух кристаллических форм ― α и γ. Кроме того, известен аморфный гидроксид алюминия пе- ременного состава Al2O3·nH2O. Химическая актив- ность убывает в ряду: аморфный гидроксид > байе- рит α-Al(OH)3 > гиббсит (гидраргиллит) γ-Al(OH)3 > > бёмит γ-AlO(OH) > диаспор α-AlO(OH). Корундовая керамика обладает высокой химической стойкостью как в окислительной, так и в восстановительной ат- мосфере, как в кислотной, так и в щелочной среде. Инертность Al2O3 связана, в частности, с тем, что он имеет теплоту образования выше, чем большинство других оксидов (ZrO2, Cr2O3, TiO2, SiO2, MgO и др.). Изделия из Al2O3 изготавливают обычными для технической керамики методами. Тонкоизмель- ченный порошок Al2O3 с размером частиц порядка 0,2‒5,0 мкм, обычно с органической связкой, прес- суют в форме; прессованную заготовку сушат и отжигают при 1500‒1800 °C. Временная органиче- ская связка может вводиться во всю массу порошка Al2O3 либо путем формирования и сушки гранул, которые затем отпрессовывают. Температура спе- кания повышается с увеличением чистоты Al2O3 в сырье. Для корундовой керамики высокой чистоты (> 99,7 %) температура спекания может достигать 1900‒2000 °C. Часто для удаления связки и следов воды и других летучих примесей применяют мед- ленный нагрев или выдержку при промежуточной температуре (кальцинирование). Вообще, методы изготовления керамики из Al2O3 могут быть самы- ми разными: экструзия, сухое прессование, литье под давлением, шликерное литье, изостатическое прессование (сухое или влажное), горячее изо- статическое прессование. Монокристаллический Al2O3 (сапфир) изготавливают методом выращи- вания кристаллов из расплава при более высокой температуре (выше точки плавления 2054 °C), ис- пользуя сырье более высокой чистоты, чем для по- лучения корундовой керамики, которая обладает поликристаллической структурой. 2 2 3 Некоторые примеси вводят как специальные до- бавки к Al2O3. Введение добавок приводит к сниже- нию температуры спекания, что облегчает процесс изготовления керамики и уменьшает его стоимость. Наиболее популярным из модификаторов, вводимых в малых количествах (1‒3 мас. % или менее, в коли- честве до сотых долей процента), является MgO, ко- торый подавляет аномальный рост зерен корунда. Аномальный рост зерна вызывают многие примеси, когда их концентрация превышает предел раствори- мости в корунде (~0,03 мас. % SiO2, ~0,003 мас. % CaO и т. п.). В отличие от MgO другие модифицирующие добавки обычно обеспечивают жидкофазное спека- ние. Часто это происходит за счет непосредственного введения или образования в ходе спекания относи- тельно легкоплавких эвтектик, например эвтектики системы Al2O3‒ZrO2. Предельное снижение темпера- туры спекания корунда до 1300 °C при жидкофазном механизме обеспечивает добавка MnO‒TiO2 [13]. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Ряд свойств алюмооксидной керамики очень сильно зависит от примесей в исходном порош- ке. Как правило, промышленно выпускаемые по- рошки Al2O3 содержат примеси оксидов кремния, кальция, магния, натрия, калия. SiO2, CaО, Na2O, K2O плавятся при более низкой температуре, чем Al2O3, и при спекании образуют жидкости. Эти жидкости способствуют формированию более плотной керамики, но ухудшают ee механиче- ские свойства при высоких температурах (проч- ность, сопротивление ползучести). Самой распро- страненной примесью является SiO2 (кремнезем). Стекла, образующиеся из оксидных примесей, как правило, сегрегируются на границах зерен спеченного материала. Такие примеси из исходно- го минерального сырья, как Na2O, однозначно яв- ляются нежелательными, так как ухудшают свой- ства алюмооксидной керамики (механические, теплопроводность и т. п.). Чистый Al2O3 спекается по твердофазному механизму. Только очень огра- ниченное количество коммерческих продуктов может быть классифицировано как полученные в условиях твердофазного спекания. Показано, что корундовая керамика чистотой 99,98 % и ниже содержит на границах зерен тонкие аморфные пленки состава SiO2‒CaO‒Al2O3. Стабильная кристаллическая фаза α-Al2O3 (ко- рунд) имеет гексагональную структуру. В ней анио- ны кислорода плотно упакованы в гексагональную подрешетку, а гораздо меньшие по размеру катионы алюминия занимают 2/3 октаэдрических центров внедрения в кислородную подрешетку. Природное сырье с содержанием Al2O3 примерно до 98 % или чи- стый корунд называют также глиноземом. При кри- сталлизации из водных растворов или дегидратации гидроксидов алюминия образуются метастабильные кристаллические фазы (кубический γ-Al2O3 и др.). Выше 1000 °C метастабильные фазы превращаются в α-Al2O3, при этом для завершения перекристаллиза- ции может требоваться температура до 1450 °C. Ме- тастабильные фазы Al2O3 имеют ограниченное при- менение преимущественно как сорбенты, носители катализаторов, сырье для получения α-Al2O3. Гидроксид алюминия имеет два стехиометри- ческих состава: Al(OH)3 = Al2O3·3H2O и AlO(OH) = = Al2O3·H2O (последний называют также оксиги- дроксидом). Каждое из этих соединений существу- ет в виде двух кристаллических форм ― α и γ. Кроме того, известен аморфный гидроксид алюминия пе- ременного состава Al2O3·nH2O. Химическая актив- ность убывает в ряду: аморфный гидроксид > байе- рит α-Al(OH)3 > гиббсит (гидраргиллит) γ-Al(OH)3 > > бёмит γ-AlO(OH) > диаспор α-AlO(OH). Корундовая керамика обладает высокой химической стойкостью как в окислительной, так и в восстановительной ат- мосфере, как в кислотной, так и в щелочной среде. Инертность Al2O3 связана, в частности, с тем, что он имеет теплоту образования выше, чем большинство других оксидов (ZrO2, Cr2O3, TiO2, SiO2, MgO и др.). И Al O б Гидроксид алюминия имеет два стехиометри- ческих состава: Al(OH)3 = Al2O3·3H2O и AlO(OH) = = Al2O3·H2O (последний называют также оксиги- дроксидом). ОКСИД АЛЮМИНИЯ И ЕГО ПРОМЫШЛЕННОЕ ПРОИЗВОДСТВО [1‒5] Тем не менее известны типы конденсаторов с диэлектрическим слоем Al2O3 (тонкопленочные, электролитиче- ские). Кроме того, Al2O3 представляет интерес в СВЧ-технике, поскольку сапфир обладает мини- мальными значениями тангенса угла диэлектри- ческих потерь tgδ среди известных веществ [11]. Это позволяет использовать непористый мелко- зернистый Al2O3 высокой чистоты для резонаторов высокой добротности в низкошумных осциллято- рах. Некоторые добавки (например, TiO2 в количе- стве 0,25 мас. %) улучшают диэлектрические свой- ства Al2O3 ― уменьшают диэлектрические потери. Те о ро о ос λ Al O р о а ой е сопротивлением ρ (см. табл. 1). Приводимые в разных источниках значения многих свойств мо- гут различаться. В первую очередь это касается таких структурно-чувствительных механических свойств, как прочность и трещиностойкость и осо- бенно Ebr. Величина Ebr зависит от рода электриче- ского напряжения (переменное или постоянное) и до определенного предела от толщины слоя диэ- лектрика [9]. Поэтому сопоставление данных по Ebr из разных источников часто затруднительно или некорректно. Диэлектрическая проницаемость εr Al2O3, со- ставляющая около 10, хотя и выше, чем у боль- шинства диэлектриков, но все же относительно невысока по сравнению с TiO2 (~102), титанатом бария (~103) и т. п. По значению εr < 15 Al2O3 по- падает в группу материалов с низкой диэлек- трической проницаемостью [10]2. Это несколько ограничивает применение Al2O3 в электрических Теплопроводность λ Al2O3 при комнатной тем- пературе заметно выше, чем у большинства неме- таллов. Среди оксидов Al2O3 уступает в теплопро- водности только MgO (~60 Вт/(м·К)) и BeO (230‒370 Вт/(м·К)) [12]. При повышенных требованиях к те- плоотводу для диэлектрических подложек вместо Al2O3 используют AlN (λ от 140‒200 Вт/(м·К) для поликристаллической керамики и до 320‒350 Вт/(м·К) для монокристаллов). ТКЛР Al2O3 выше, чем у керамических материалов (спеченные кар- биды, нитриды, муллит, кордиерит и др.). 2 Имеется альтернативная классификация, согласно ко- торой материалы с εr > 7 (больше, чем у нитрида крем- ния), в том числе Al2O3, попадают в группу высокой диэ- лектрической проницаемости. 17 ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 1 2019 ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Корундовая керамика пре- восходит циркониевую по электроизоляционным свойствам (ρ, Еbr) и λ, а также по Е и Н, но уступает ей по σf и К1с. Следует отметить, что обеспечение мел- козернистой прочной микроструктуры корундовой керамики представляет собой сложную технологи- ческую задачу ввиду естественной тенденции роста кристаллитов α-Al2O3 при высоких температурах. Для коммерчески доступной прочной конструкци- онной керамики (см. раздел «Производители алюмо- оксидной керамики») размер зерен (кристаллитов) составляет от одного до нескольких микрометров. У прозрачной керамики оптического назначения раз- мер кристаллов α-Al2O3 обычно составляет десятки микрометров, хотя основным условием прозрачно- сти корундовой керамики является низкая остаточ- ная пористость (<0,1 %). Так, возможно получение прозрачных образцов Al2O3 с субмикронной зерни- стостью [5]. О достаточной отработанности техноло- гий изготовления алюмооксидной керамики свиде- тельствует то, что в международных стандартах ISO 6474-1:2010 и ISO 6474-2:2012 на изделия медицинско- 2 д р щ р Возможность получения композитов Al2O3‒ ZrO2 обусловлена тем, что Al2O3 не образует с ZrO2 соединений. В отличие от этого в других имеющих практическое значение двойных системах Al2O3‒ оксид обычно существуют стабильные соедине- ния. Для систем Al2O3‒SiO2, Al2O3‒MgO, Al2O3‒TiO2 ― это соответственно муллит 3Al2O3·2SiO2 (метаси- ликат алюминия3, Tm = 1890 °C), алюмомагнезиаль- ная шпинель MgAl2O4 (Tm = 2135 °C), титанат алю- миния Al2TiO5 (Tm = 1860 °C). Все эти соединения имеют самостоятельное значение, например изго- тавливаются в виде керамики. При введении доба- вок оксидов в ходе спекания Al2O3 эти соединения могут образовываться в виде отдельных фаз. Кроме того, важное практическое значение имеют алю- минат кальция CaAl2O4 (Tm = 1870 °C, огнеупорный цемент) и алюминат иттрия Y3Al5O12 (алюмоиттрие- вый гранат, Tm = 1970 °C). Во всех рассмотренных бинарных системах существуют эвтектики. Среди них эвтектики с наименьшей в системе температу- рой плавления Tm: 1390 °C (C12A7‒CA), 1590 °C (SiO2‒ муллит), 1725 °C (TiO2‒Al2TiO5), 1870 °C (Al2O3‒ZrO2, 40 мас. % ZrO2), 1995 °C (MgO‒шпинель) [4, 16, 17]. Другой тип фазовой диаграммы характерен для 3 Известны также метастабильные ортосиликаты алюми- ния Al2SiO5 (силлиманит, андалузит, кианит). Таблица 2. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ В отличие от этого в других имеющих практическое значение двойных системах Al2O3‒ оксид обычно существуют стабильные соедине- ния. Для систем Al2O3‒SiO2, Al2O3‒MgO, Al2O3‒TiO2 ― это соответственно муллит 3Al2O3·2SiO2 (метаси- ликат алюминия3, Tm = 1890 °C), алюмомагнезиаль- ная шпинель MgAl2O4 (Tm = 2135 °C), титанат алю- миния Al2TiO5 (Tm = 1860 °C). Все эти соединения имеют самостоятельное значение, например изго- тавливаются в виде керамики. При введении доба- вок оксидов в ходе спекания Al2O3 эти соединения могут образовываться в виде отдельных фаз. Кроме того, важное практическое значение имеют алю- минат кальция CaAl2O4 (Tm = 1870 °C, огнеупорный цемент) и алюминат иттрия Y3Al5O12 (алюмоиттрие- вый гранат, Tm = 1970 °C). Во всех рассмотренных бинарных системах существуют эвтектики. Среди них эвтектики с наименьшей в системе температу- рой плавления Tm: 1390 °C (C12A7‒CA), 1590 °C (SiO2‒ муллит), 1725 °C (TiO2‒Al2TiO5), 1870 °C (Al2O3‒ZrO2, 40 мас. % ZrO2), 1995 °C (MgO‒шпинель) [4, 16, 17]. Другой тип фазовой диаграммы характерен для мики Al2O3‒ZrO2 может варьироваться в полном диапазоне от Al2O3 до ZrO2. Примером могут слу- жить композиты с наполнителем из частиц Al2O3 в количестве 0‒30 мол. % в матрице полностью стабилизированного ZrO2, содержащего 10 мол. % Y2O3 [14]. Керамика системы Al2O3‒ZrO2 исполь- зуется для производства режущего инструмента и абразивов. В последнее время японскими иссле- дователями получена керамика состава 75 мол. % ZrO2 (с 1,5 мол. % Y2O3) ‒ 25 мол. % Al2O3 с очень высокими показателями механических свойств (σf ≥ 1350 МПа и одновременно K1с ≥ 15,5 МПа·м0,5) [15]. Однако рассмотрение цирконийкорундовой керамики с доминирующей по содержанию фа- зой ZrO2 выходит за рамки настоящего обзора. системы Al2O3‒Cr2O3, в которой имеется непрерыв- ный ряд твердых растворов [18]. Алюмооксидная керамика выпускается в очень широком диапазоне чистоты (содержания базового Al2O3) в зависимости от специфики применения. Так, корундовая керамика высокой чистоты (99,5‒99,8 %) может эксплуатироваться при предельных темпе- ратурах Tmax от 1750 до 1900 °C [19, 20]. Содержащая 94‒97 % Al2O3 керамика легко металлизируется. В табл. 2 приведены свойства керамики из Al2O3 в за- висимости от чистоты и для сравнения керамики из частично стабилизированного оксида циркония (PSZ) по немецкому стандарту «Керамические и сте- клянные изолирующие материалы» («Ceramic and glass insulating materials») 1999 г. В корундовой кера- мике со снижением содержания примесей увеличи- ваются плотность и теплопроводность, улучшаются механические свойства (E, σ, К1с, Н). Непосредствен- но свойства керамики, в особенности механические, определяются микроструктурой материала, так что корреляция свойств с составом материала может но- сить сложный характер. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Вве- денные модификаторы обычно концентрируются на границах зерен спекаемого корунда, что ограничи- вает рост зерен изготавливаемой керамики и, таким образом, обеспечивает высокие механические пока- затели. Хотя, например, такая добавка, как TiO2, при- водит не к снижению, а к увеличению размера зерен корундовой керамики. дру д ( 2, 2 3, 2, 2, g др) Изделия из Al2O3 изготавливают обычными для технической керамики методами. Тонкоизмель- ченный порошок Al2O3 с размером частиц порядка 0,2‒5,0 мкм, обычно с органической связкой, прес- суют в форме; прессованную заготовку сушат и отжигают при 1500‒1800 °C. Временная органиче- ская связка может вводиться во всю массу порошка Al2O3 либо путем формирования и сушки гранул, которые затем отпрессовывают. Температура спе- кания повышается с увеличением чистоты Al2O3 в сырье. Для корундовой керамики высокой чистоты (> 99,7 %) температура спекания может достигать 1900‒2000 °C. Часто для удаления связки и следов воды и других летучих примесей применяют мед- ленный нагрев или выдержку при промежуточной температуре (кальцинирование). Вообще, методы изготовления керамики из Al2O3 могут быть самы- ми разными: экструзия, сухое прессование, литье под давлением, шликерное литье, изостатическое прессование (сухое или влажное), горячее изо- статическое прессование. Монокристаллический Al2O3 (сапфир) изготавливают методом выращи- вания кристаллов из расплава при более высокой температуре (выше точки плавления 2054 °C), ис- пользуя сырье более высокой чистоты, чем для по- лучения корундовой керамики, которая обладает поликристаллической структурой. ZrO2 в виде частично стабилизированного (обычно примесью порядка 3 мас. % Y2O3) тетра- гонального ZrO2 может вводиться в Al2O3 в зна- чительных количествах (10‒30 мас. %), так что продукт спекания представляет собой композит Al2O3‒ZrO2 и обычно обозначается ZTA. Добав- ки ZrO2 существенно увеличивают прочность и ударную вязкость. Вообще говоря, состав кера- ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 1 2019 18 ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ мики Al2O3‒ZrO2 может варьироваться в полном диапазоне от Al2O3 до ZrO2. Примером могут слу- жить композиты с наполнителем из частиц Al2O3 в количестве 0‒30 мол. % в матрице полностью стабилизированного ZrO2, содержащего 10 мол. % Y2O3 [14]. Керамика системы Al2O3‒ZrO2 исполь- зуется для производства режущего инструмента и абразивов. В последнее время японскими иссле- дователями получена керамика состава 75 мол. % ZrO2 (с 1,5 мол. % Y2O3) ‒ 25 мол. % Al2O3 с очень высокими показателями механических свойств (σf ≥ 1350 МПа и одновременно K1с ≥ 15,5 МПа·м0,5) [15]. Однако рассмотрение цирконийкорундовой керамики с доминирующей по содержанию фа- зой ZrO2 выходит за рамки настоящего обзора. Возможность получения композитов Al2O3‒ ZrO2 обусловлена тем, что Al2O3 не образует с ZrO2 соединений. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ На се- годня порядка 90 или 95 % мирового гидроксида алюминия извлекается по процессу Байера, пред- ложенному в 1887 г. Бокситная руда измельчается и обрабатывается щелочным раствором Na(OH), Ca(OH)2. На первой стадии гидроксиды алюминия превращаются в алюминат натрия NaAlO2, при этом попутно растворяется только кремнезем, а CaО, Fe2O3, TiО2 остаются в нерастворимом осадке. Кремнезем удаляется последующим медленным нагревом, когда выпадает в осадок Na2Si(OH)6. Оставшийся чистый раствор NaAl(OH)4 охлаждают, разбавляют водой и нейтрализуют углекислым га- зом. В результате из раствора избирательно осаж- дается Al(OH)3 без остатков растворенного крем- незема. Тригидрат алюминия Al(OH)3 (гиббсит) прокаливают при 250‒1300 °C, получая безводный Al2O3. Основная часть мирового производства ги- дроксида алюминия идет на выплавку металли- ческого алюминия (из металлургического Al2O3). Частично Al(OH)3 используется для наполнителей в композитах, огнезащитных составов, химикатов Таблица 3. Требования ИСО 6474-1 к керамике для хирургических имплантатов из корунда вы- сокой чистоты [21] Свойство1 Тип А Тип В Состав, мас. %: базовый Al2O3 добавка MgO примеси (SiO2 + CaO + Na2O) γ, г/см3 d, мкм СКО d, % σf, МПа Модуль Вейбулла для σf K1с3, МПа·м0,5 HV, ГПа (при нагрузке 1 кг) ≥99,7 ≤0,2 ≤0,1 ≥3,94 ≤2,5 ≤25 ⎛≥300⎞2 ⎝≥500⎠ ≥8 ≥2,5 ≥18 ≥99,5 ≤0,2 ≤0,3 ≥3,90 ≤3,5 ≤25 ⎛≥150⎞2 ⎝≥250⎠ ≥8 ‒ ≥17 1 d ― cредний размер зерен (кристаллов); СКО ― средне- квадратичное отклонение. 2 В числителе ― при испытании пластины двумя коакси- альными кольцами, в знаменателе ― при четырехточеч- ном изгибе. 3 При испытании методом SEVNB, или SEPB, или SCF, т. е. с предварительным нанесением на одну сторону образца надреза, трещины или углубления. Таблица 4. Требования ИСО 6474-2 к керамике для хирургических имплантатов из ZTA [22] Таблица 4. Требования ИСО 6474-2 к керамике для хирургических имплантатов из ZTA [22] Свойство Тип X Тип S Состав, мас. %: Al2O3 (ZrO2 + HfO2) HfO2 в составе ZrO2 заданные добавки примеси Относительная плотность, % ZrO2: d, мкм СКО d, % Al2O3: d, мкм СКО d, % σf, МПа Модуль Вейбулла для σf K1с2, МПа·м0,5 HV, ГПа (при нагрузке 1 кг) 60‒90 10‒30 ≤5 ≤10 ≤0,2 ≥99 ≤1,5 ≤25 ≤0,6 ≤40 ⎛ ≥600 ⎞1 ⎝≥1000⎠ ≥8 ≥4,0 ≥16,0 60‒90 10‒30 ≤5 ≤10 ≤0,2 ≥99 ≤1,5 ≤25 ≤0,6 ≤40 ⎛≥450⎞1 ⎝≥750⎠ ≥8 ≥3,5 ≥15,5 1 В числителе ― при испытании пластины двумя коакси- альными кольцами, в знаменателе ― при четырехточеч- ном изгибе. 2 При испытании методом SEVNB, или SEPB, или SCF, т. е. с предварительным нанесением на одну сторону образца надреза, трещины или углубления. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Свойства алюмооксидной керамики с разным содержанием Al2O3 и PSZ по DIN EN 60672 [1] (открытая пористость W всех материалов равна нулю) Свойство <90 % Al2O3 92‒96 % Al2O3 99 % Al2O3 >99 % Al2O3 PSZ γ, г/см3 Tmax1, °C σf, МПа E, ГПа K1c, МПа·м0,5 HV2, ГПа ТКЛР3, ppm/К λ4, Вт/(м·К) ρ, Ом·см Ebr, кВ/мм εr tgδ (при 1 МГц) >3,2 1400‒1500 >200 >200 3,5‒4,5 12‒15 6‒8 10‒16 1012‒1013 10 9 1·10‒3 3,4‒3,8 1400‒1500 230‒400 220‒340 4‒4,2 12‒15 6‒8 14‒25 1012‒1014 15‒25 9‒10 1·10‒3 3,5‒3,9 1400‒1500 280‒400 220‒350 4‒4,2 12‒20 6–8 16‒28 1012‒1015 15 9 1·10‒3 3,75‒3,98 1400‒1700 300‒580 300‒380 4‒5,5 17‒23 7‒8 19‒30 1012‒1015 17 9 1·10‒3 ≥5‒6 900‒1600 500‒1000 200‒210 5,8‒10,5 11‒12,5 10‒12,5 1,5‒3,0 108‒1013 ‒ ‒ ‒ 1 Типичные значения. 2 При нагрузке 100 г. 3 При 30‒600 °C. 4 При 30‒100 °C. Таблица 2. Свойства алюмооксидной керамики с разным содержанием Al2O3 и PSZ по DIN (открытая пористость W всех материалов равна нулю) ойства алюмооксидной керамики с разным содержанием Al2O3 и PSZ по DIN EN 60672 [1] ристость W всех материалов равна нулю) ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 1 2019 19 ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ стоимости. Действительно, в среднем стоимость обычной корундовой керамики в несколько раз ниже, чем другой керамики (AlN, Si3N4, SiC) и мате- риалов, полученных методом CVD ― химическим осаждением из газовой фазы (пиролитические BN, графит). Однако стоимость корундовой кера- мики высокой чистоты близка или даже выше, чем у монокристаллического Al2O3 ― сапфира (из расчета на единицу объема материала), притом что чистота сапфира и температура его получе- ния выше, чем у любой алюмооксидной керамики. го назначения нормируются показатели состава, ми- кроструктуры и механических свойств корундовой керамики и ZTA. В табл. 3, 4 приведены данные из этих стандартов. В литературе встречается утверждение о де- шевизне алюмооксидной керамики. Для коли- чественной оценки ее стоимости и сравнения со стоимостью других материалов автором исполь- зован ресурс [23], в котором были найдены цены на изделия одного формата и одинаковых либо сопоставимых размеров для подложек из плотной керамики и других технических материалов. Ре- зультаты такого сравнения приведены в табл. 5, в которой материалы ранжированы по уровню их Основным сырьевым источником Al2O3 явля- ется боксит, который состоит главным образом из гидратированных форм Al2O3 (гиббсит, бёмит, диа- спор). В среднем боксит содержит от 45 до 60 мас. % Al2O3 (в пересчете из гидроксидов), 10‒30 мас. % Fe2O3, остальное ― SiO2, CaO, TiO2 и H2O. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Характерными свойствами их являются высокая удельная поверхность, достигающая 400 м2/г, и внутренняя пористость. При этом переходные оксиды алюминия являются неселективными ад- сорбентами. Трансформация переходных оксидов в α-Al2O3 при термообработке является необратимой; ‒ переходные оксиды алюминия (transition aluminas), т. е. метастабильные формы Al2O3 (все, кро- ме α-Al2O3). Характерными свойствами их являются высокая удельная поверхность, достигающая 400 м2/г, и внутренняя пористость. При этом переходные оксиды алюминия являются неселективными ад- сорбентами. Трансформация переходных оксидов в α-Al2O3 при термообработке является необратимой; ‒ обожженный (кальцинированный, прока- ленный) оксид алюминия (calcined alumina, burned alumina) для производства керамики и огнеупоров, который является конечным продуктом термическо- го разложения гидроксидов алюминия. Процесс об- жига ведут в барабанных и туннельных печах, в псев- доожиженном слое, в результате образуется хрупкий поликристаллический α-Al2O3. Существует широкий набор технических сортов обожженного Al2O3 ― от чистого до сортов, содержащих натрий в большом ко- личестве (так называемый β-глинозем Na2O·11Al2O3). Морфология кристаллитов α-Al2O3 сильно зависит от природы минерализатора. Так, фтор дает плоские кристаллиты гексагональной формы, бор ― окру- глые кристаллиты, а хлорид бора ― круглые плотные кристаллы. В отличие от переходного Al2O3 частицы обожженного Al2O3 непористые, так что удельная поверхность материала совпадает с внешней по- верхностью частиц. Технический обожженный Al2O3 ‒ обожженный (кальцинированный, прока- ленный) оксид алюминия (calcined alumina, burned alumina) для производства керамики и огнеупоров, который является конечным продуктом термическо- го разложения гидроксидов алюминия. Процесс об- жига ведут в барабанных и туннельных печах, в псев- доожиженном слое, в результате образуется хрупкий поликристаллический α-Al2O3. Существует широкий набор технических сортов обожженного Al2O3 ― от чистого до сортов, содержащих натрий в большом ко- личестве (так называемый β-глинозем Na2O·11Al2O3). Морфология кристаллитов α-Al2O3 сильно зависит от природы минерализатора. Так, фтор дает плоские кристаллиты гексагональной формы, бор ― окру- глые кристаллиты, а хлорид бора ― круглые плотные кристаллы. В отличие от переходного Al2O3 частицы обожженного Al2O3 непористые, так что удельная поверхность материала совпадает с внешней по- верхностью частиц. Технический обожженный Al2O3 ‒ белый плавленый оксид алюминия (white fused alumina). Выше 2050 °C чистый Al2O3 плавит- ся, образуя непроводящую жидкость, которая при охлаждении затвердевает в массивный корунд. Пе- реплавленный α-Al2O3 называют белым плавленым Al2O3. Он обладает мелкозернистой микрострукту- рой, в которой кристаллы имеют отчетливо выражен- ‒ белый плавленый оксид алюминия (white fused alumina). Выше 2050 °C чистый Al2O3 плавит- ся, образуя непроводящую жидкость, которая при охлаждении затвердевает в массивный корунд. Пе- реплавленный α-Al2O3 называют белым плавленым Al2O3. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ 280 °С 800 °С 1000 °С γ-Al(OH)3 → χ-Al2O3 → κ-Al2O3 → α-Al2O3 280 °С 830 °С 1000 °С α-Al(OH)3 → η-Al2O3 → θ-Al2O3 → α-Al2O3 450 °С 800 °С 920 °С 1050 °С γ-AlO(OH) → γ-Al2O3 → δ-Al2O3 → θ-Al2O3 → α-Al2O3 500 °С α-AlO(OH) → α-Al2O3 Гиббсит.... Байерит... Бёмит….... Диаспор... 280 °С 800 °С 1000 °С γ-Al(OH)3 → χ-Al2O3 → κ-Al2O3 → α-Al2O3 280 °С 830 °С 1000 °С α-Al(OH)3 → η-Al2O3 → θ-Al2O3 → α-Al2O3 450 °С 800 °С 920 °С 1050 °С γ-AlO(OH) → γ-Al2O3 → δ-Al2O3 → θ-Al2O3 → α-Al2O3 500 °С α-AlO(OH) → α-Al2O3 Главной примесью в оксиде алюминия, полу- ченном по методу Байера, является натрий, кото- рый ограничивает ряд технических применений продукта. Для катализа кристаллизации α-Al2O3 используют галогенсодержащие соединения (BF3, BCl3), которые называют минерализаторами. Кро- ме того, минерализаторы образуют летучий NaCl, который, испаряясь, выводит натрий. Размер кри- сталлитов образующегося α-Al2O3 варьируется обычно от 0,5 до 10 мкм (больше при более высо- кой температуре отжига). Продукты промышлен- ного производства Al2O3 подразделяются на: р ( ); ‒ пластинчатый (табулярный) оксид алю- миния, называемый также спеченным (tabular alumina, sintered alumina), получают спеканием ото- жженного Al2O3 выше 1600 °C. В промышленном масштабе спекание проводят обычно в высоких шахтных печах с газовыми горелками в средней зоне. Первоначально заготавливают шары диаме- тром 20 мм путем комкования смеси измельченного обожженного Al2O3, активного микропорошка Al2O3 и подходящей органической связки для обеспече- ния наивысшей плотности сырца. Обычно добав- ляют также трихлорид бора для удаления натрия в виде NaCl при нагреве. Перед подачей в печь шары высушивают. Спекание ведут в непрерывном режи- ме при рабочей температуре 1900‒1950 °C (всегда ниже точки плавления α-Al2O3 2050 °C); процесс длится около 15 ч до выхода шаров из основания печи. Достигаются кажущаяся плотность спека 3,55 г/см3 и остаточная пористость до 5 об. %, усадка шаров 20 об. %. Спеченные шары дробят и измель- чают, обогащенный железом материал удаляют магнитной сепарацией, продукт классифициру- ют по размеру частиц. Пластинчатый Al2O3 может иметь чистоту примерно до 99,8 мас. % и низкое со- держание натрия (Na2O < 0,1 мас. %). Продукт явля- ется поликристаллическим материалом с крупны- ми пластинчатыми кристаллами гексагональной формы размерами 200‒300 мкм, обладает малой остаточной пористостью. Промышленный продукт может содержать добавки, снижающие температу- ру плавления Al2O3 до 1700‒1850 °C, а также измель- чаться до мелких фракций; ‒ переходные оксиды алюминия (transition aluminas), т. е. метастабильные формы Al2O3 (все, кро- ме α-Al2O3). ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Таблица 5. Стоимость подложек из различных не- металлических материалов (выборочные ориен- тировочные данные [23]) Материал Размеры подложки, мм Цена за 1 шт., USD Al2O3 Al2O3 (высокой чистоты 99,9 %) Al2O3 (сапфир) AlN SiO2 (кварцевое стекло) Si3N4 SiC BN (пиролитиче- ский) С (пирографит) С (высокоори- ентированный пирографит) 10×10×0,5 25,4×25,4×0,5 50,8×50,8×0,5 10×10×0,5 12,7×12,7×0,5 25,4×25,4×0,5 10×10×0,5 10×10×1 25,4×25,4×1 10×10×1 10×10×0,5 10×10×1 10‒13 20 300‒350 22‒35 35‒50* 25‒40 18 70 85 80 50 300‒600 * Варьируется в зависимости от кристаллографической ориентации пластины (в плоскости A, C, M или R). Таблица 5. Стоимость подложек из различных не- металлических материалов (выборочные ориен- тировочные данные [23]) ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 1 2019 20 ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ и т. п., остальная часть Al(OH)3 служит сырьем для получения неметаллургического Al2O3, т. е. алюмо- оксидной керамики, огнеупоров и т. п. Получение оксида из гидроксида сводится к термообработке. Характеристики прокаленного Al2O3 варьируются очень сильно в зависимости от условий отжига. При низких температурах отжига (300‒800 °C) об- разуются переходные формы Al2O3 (метастабиль- ные кристаллические модификации δ-, γ-, η-, κ-, χ- и θ-Al2O3). В результате высокотемпературного от- жига при 1000‒1300 °C получается корунд α-Al2O3. Химические и фазовые переходы от гидроксидов алюминия к корунду [2] приведены ниже: классифицируют по размеру частиц, морфологии кристаллитов (угловатые, округлые, плоские), по со- держанию натрия и в меньшей степени других при- месей. Сорта обожженного Al2O3 в зависимости от со- держания примесного натрия: стандартный с 0,3‒0,7 мас. % Na2O; промежуточный с 0,1‒0,3 мас. % Na2O; с низким содержанием натрия (0,03‒0,10 мас. % Na2O), высокой чистоты с экстранизким содержанием на- трия (<0,01 мас. % Na2O; такой Al2O3 получают из гидроксида алюминия по иным, чем способ Байера, технологиям). Основная область применения обо- жженного Al2O3 ― сырье для изготовления керами- ки, огнеупоров, стекла, эмалей, плитки, фарфора. Немолотый кальцинированный Al2O3 обычно имеет размер частиц 50‒150 мкм, более тонкодисперсные сорта получают путем дополнительного помола. Тонкомолотый обожженный Al2O3 для изготовления керамики и огнеупоров, который хорошо спекается (позволяет получать плотную керамику при относи- тельно невысокой температуре спекания ~1600 °С), называют реактивным (reactive alumina); Гиббсит.... Байерит... Бёмит….... Диаспор... 280 °С 800 °С 1000 °С γ-Al(OH)3 → χ-Al2O3 → κ-Al2O3 → α-Al2O3 280 °С 830 °С 1000 °С α-Al(OH)3 → η-Al2O3 → θ-Al2O3 → α-Al2O3 450 °С 800 °С 920 °С 1050 °С γ-AlO(OH) → γ-Al2O3 → δ-Al2O3 → θ-Al2O3 → α-Al2O3 500 °С α-AlO(OH) → α-Al2O3 Гиббсит.... Байерит... Бёмит….... Диаспор... ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Сообщается [2], что после закалки распла- ва прочность циркониевого электрокорунда впятеро превышает прочность коричневого. ные грани. В небольшом промышленном масштабе с получением одиночных кристаллов килограммового веса переплавка Al2O3 может осуществляться мето- дом Вернейля (плавление порошка оксида в пламени водородно-кислородной горелки с подачей расплава на расположенный ниже вращающийся пьедестал). Но в основном в крупнотоннажном производстве ис- пользуют наклонные электродуговые печи с тремя электродами переменного тока, когда нагрев осу- ществляется теплом электрического разряда. После расплавления и гомогенизации расплав Al2O3 раз- ливается по формам и медленно остывает до отделе- ния от стенок форм. β-Al2O3 (NaAl11O17) представляет собой основную примесь в белом плавленом оксиде алюминия вследствие концентрирования натрия в определенных областях. Однако при 2100 °C идет испарение натрия, и образующиеся поры являются полезными. В отечественной литературе произве- денный в электропечах синтетический корунд назы- вают электрокорундом; й ‒ коричневый электрокорунд (brown fused alumina, brown corundum). Помимо термической дегидратации гидроксидов алюминия существует другой промышленный способ получения α-Al2O3 ― непосредственно из бокситовой руды, когда боксит в смеси с восстановителем (антрацит, нефтяной кокс) и осадителем (железные опилки) плавят при 2100 °C в электродуговых печах. Процесс включает восста- новление оксидов железа, кремния и в меньшей сте- пени титана; при этом в качестве ценного побочно- го продукта образуется титаносодержащий сплав железа с кремнием ― ферросилиций. Основным продуктом является коричневый электрокорунд, представляющий собой корунд со значительной примесью титана. Различают такие сорта коричне- вого электрокорунда, как хрупкий (friable grade, 1,5 мас. % TiO2) и стандартный (standard grade, 3 мас. % TiO2). Трещиностойкость коричневого электро- корунда больше, чем белого, поскольку из-за при- сутствия титана уменьшается размер кристаллитов Al2O3; стандартный коричневый корунд превосходит по прочности хрупкий. В отечественной традиции коричневый электрокорунд называют нормальным. Оксид алюминия непосредственно в виде по- рошка используется как абразив. Согласно [27, 28] изготавливают абразивы (в том числе Al2O3) с раз- мерами частиц примерно от 5 мкм до 2 мм (в виде достаточно узких по зерновому составу фракций). Абразивный электрокорунд мало пригоден для спекания плотной керамики, поскольку обладает недостаточной тонкодисперсностью и низкой ак- тивностью. В описании технологий производства дисперсных корундовых материалов абразивного назначения, разработанных в России (СССР) [18], 4 Согласно [18] состав, плотность и твердость указанных корундовых абразивов могут несколько отличаться от данных табл. 6. Электрокорунд используется преимуществен- но для изготовления абразивного инструмента на Таблица 6. Отечественные марки электрокорунда абразивного применения [24, 25] Электрокорунд Обозначение Содержание Al2O3, мас. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ Он обладает мелкозернистой микрострукту- рой, в которой кристаллы имеют отчетливо выражен- ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 21 ¹ 1 2019 ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ связке, в сыпучем виде для шлифования и песко- струйной обработки и в меньшей степени для изго- товления огнеупоров. Соответственно, на последней стадии его производства осуществляются дробление слитков и классификация по размеру частиц. Для улучшения параметров электрокорунда (твердость, прочность, термостойкость) на стадии плавки в ма- териал могут вводиться легирующие добавки, в ка- честве которых могут быть использованы Ti, Cr, Zr, Mg, Si. В табл. 6 приведены марки электрокорунда по нормативно-технической документации 1970-х годов [18]4, которые тем не менее продолжают использо- ваться российскими поставщиками абразивов. Со- гласно [26] содержание примесей, мас. %: в нормаль- ном электрокорунде марок 13А и 14А TiO2 ≥1,8, Fe2O3 ≤0,3‒1,3, CaO ≤0,5–1,3 (диапазоны соответствуют разным зернистостям), в белом электрокорунде мар- ки 25А Fe2O3 ≤0,03‒0,05, SiO2 ≤0,1‒0,2, Na2O ≤0,2‒0,3. Для улучшения механической прочности продукта в расплав Al2O3 добавляют около 2 мас. % Cr2O3. Трехвалентный хром замещает Al3+ в решетке и уве- личивает трещиностойкость корунда. Аналогично процессу получения нормального электрокорунда, но с добавкой в шихту с бокситом цирконового песка выплавляют циркониевый электрокорунд, который может содержать гораздо большее количество при- месного оксида ― примерно до 30 мас. % ZrO2 (см. табл. 6). Сообщается [2], что после закалки распла- ва прочность циркониевого электрокорунда впятеро превышает прочность коричневого. связке, в сыпучем виде для шлифования и песко- струйной обработки и в меньшей степени для изго- товления огнеупоров. Соответственно, на последней стадии его производства осуществляются дробление слитков и классификация по размеру частиц. Для улучшения параметров электрокорунда (твердость, прочность, термостойкость) на стадии плавки в ма- териал могут вводиться легирующие добавки, в ка- честве которых могут быть использованы Ti, Cr, Zr, Mg, Si. В табл. 6 приведены марки электрокорунда по нормативно-технической документации 1970-х годов [18]4, которые тем не менее продолжают использо- ваться российскими поставщиками абразивов. Со- гласно [26] содержание примесей, мас. %: в нормаль- ном электрокорунде марок 13А и 14А TiO2 ≥1,8, Fe2O3 ≤0,3‒1,3, CaO ≤0,5–1,3 (диапазоны соответствуют разным зернистостям), в белом электрокорунде мар- ки 25А Fe2O3 ≤0,03‒0,05, SiO2 ≤0,1‒0,2, Na2O ≤0,2‒0,3. Для улучшения механической прочности продукта в расплав Al2O3 добавляют около 2 мас. % Cr2O3. Трехвалентный хром замещает Al3+ в решетке и уве- личивает трещиностойкость корунда. Аналогично процессу получения нормального электрокорунда, но с добавкой в шихту с бокситом цирконового песка выплавляют циркониевый электрокорунд, который может содержать гораздо большее количество при- месного оксида ― примерно до 30 мас. % ZrO2 (см. табл. 6). Библиографический список Harris Science Review of Doshisha University. ― 2017. ― Vol. 58, № 2. ― P. 51‒62. 1. Springer handbook of condensed matter and materials data ; ed. by W. Martienssen, H. Warlimont. ― Berlin : Springer, 2005. ― Ch. 3.2. ― P. 431‒476. 16. Ilatovskaia, M. Thermodynamic description of the Ti‒Al‒O system based on experimental data / M. Ilatovskaia, G. Savinykh, O. J. Fabrichnaya // Journal of Phase Equilibria and Diffusion. ― 2017. ― Vol. 38, № 3. ― P. 175‒184. 2. Cardarelli, F. Materials handbook: a concise desktop reference ; 2nd ed. / F. Cardarelli. ― London : Springer-Verlag, 2008. ― P. 600‒609. 3. Doremus, R. H. Alumina-silica system / R. H. Doremus // Handbook of ceramics and composites. Vol. 1 : Synthesis and properties ; ed. by N. P. Cheremisinoff. ― New York, Basel : Marcel Dekker, 1990. ― P. 23‒34. 17. Jerebtsov, D. A. Phase diagram of the system: Al2O3‒ZrO2 / D. A. Jerebtsov, G. G. Mikhailov, S. V. Sverdina // Ceram. Int. ― 2000. ― Vol. 26, № 8. ― P. 821‒823. 18. Гаршин, А. П. Абразивные материалы и инструменты: техно- логия производства / А. П. Гаршин, С. М. Федотова. ― СПб. : Изд-во политехн. ун-та, 2008. ― 1009 с. 4. Ceramic and glass materials: structure, properties and processing ; ed. by J. F. Shackelford, R. H. Doremus. ― New York : Springer, 2008. ― 201 p. 19. CoorsTek. Alumina overview // [Электронный ресурс]. Режим доступа : https://www.coorstek.com/english/solutions/materials/ technical-ceramics/alumina. 5. Galusek, D. Ceramic oxides / D. Galusek, K. Ghillányová // Ceramics science and technology. Vol. 2 : Materials and properties ; ed. by R. Riedel, I.-W. Chen. ― Darmstadt : Wiley-VCH, 2010. ― Ch. 1. ― P. 3‒58. 20. Accuratus. Aluminum oxide, Al2O3 ceramic properties // [Электрон- ный ресурс]. Режим доступа : http://accuratus.com/alumox.html. 6. AZoM // [Электронный ресурс]. Режим доступа : https://www. azom.com. 21. ГОСТ Р ИСО 6474-1‒2014. Имплантаты для хирургии. Ке- рамические материалы. Часть 1. Керамические материалы на основе оксида алюминия высокой чистоты. ― М. : Стандартин- форм, 2015. ― 11 с. 7. Reade. Mohs' hardness (typical) of abrasives // [Электронный ресурс]. Режим доступа : http://www.reade.com/reade-resources/ reference-educational/reade-reference-chart-particle-property- briefings/32-mohs-hardness-of-abrasives. briefings/32-mohs-hardness-of-abrasives. 22. ГОСТ Р ИСО 6474-2‒2014. Имплантаты для хирургии. Ке- рамические материалы. Часть 2. Композитные материалы на основе оксида алюминия высокой чистоты с усилением цирко- нием. ― М. : Стандартинформ, 2015. ― 12 с. 23. MTI // [Электронный ресурс]. Режим доступа : http://www. mtixtl.com. 22. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ (Продолжение следует) * * * * щелочной процесс. Чистый алюминий раство- ряют в водном растворе NaOH, из которого осаждают гиббсит Al(OH)3 путем нейтрализации или по способу Байера. Натрий удаляют гидротермальной обработ- кой. На последней стадии проводят отжиг гиббсита. (Продолжение следует) * * * Работа выполнена в рамках государственного задания Минобрнауки России № 10.8003.2017/8.9. ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ % γ, г/см3 H, ГПа Цвет Нормальный Белый Монокристалличе- ский (монокорунд) Титанистый Хромистый Циркониевый Хромотитанистый Магниево- кремниевый 12А, 13А, 14А, 15А, 16А 22А, 23А, 24А, 25А 43А, 44А, 45А 37А 32А, 33А, 34А 38А 91А, 92А, 93А, 94А, 95А 96А 91‒96 96‒99 98‒99 91‒98 91‒98 60‒75 60‒75 60‒75 3,8‒3,9 3,9‒3,95 3,94‒4,00 3,96‒4,00 3,95‒4,00 4,05‒4,15 ‒ ‒ 18,6‒19,6 19,6‒24,5 22,6‒23,5 19,6‒22,6 19,6‒22,6 22,6‒23,5 19,6‒22,6 ‒ Коричневый, от светлого до темного Белый » Серо-голубой От розового до рубинового Серо-розовый ‒ ‒ Микротвердость. Таблица 6. Отечественные марки электрокорунда абразивного применения [24, 25] ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 1 2019 ¹ 1 2019 22 ÍÀÓ×ÍÛÅ ÈÑÑËÅÄÎÂÀÍÈß È ÐÀÇÐÀÁÎÒÊÈ рассмотрены также монокорунд (крупнокристал- лический Al2O3, получаемый из оксисульфидного шлака), формкорунд (с зернами преимущественно цилиндрической и призматической формы) и сфе- рокорунд (состоящий из шарообразных частиц); * гель-процесс. Металлический алюминий вы- сокой чистоты растворяют в спиртовом (изопропа- нол) растворе KOH. Образовавшийся пропанолат алюминия очищают перегонкой и гидролизуют с образованием геля, который затем прокаливают; * хлоридный процесс. Чистый алюминий рас- творяют в концентрированной соляной кислоте и осаждают гексагидрат AlCl3·6H2O, отжиг которо- го при 1000 °C дает чистый Al2O3; ‒ оксид алюминия высокой чистоты со- держит не менее 99,99 мас. % Al2O3, имеет кри- сталлиты малого размера. Примерно половина его используется для производства сапфира и в меньшей степени для полировки (металлография, оптика). Производится следующими способами: * процесс с квасцами. Гиббсит (продукт байе- ровского процесса) растворяют в серной кислоте, затем раствор нейтрализуют водным аммиаком и охлаждают для осаждения алюмоаммонийных квасцов NH4Al(SO4)2·12H2O. Высушенные кристал- лы соли прокаливают выше 1000 °C, получая по- рошок чистого Al2O3; ‒ оксид алюминия высокой чистоты со- держит не менее 99,99 мас. % Al2O3, имеет кри- сталлиты малого размера. Примерно половина его используется для производства сапфира и в меньшей степени для полировки (металлография, оптика). Производится следующими способами: * процесс с квасцами. Гиббсит (продукт байе- ровского процесса) растворяют в серной кислоте, затем раствор нейтрализуют водным аммиаком и охлаждают для осаждения алюмоаммонийных квасцов NH4Al(SO4)2·12H2O. Высушенные кристал- лы соли прокаливают выше 1000 °C, получая по- рошок чистого Al2O3; * щелочной процесс. Чистый алюминий раство- ряют в водном растворе NaOH, из которого осаждают гиббсит Al(OH)3 путем нейтрализации или по способу Байера. Натрий удаляют гидротермальной обработ- кой. На последней стадии проводят отжиг гиббсита. (Продолжение следует) * * * * щелочной процесс. Чистый алюминий раство- ряют в водном растворе NaOH, из которого осаждают гиббсит Al(OH)3 путем нейтрализации или по способу Байера. Натрий удаляют гидротермальной обработ- кой. На последней стадии проводят отжиг гиббсита. Библиографический список ГОСТ Р ИСО 6474-2‒2014. Имплантаты для хирургии. Ке- рамические материалы. Часть 2. Композитные материалы на основе оксида алюминия высокой чистоты с усилением цирко- нием. ― М. : Стандартинформ, 2015. ― 12 с. 8. CRC handbook of chemistry and physics ; ed. by W. M. Haynes ; 97th ed. ― Boca Raton : CRC Press, 2017. ― P. 12‒48, 15‒43. 9. Neusel, C. Thickness-dependence of the breakdown strength: analysis of the dielectric and mechanical failure / C. Neusel, H. Jelitto, D. Schmidt [et al.] // J. Eur. Ceram. Soc. ― 2015. ― Vol. 35, № 1. ― P. 113‒123. 23. MTI // [Электронный ресурс]. Режим доступа : http://www. mtixtl.com. 10. Nanni, P. Synthesis of dielectric ceramic materials / P. Nanni, M. Viviani, V. Buscaglia // Handbook of low and high dielectric constant materials and their applications ; ed. by H. S. Nalwa. ― San Diego : Academic Press, 1999. ― Vol. 1, Ch. 9. ― P. 431. 24. Техстрой. Абразивные и пескоструйные материалы на осно- ве оксида алюминия – электрокорунд и его разновидности // [Электронный ресурс]. Режим доступа : http://www.teh-stroy. ru/st_elektrokorunda-vidy-modifikatsii-oksida-alyuminiya-al2o3- abrazivnogo-korunda.php. 11. Penn, S. Ceramic dielectrics for microwave applications / S. Penn, N. Alford // Handbook of low and high dielectric constant materials and their applications ; ed. by H. S. Nalwa. ― San Diego : Academic Press, 1999. ― Vol. 2. ― Ch. 10. ― P. 496. 25. Стратиевский, И. Х. Абразивная обработка : справочник / И. Х. Стратиевский, В. Г. Юрьев, Ю. М. Зубарев. ― М. : Машино- строение, 2010. ― С. 7. 26. ГОСТ 28818‒90. Материалы шлифовальные из электрокорун- да. Технические условия. ― М. : Изд-во стандартов, 1991. ― 7 с. 12. High thermal conductivity materials ; ed. by S. L. Shinde, J. S. Goela. ― New York : Springer, 2006. ― 271 p. 27. ГОСТ 3647‒80. Материалы шлифовальные. Зернистость и зерновой состав. Методы контроля. ― М. : ИПК Изд-во стандар- тов, 2004. ― 18 с. 13. Лукин, Е. С. Новые керамические материалы на основе ок- сида алюминия / Е. С. Лукин, Н. А. Макаров, И. В. Додонова [и др.] // Огнеупоры и техническая керамика. ― 2001. ― № 7. ― С. 2‒10. 28. ГОСТ Р 52381‒2005. Материалы абразивные. Зернистость и зерновой состав шлифовальных порошков. Контроль зерново- го состава. ― М. : Стандартинформ, 2005. ― 11 с. ■ Получено 22 0618 28. ГОСТ Р 52381‒2005. Материалы абразивные. Зернистость и зерновой состав шлифовальных порошков. Библиографический список Контроль зерново- М С ф 2005 11 ■ 28. ГОСТ Р 52381‒2005. Материалы абразивные. Зернистость и зерновой состав шлифовальных порошков. Контроль зерново- го состава. ― М. : Стандартинформ, 2005. ― 11 с. ■ Получено 22.06.18 © А. М. Абызов, 2019 г. 14. Handbook of ceramic composites ; ed. by N. P. Bansal. ― Boston, Dordrecht, London : Kluwer Academic Publishers, 2005. ― 558 p. 5. ― 11 с. ■ Получено 22.06.18 © А. М. Абызов, 2019 г. 15. Hirota, K. Fabrication of dense ZrO2‒Al2O3 composite ceramics by pulsed electric-current pressure sintering of neutralization co- precipitated powders / K. Hirota, K. Yamamoto, K. Sasai [et al.] // The 23 ¹ 1 2019 ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 1 2019
https://openalex.org/W3101237195	https://link.springer.com/content/pdf/10.1007%2FJHEP02%282016%29176.pdf	English	null	A case study of the sensitivity to LFV operators with precision measurements and the LHC	The Journal of high energy physics/The journal of high energy physics	2,016	cc-by	12,508	Received: October 19, 2015 Revised: February 3, 2016 Accepted: February 13, 2016 Published: February 26, 2016 Received: October 19, 2015 Revised: February 3, 2016 Accepted: February 13, 2016 Published: February 26, 2016 JHEP02(2016)176 Open Access, c⃝The Authors. Article funded by SCOAP3. A case study of the sensitivity to LFV operators with precision measurements and the LHC Yi Caia and Michael A. Schmidtb aARC Centre of Excellence for Particle Physics at the Terascale, School of Physics, University of Melbourne, Tin Alley, Melbourne, Victoria, 3010 Australia bARC Centre of Excellence for Particle Physics at the Terascale, School of Physics, University of Sydney, Physics Road, Sydney, NSW 2006 Australia E-mail: yi.cai@unimelb.edu.au, michael.schmidt@sydney.edu.au Yi Caia and Michael A. Schmidtb aARC Centre of Excellence for Particle Physics at the Terascale, School of Physics, University of Melbourne, Tin Alley, Melbourne, Victoria, 3010 Australia bARC Centre of Excellence for Particle Physics at the Terascale, School of Physics, University of Sydney, Physics Road, Sydney, NSW 2006 Australia E-mail: yi.cai@unimelb.edu.au, michael.schmidt@sydney.edu.au Abstract: We compare the sensitivity of precision measurements of lepton ﬂavour ob- servables to the reach of the LHC in a case study of lepton-ﬂavour violating operators of dimension six with two leptons and two quarks. For light quarks precision measurements always yield the more stringent constraints. The LHC complements precision measure- ments for operators with heavier quarks. Competitive limits can already be set on the cutoﬀscale Λ > 600–800 GeV for operators with right-handed τ leptons using the LHC run 1 data. Keywords: Beyond Standard Model, Rare Decays, LEP HERA and SLC Physics ArXiv ePrint: 1510.02486 ArXiv ePrint: 1510.02486 Open Access, c⃝The Authors. Article funded by SCOAP3. doi:10.1007/JHEP02(2016)176 doi:10.1007/JHEP02(2016)176 JHEP02(2016)176 Contents 1 Introduction 1 2 Eﬀective operators 3 3 Renormalization group running 5 4 Existing ﬂavour physics constraints 5 4.1 µ-e conversion 6 4.2 Semi-leptonic τ-decays 8 4.3 Leptonic neutral meson decays 9 4.4 Leptonic charged meson decays 9 5 LHC search 12 5.1 Signal and background 13 5.2 Event selection 14 5.3 Limit setting and results 15 6 Discussion 16 7 Conclusions 18 A Mesons 18 Contents 1 Introduction 1 2 Eﬀective operators 3 3 Renormalization group running 5 4 Existing ﬂavour physics constraints 5 4.1 µ-e conversion 6 4.2 Semi-leptonic τ-decays 8 4.3 Leptonic neutral meson decays 9 4.4 Leptonic charged meson decays 9 5 LHC search 12 5.1 Signal and background 13 5.2 Event selection 14 5.3 Limit setting and results 15 6 Discussion 16 7 Conclusions 18 A Mesons 18 JHEP02(2016)176 6 Discussion 7 Conclusions A Mesons 1 Introduction The discovery of the 125 GeV Higgs boson [1, 2] in 2012 at the Large Hadron Collider (LHC) has completed the description of the highly successful Standard Model (SM) of particle physics. However, a number of experimental observations and theoretical arguments, such as the origin of neutrino masses, the existence of dark matter, the hierarchy problem and the strong CP problem, can not be accommodated within the SM. Many theoretical proposals addressing these issues generally lead to lepton ﬂavour violating (LFV) processes which are theoretically forbidden in the SM by accidental symmetries. The prime examples are models of neutrino mass. The observation of neutrino oscillations [3] undeniably showed that individual lepton number is not conserved. Thus LFV processes, such as µ →eγ, may exist. In the minimal type-I seesaw model [4], these processes are suppressed by unitarity and far out of current and future experimental reach. However, in the other two seesaw – 1 – models [5, 6] and also in radiative neutrino mass models [7], LFV processes enjoy more freedom and their rates can be large enough to be tested. Other examples include but are not limited to (R-parity violating) supersymmetric models [8] and Z′ models [9]. The observation of these LFV processes will deﬁnitely shed light on the deeper under- lying physics, while the non-observation surely places stringent constraints on the model parameters of the proposed theories. The classical experiments try to search for very rare processes such as µ−→e−γ, µ−→e−e+e−, µ-e conversion in nuclei and rare τ and LFV meson decays at MEG [10], Mu3e [11], Mu2E [12, 13], COMET [14, 15], SINDRUM [16], B-factories [17, 18], et al. We will refer to these experiments as precision measurements due to the ultra-high experimental sensitivities. Meanwhile, LFV processes can also occur at collider experiments with a relatively low SM background. For example, in supersymmetric models squarks and gluinos can be produced at the Tevatron or the LHC with subsequent LFV decays in a cascade decay chain via sleptons. Of this type of collider tests, we will focus on the LHC since the results are generally the best ones. JHEP02(2016)176 So far such LFV processes have not been observed from precision measurements. At the LHC, the CMS experiment recently reported a 2.4 σ anomaly in the h →µτ decay [19], while the analysis of ATLAS [20] is consistent with the SM and the CMS result. 2 Eﬀective operators JHEP02(2016)176 Following the general classiﬁcation of dimension six operators [21, 22], there are 10 dimen- sion six operators with two quark and two lepton ﬁelds neglecting the ﬂavour structure Q(1) lq = (¯LγµL)( ¯QγµQ), Q(3) lq = (¯Lγµτ IL)( ¯Qγµτ IQ), (2.1) Qeu = (¯ℓγµℓ)(¯uγµu), Qed = (¯ℓγµℓ)( ¯dγµd), (2.2) Qlu = (¯LγµL)(¯uγµu), Qld = (¯LγµL)( ¯dγµd), Qqe = ( ¯QγµQ)(¯ℓγµℓ), (2.3) Qledq = (¯Lαℓ)( ¯dQα), Q(1) lequ = (¯Lαℓ)ϵαβ( ¯Qβu), (2.4) Q(3) lequ = (¯Lασµνℓ)ϵαβ( ¯Qβσµνu) , (2.5) Q(3) lq = (¯Lγµτ IL)( ¯Qγµτ IQ), (2.1) Qed = (¯ℓγµℓ)( ¯dγµd), (2.2) Qld = (¯LγµL)( ¯dγµd), Qqe = ( ¯QγµQ)(¯ℓγµℓ), (2.3) Q(1) lequ = (¯Lαℓ)ϵαβ( ¯Qβu), (2.4) , (2.5) (2.5) where α, and β are SU(2)L indices. where α, and β are SU(2)L indices. In general, the quark bilinears can be any combination of ﬂavours and the leptonic bilinear has to be ﬂavour oﬀ-diagonal to explain LFV. Various combinations of quark ﬂavours will involve diﬀerent mesons in the analysis. To cover the whole spectra of mesons is deﬁnitely a mission that can not be contained in this single work. Thus we will only start with quark bilinears of same ﬂavours, where we expect the weakest constraints from precision experiments. Among those, the operator with the top quark pair bilinear can only contribute at one-loop at the LHC as shown in ﬁgure 1, which leads to an eﬀective dimension 7 operator at low energies with two gluons ﬁeld strength tensors coupled to a lepton bilinear. This operator has completely diﬀerent ﬂavour constraints from other operators with lighter quark bilinears. Thus we will restrict ourselves to a study of eﬀective operators with the ﬁrst ﬁve ﬂavour quarks and leave the operator with top quarks for future study. Both t-channel scalar exchange and s-channel vector boson exchange generate oper- ators with vector bilinears, which have been studied intensively in refs. [23, 24] in terms of eﬀective four fermion interactions. Thus, we will take the operators generated via an s-channel scalar exchange Qledq and Q(1) lequ in eq. (2.4) with Wilson coeﬃcients Ξd and Ξu, respectively, as a fresh example to demonstrate our study of the sensitivity with precision measurements and the LHC −L = Ξd ij,kl (Qledq)ij,kl + Ξu ij,kl Q(1) lequ ij,kl + h.c. . (2.6) (2.6) They, for instance, are generated in two Higgs doublet models with tree-level ﬂavour viola- tion [25–28]. 1 Introduction All these experimental results suggested that the energy scale Λ where new physics emerges are rather high and much larger than the electroweak scale. Therefore we can adopt a simple formalism to interpret the experimental results, namely the eﬀective operators. In light of the LHC particularly interesting operators are the ones with two quarks and two leptons, because they allow for relatively large cross sections and clean signatures with low SM background. There are ten diﬀerent gauge invariant operators with two quarks and two leptons, denoted by representations of SU(2)L, following the discussion in refs. [21, 22]. After electroweak symmetry breaking, the gauge-invariant operators induce diﬀerent contributions to the four-fermion interactions of neutrinos, charged leptons and quarks, which directly enter the relevant physical processes. Constraints obtained for the individual four-fermion interactions can be translated to constraints on the gauge-invariant eﬀective operators by using the most stringent constraint of the generated four-fermion interactions of quarks and leptons. We consider the SU(2)L invariant operators, obtain the corresponding eﬀective four fermion interactions and determine the most stringent constraints both from precision experiments and the LHC. Previous studies [23, 24] of eﬀective operators with two quarks and two leptons focused on constraints from precision experiments and did not aim to explore the potential of the LHC. The paper is organised as follows: in section 2 we discuss the LFV eﬀective operators and choose one type for our case study. Although we restricted ourselves to one operator, operator mixing will induce other operators. We discuss QCD renormalization group (RG) corrections in section 3. Then we study the constraints on the chosen operator from precision measurements in section 4. In section 5, we recast the relevant study on the LFV processes from the LHC and draw the current limits and also the future projection at Run 2. We summarise and discuss our results in section 6. Section 7 is devoted to the conclusion. Technical details are collected in the appendix. – 2 – g g ℓi ℓj t t t g g ℓi ℓj t t t g g ℓi ℓj t t t Figure 1. Feynman diagram for the operator with top-quark bilinear to generate LFV ﬁnal states at the LHC. 2 Eﬀective operators We will, however, be agnostic about the underlying UV completion and will study the eﬀective operators without any theoretical prejudice. – 3 – Speciﬁcally, we exemplify the possibility to test eﬀective operators Qledq, Q(1) lequ with two leptons ℓi,j and two same-ﬂavour quarks qk besides the top quark, t, at the LHC and in precision experiments. It is straightforward to extend the study to operators with a diﬀerent Lorentz structure. Writing the SU(2)L structure explicitly, the operators read Writing the SU(2)L structure explicitly, the operators read (Qledq)ij,kl = (¯Lα i ℓj)( ¯dkQα l ) = (¯νLiℓRj)( ¯dRkuLl) + (¯ℓLiℓRj)( ¯dRkdLl), (2.7) Q(1) lequ ij,kl = (¯Lα i ℓj)ϵαβ( ¯Qβ kul) = (¯νLiℓRj)( ¯dLkuRl) −(¯ℓLiℓRj)(¯uLkuRl) (2.8) (2.7) (2.8) and thus lead to two eﬀective four fermion interactions. We deﬁne the Wilson coeﬃcients of the eﬀective four fermion interactions ΞNu, ΞNd, ΞCu, ΞCd as follows and thus lead to two eﬀective four fermion interactions. We deﬁne the Wilson coeﬃcients of the eﬀective four fermion interactions ΞNu, ΞNd, ΞCu, ΞCd as follows JHEP02(2016)176 L4f = ΞCd ij,kl(¯νLiℓRj)( ¯dRkuLl) + ΞNd ij,kl(¯ℓLiℓRj)( ¯dRkdLl) (2.9) + ΞCu ij,kl(¯νLiℓRj)( ¯dLkuRl) + ΞNu ij,kl(¯ℓLiℓRj)(¯uLkuRl) . (2.9) They are related to the Wilson coeﬃcients in the unbroken theory via ΞNd ij,kl = U ℓ∗ ii′ V d ll′ Ξd i′j,kl′, ΞCd ij,kl = U ν∗ ii′ V u ll′ Ξd i′j,kl′, (2.10) ΞNu ij,kl = −U ℓ∗ ii′ V u∗ kk′ Ξu i′j,k′l, ΞCu ij,kl = U ν∗ ii′ V d∗ kk′ Ξu i′j,k′l, (2.11) (2.10) (2.11) (2.10) (2 11) (2.11) ΞNu ij,kl = −U ℓ∗ ii′ V u∗ kk′ Ξu i′j,k′l, where the unitary matrices U ℓ,ν and V u,d relate the quark and lepton states in the ba- sis where the dimension six operator is deﬁned to their mass eigenstates denoted by subscript m, i.e. where the unitary matrices U ℓ,ν and V u,d relate the quark and lepton states in the ba- sis where the dimension six operator is deﬁned to their mass eigenstates denoted by subscript m, i.e. ν = U ννm, ℓ= U ℓℓm, u = V dum, d = V ddm . (2.12) (2.12) ν = U ννm, In the following discussion we choose the charged leptons to be diagonal, i.e. U ℓ= 1 and thus U ν becomes the PMNS matrix U. Furthermore, we choose the Wilson coeﬃcients ΞNu,Nd to be diagonal in the quark sector. 3 Renormalization group running JHEP02(2016)176 Particularly QCD corrections to the operators are important due to the size of the strong interactions. We follow the discussion in ref. [31] to include QCD corrections at next-to leading order to the operators. We take into account the mass thresholds of the quarks and match the eﬀective theories with nF quark ﬂavours at the pole mass of each quark. As there is no operator mixing for the two quark-two lepton operators from QCD running, the next-to leading order QCD correction simpliﬁes tremendously and the Wilson coeﬃcients at a scale µ are related to the ones at a scale µ0 via Ξ(µ) = Ξ(µ0) αs(µ) αs(µ0) γ0 2β0 . (3.1) (3.1) The relevant coeﬃcients β0 and γ0 can be directly read from the beta functions of Ξ dΞ d ln µ = −γ0 αs 4πΞ (3.2) (3.2) and the one-loop beta function of the strong coupling dαs d ln µ = −2β0 α2 s 4π . (3.3) (3.3) A straightforward calculation shows β0 = 11 −2nF /3 and γ0 = 6C2(3) , (3.4) (3.4) where C2(3) = 4/3 is the quadratic Casimir invariant of the fundamental representation. The relevant diagrams of QCD corrections to the quark propagator and the eﬀective vertex are shown in ﬁgure 2. We use the Mathematica code RunDec [32] to obtain the strong coupling at the diﬀerent mass scales, which we use to evaluate the running of the Wilson coeﬃcients. Given the large uncertainty of the LHC analysis, we only use the two loop QCD RG equations and match the eﬀective theories at one-loop. 2 Eﬀective operators This choice implies that there are no ﬂavour changing neutral currents at tree level. In case of operator Qledq, this implies V u = 1 and V d becomes the CKM matrix V . Similarly for operator Q(1) lequ we ﬁnd V d = 1 and V u = V †. We use the current best-ﬁt values from the UTﬁt collaboration [29] for the CKM matrix and the ones of the nu-ﬁt collaboration [30] for the PMNS matrix assuming that all leptonic CP phases vanish. Generally, however, those operators are accompanied by operators with neutral current quark-ﬂavour-violating (QFV) operators. We will also quote limits from these induced operators. In particular, we parameterize the Wilson coeﬃcients of the accompanying QFV operators by (no summation on the right-hand side) Ξu ij,kl = λ Ξu ij,llVkl , Ξd ij,kl = λ Ξd ij,kkVkl (2.13) Ξd ij,kl = λ Ξd ij,kkVkl (2.13) (2.13) for up-type and down-type quarks, where λ indicates the mixing induced from matching to the full theory, which is normalised to the corresponding CKM mixing matrix element. All Wilson coeﬃcients are ﬁxed at the scale µ = 1 TeV. Thus in order to make connection with results from low-energy precision experiments, we have to include RG corrections. – 4 – q q q q ℓi ℓj u d ν ℓ Figure 2. Relevant diagrams for QCD corrections, where q collectively stands for up- and down- quarks. Figure 2. Relevant diagrams for QCD corrections, where q collectively stands for up- and down- quarks. 4 Existing ﬂavour physics constraints There are already several constraints on the operators in eq. (2.6) with same-ﬂavour quarks from existing ﬂavour experiments. The main constraints are from µ-e conversion, LFV neutral meson decays, leptonic charged pseudoscalar decays and semi-leptonic τ-decays. – 5 – We do not take into account the recent hints for new physics in diﬀerent B-decays measured at LHCb [33–35] or the recent hint for lepton ﬂavour non-universality in B → D∗τν measured by BaBar [36], Belle [37], and LHCb [38]. An explanation of these hints for new physics requires operators with quark ﬂavour violation. See ref. [39] for a recent study using eﬀective operators. We deﬁne the Wilson coeﬃcients Ξu,d ij,kk with an arbitrary phase at the scale µ = 1 TeV and evolve them down to the scale of the relevant process, like the mass of the τ lepton or the heavy quark of the decaying meson. For µ-e conversion and decays of light mesons with masses below 1 GeV, we evaluate the operator at µ = 1 GeV, where the operators are matched to chiral perturbation theory, but we neglect any additional quantum corrections in chiral perturbation theory for simplicity. The masses, decay constants, and mixing angles of the considered mesons are summarised in appendix A. Unless stated otherwise we use the experimental values reported in ref. [40]. We vary the phase of the Wilson coeﬃcient in steps of 1◦and report the range of obtained limits. We quote all limits in terms of the cutoﬀscale Λ of the eﬀective operators, i.e. JHEP02(2016)176 Λ ≡Ξ−1/2 . (4.1) (4.1) 4.1 µ-e conversion The conversion of µ-e in nuclei is probed for several diﬀerent nuclei, like gold (Au), titanium (Ti), and lead (Pb). So far no observation of the process has been made. This places a stringent limit on the dimensionless µ-e conversion rate deﬁned as, The conversion of µ-e in nuclei is probed for several diﬀerent nuclei, like gold (Au), titanium (Ti), and lead (Pb). So far no observation of the process has been made. This places a stringent limit on the dimensionless µ-e conversion rate deﬁned as, R(A,Z) µe ≡ Γ (µ−+ (A, Z) →e−+ (A, Z)) Γ (µ−+ (A, Z) →νµ + (A, Z −1)) , (4.2) (4.2) where A and Z are the mass number and the atomic number of the nuclei. The denominator of eq. (4.2) denotes the well-measured muon capture rate and the numerator is the muon conversion rate calculated with Γ µ−+ (A, Z) →e−+ (A, Z) = ΞNu,Nd ij,kk 2 × F × peEe (Mp + Mn)2 2π , (4.3) (4.3) where pe and Ee is the momentum and energy of the ﬁnal electron, and Mp,n are the nuclear matrix elements. We follow ref. [41] in the analysis and list both the muon total capture rates and the nuclear matrix elements in table 1. The factor F parameterizes the interaction between the charged lepton current and the nuclei, F = α(0) SS + α(3) SS Mp −Mn Mp + Mn 2 + α(0) PS + α(3) PS Mp −Mn Mp + Mn 2 , (4.4) (4.4) where parameters with superscripts 0 and 3 are related to isospin singlet and triplet re- spectively. It can be described with two methods, i.e. direct nuclear mediation and meson exchange mediation. Currently the relative strength of the two mechanisms is not known and for simplicity we separately consider them to obtain a limit and we expect that the actual limit will lie in between. – 6 – 48Ti 197Au 208Pb pe/fm−1 0.529 0.485 0.482 Mp/fm−3/2 0.104 0.395 0.414 Mn/fm−3/2 0.127 0.516 0.566 Γ(µ−N →νµN)/106s−1 2.60 13.07 13.45 Rmax µe 4.3 × 10−11 [16] 7.0 × 10−13 [44] 4.6 × 10−11 [45] ¯uu 1100 [870] 2100 [1700] 760 [610] ¯dd 1100 [930] 2200 [1900] 780 [680] ¯ss 480 [–] 950 [–] 340 [–] ¯cc 150 [–] 290 [–] 110 [–] ¯bb 84 [–] 170 [–] 61 [–] Table 1. 4.1 µ-e conversion Parameters for calculation of µ-e conversion rate and the constraints on the cutoﬀscale Λ [TeV] from µ-e conversion in nuclei using direct nuclear mediation [meson exchange mediation]. We obtain the same constraints for right-handed and left-handed operators. Similarly the constraints are symmetric in the leptons and it does not depend on the lepton bilinear ¯µPLe vs. ¯ePLµ. 48Ti 197Au 208Pb pe/fm−1 0.529 0.485 0.482 Mp/fm−3/2 0.104 0.395 0.414 Mn/fm−3/2 0.127 0.516 0.566 Γ(µ−N →νµN)/106s−1 2.60 13.07 13.45 Rmax µe 4.3 × 10−11 [16] 7.0 × 10−13 [44] 4.6 × 10−11 [45] ¯uu 1100 [870] 2100 [1700] 760 [610] ¯dd 1100 [930] 2200 [1900] 780 [680] ¯ss 480 [–] 950 [–] 340 [–] ¯cc 150 [–] 290 [–] 110 [–] ¯bb 84 [–] 170 [–] 61 [–] JHEP02(2016)176 Table 1. Parameters for calculation of µ-e conversion rate and the constraints on the cutoﬀscale Λ [TeV] from µ-e conversion in nuclei using direct nuclear mediation [meson exchange mediation]. We obtain the same constraints for right-handed and left-handed operators. Similarly the constraints are symmetric in the leptons and it does not depend on the lepton bilinear ¯µPLe vs. ¯ePLµ. Table 1. Parameters for calculation of µ-e conversion rate and the constraints on the cutoﬀscale Λ [TeV] from µ-e conversion in nuclei using direct nuclear mediation [meson exchange mediation]. We obtain the same constraints for right-handed and left-handed operators. Similarly the constraints are symmetric in the leptons and it does not depend on the lepton bilinear ¯µPLe vs. ¯ePLµ. The direct nuclear mediation describes the interaction at the quark level, while the meson exchange mediation uses meson ﬁelds formed from the quark bilinear to mediate the interaction between the charged lepton and the nuclei. With direct nuclear mediation, the parameters α(0,3) SS,PS are given by α(0,3) rS = 1 4 × ηq rS ×        G(0) S , G(3) S q = u G(0) S , −G(3) S q = d Gq S, 0 q = c, s, b , (4.5) (4.5) where r = S, P, G(0,3) S = (Gu S ± Gd S)/2, and the factor of 1/4 in α(0,3) rS comes from the two projection operators in the quark bilinear and the lepton bilinear. ηq rS takes −1 for r = P with Γl = PL and takes 1 in all other cases. where the parameters are estimated to be βf0 = 1.58 and βa0 = 2.24 as in [41]. 4.1 µ-e conversion (2.13) and ϕm is the mixing angle between f0(500) and f0(980) and is deﬁned in eq. (A.9). The current best limits on the conversion in these nuclei are Rµν ≤4.3 × 10−11, 4.6 × 10−11, 7.0 × 10−13 in 48Ti [16], 208Pb [44], and 197Au [45]. Following ref. [41] we cal- culate the constraint for the diﬀerent quark ﬂavours and summarise the results in table 5. Given the experimental constraints, µ-e conversion in gold leads to the most stringent con- straint on the cutoﬀscale Λ. Direct nuclear mediation generally gives stronger constraints, particularly for the heavier quarks. If it would be entirely described by meson exchange mediation, the eﬀective operators with heavier quarks are not constrained, because the form factors of all considered mesons vanish. 4.1 µ-e conversion The nucleon form factors take the following values [42, 43] Gu S = 3.74, Gd S = 2.694, Gc S = 0.06, Gs S = 0.64, Gb S = 0.02 . (4.6) (4.6) Note, however, that there is signiﬁcant uncertainty and the values might be up to a factor of 2–4 larger as other calculations suggest [41]. With the meson mediation method, the lepton bilinear will couple to an intermediate meson which also couples to the nuclei. Because of the Lorentz structure of the eﬀective operators considered in this work, the only relevant mesons scalar mesons isosinglet f0(500) and isotriplet a0(980). The relevant parameters are α(0,3) rS = 1 4 × ηq rS        βf0, βa0 q = u βf0, −βa0 q = d 0 q = c, s, b , (4.7) (4.7) where the parameters are estimated to be βf0 = 1.58 and βa0 = 2.24 as in [41]. – 7 – decay Brmax i cutoﬀscale Λ [TeV] Ξu ij,uu Ξd ij,dd Ξd ij,ss τ −→e−π0 8.0 × 10−8 10 10 – τ −→e−η 9.2 × 10−8 34 34 7.9 τ −→e−η′ 1.6 × 10−7 42 42 12 τ −→e−K0 S 2.6 × 10−8 – 7.8 √ λ 7.8 √ λ τ −→e−(f0(980) →π+π−) 3.2 × 10−8 13 √sin ϕm 13 √sin ϕm 16 √cos ϕm τ −→µ−π0 1.1 × 10−7 9.0–9.6 9.0–9.6 – τ −→µ−η 6.5 × 10−8 36–38 36–38 8.4–8.9 τ −→µ−η′ 1.3 × 10−7 42–46 42–46 12–13 τ −→µ−K0 S 2.3 × 10−8 – (7.8–8.3) √ λ (7.8–8.3) √ λ τ −→µ−(f0(980) →π+π−) 3.4 × 10−8 (12–14) √sin ϕm (12–14) √sin ϕm (15–16)√cos ϕm Table 2. Semi-leptonic τ-decays. Experimental constraint on the cutoﬀscale Λ [TeV] of the eﬀective operators. λ denotes the mixing angle inducing operator mixing as deﬁned in eq. (2.13) and ϕm is the mixing angle between f0(500) and f0(980) and is deﬁned in eq. (A.9). JHEP02(2016)176 Table 2. Semi-leptonic τ-decays. Experimental constraint on the cutoﬀscale Λ [TeV] of the eﬀective operators. λ denotes the mixing angle inducing operator mixing as deﬁned in eq. (2.13) and ϕm is the mixing angle between f0(500) and f0(980) and is deﬁned in eq. (A.9). Table 2. Semi-leptonic τ-decays. Experimental constraint on the cutoﬀscale Λ [TeV] of the eﬀective operators. λ denotes the mixing angle inducing operator mixing as deﬁned in eq. 4.2 Semi-leptonic τ-decays Semi-leptonic τ-decays impose another important constraint on operators with τ leptons and light quarks. For the operators considered in this work, the only relevant and well- measured τ-decay modes are decays to pseudoscalar mesons π0, η, η′ and K0 S and to the scalar meson f0(980) which subsequently decays to pions. We list the kinematically allowed channels and the limit on the branching ratios in table 2, where we quote the current experimental limit on the branching ratios [40]. The decay width for a τ +-lepton to a lighter lepton ℓ+ with mass mℓand a neutral meson M0 kk = (¯qkqk) is given by Γ(τ + →ℓ+M0 kk) = kM 32π m2 M ¯f2 M m2τ m2 τ + m2 ℓ−m2 M \|Ξ±\|2 + 2mτmℓRe Ξ2 ± , (4.8) (4.8) where kM is the magnitude of the meson 3-momentum in the centre-of-momentum frame k2 M = (m2 τ −(mℓ+ mM)2)(m2 τ −(mℓ−mM)2) 4m2τ (4.9) (4.9) – 8 – – 8 – and the eﬀective coupling Ξ± is deﬁned as and the eﬀective coupling Ξ± is deﬁned as Ξ± ≡ΞNu ij,kl cos ϕ ± ΞNd ij,kl sin ϕ . (4.10) (4.10) Ξ+ is the coupling for a scalar meson and Ξ−is the coupling for a pseudo-scalar meson in the ﬁnal state. The up-type (down-type) quark content of the meson is cos ϕ (sin ϕ). The scale-dependent scalar meson decay constant ¯fM is deﬁned in eq. (A.1). The partial decay width will be compared with the total decay width Γτ = τ −1 τ = 2.27 × 10−9 MeV . Besides the pseudoscalar mesons, we consider the scalar meson f0(980), which domi- nantly decays to two pions with a branching ratio Br(f0(980) →π+π−) = 0.46 [46]. We parameterize its quark content by the mixing angle ϕm, which is deﬁned in eq. (A.9). JHEP02(2016)176 Our limits are quoted in table 5. The result only very weakly depends on the phase of the of the Wilson coeﬃcient ΞNu,Nd ij,kl for hierarchical lepton masses and generally leads to a correction at the level of 4mℓimℓj m2 ℓi + m2 ℓj ∼4 min{mℓk} max{mℓk} (4.11) (4.11) percent compared to the total decay width, which amounts to about 1% (10%) in case of an electron (muon) ﬁnal state. Thus it is below the precision for an electron in the ﬁnal state, but we quote the range in case of a muon in the ﬁnal state. 4.3 Leptonic neutral meson decays Another important class of constraints comes from LFV neutral meson decays. The decay width of a meson M0 kl = (¯qkql) can be expressed as Another important class of constraints comes from LFV neutral meson decays. The decay width of a meson M0 kl = (¯qkql) can be expressed as Γ(M0 kl →ℓiℓj) = kℓ 16π ¯f2 M h m2 M −m2 ℓi −m2 ℓj \|Ξ−\|2 + 2mℓimℓjRe Ξ2 − i , (4.12) (4.12) here kℓis the magnitude of the lepton 3-momentum in the centre-of-momentum frame, k2 ℓ= (m2 M −(mℓi + mℓj)2)(m2 M −(mℓi −mℓj)2) 4m2 M (4.13) (4.13) and the eﬀective coupling Ξ−is deﬁned in eq. (4.10). The experimental constraints on the cutoﬀscale Λ [TeV] of the eﬀective operators are collected in table 3. The top part of the table lists the direct constraints on the operators with the same quarks in eq. (2.6), while the lower part summarises indirect constraints on the operators from operator mixing induced by their creation from gauge invariant operators. These constraints are parameterised by λ, which is deﬁned in eq. (2.13). It is clear that we can place the strongest limit on operators with eµ. 4.2 Semi-leptonic τ-decays The strongest limits are from decays to η(′) and f0(980) mesons because the product mM ¯fM is relatively large. 4.4 Leptonic charged meson decays As discussed in section 2 there are also eﬀective four fermion interactions which contribute to diﬀerent charged meson decays. Many charged meson decays have already been measured – 9 – decay Brmax i cutoﬀscale Λ [TeV] Ξu ij,uu Ξd ij,dd Ξd ij,ss Ξu ij,cc Ξd ij,bb π0 →µ+e− 3.8 × 10−10 2.2 2.2 – – – π0 →µ−e+ 3.4 × 10−9 1.2 1.2 – – – π0 →µ+e−+ µ−e+ 3.6 × 10−10 2.6 2.6 – – – η →µ+e−+ µ−e+ 6 × 10−6 0.52 0.52 0.12 – – η′ →eµ 4.7 × 10−4 0.091 0.091 0.026 – – K0 L →e±µ∓ 4.7 × 10−12 – 86 √ λ 86 √ λ – – D0 →e±µ∓ 2.6 × 10−7 6.4 √ λ – – 6.4 √ λ – B0 →e±µ∓ 2.8 × 10−9 – 10 √ λ – – 6.6 √ λ B0 →e±τ ∓ 2.8 × 10−5 – 0.97 √ λ – – 0.62 √ λ B0 →µ±τ ∓ 2.2 × 10−2 – 0.18 √ λ – – 0.12 √ λ JHEP02(2016)176 Table 3. Leptonic LFV meson decays. Experimental constraint on the cutoﬀscale Λ [TeV] of the eﬀective operators. The processes listed in the top part of the table directly constrain the operators with the same quarks in eq. (2.6), while the ones in the lower part indirectly constrain the operators with the same quarks via the operators generated by operator mixing as deﬁned in eq. (2.13). Table 3. Leptonic LFV meson decays. Experimental constraint on the cutoﬀscale Λ [TeV] of the eﬀective operators. The processes listed in the top part of the table directly constrain the operators with the same quarks in eq. (2.6), while the ones in the lower part indirectly constrain the operators with the same quarks via the operators generated by operator mixing as deﬁned in eq. (2.13). Table 3. Leptonic LFV meson decays. Experimental constraint on the cutoﬀscale Λ [TeV] of the eﬀective operators. The processes listed in the top part of the table directly constrain the operators with the same quarks in eq. (2.6), while the ones in the lower part indirectly constrain the operators with the same quarks via the operators generated by operator mixing as deﬁned in eq. (2.13). and can be used to indirectly constrain the operators in eq. (2.6). 4.4 Leptonic charged meson decays – 10 – decay constraint cutoﬀscale Λ [TeV] Wilson coeﬃcients Λµe,eµ,eτ Λτe,τµ,µτ Ξu ij,uu Ξd ij,dd Ξd ij,ss Ξu ij,cc Ξd ij,bb Rπ Rexp π ± 5% 25–280 25–260 ✓ ✓ – – – RK Rexp K ± 5% 24–160 24–150 ✓ – ✓ – – Br(D+ →e+ν) < 8.8 × 10−6 2.8–2.9 2.9 – ✓ – ✓ – Br(D+ s →e+ν) < 8.3 × 10−5 3.2–3.3 3.2–3.3 – – ✓ ✓ – Br(B+ →e+ν) < 9.8 × 10−7 2.0 2.0 ✓ – – – ✓ Br(π+ →µ+ν) Brexp ± 5% 1.9–7.4 1.9–9.4 ✓ ✓ – – – Br(K+ →µ+ν) Brexp ± 5% 1.7–5.8 1.7–7.4 ✓ – ✓ – – Br(D+ →µ+ν) (3.82 ± 0.33) × 10−4 1.1–2.7 1.1–3.4 – ✓ – ✓ – Br(D+ s →µ+ν) (5.56 ± 0.25) × 10−3 1.3–4.3 1.3–5.3 – – ✓ ✓ – Br(B+ →µ+ν) < 1.0 × 10−6 1.9–2.7 1.7–3.0 ✓ – – – ✓ Br(D+ →τ +ν) < 1.2 × 10−3 0.21–0.78 0.23–0.73 – ✓ – ✓ – Br(D+ s →τ +ν) (5.54 ± 0.24) × 10−2 0.33–1.2 0.33–1.1 – – ✓ ✓ – Br(B+ →τ +ν) (1.14 ± 0.27) × 10−4 0.49–1.3 0.49–1.2 ✓ – – – ✓ Table 4. Experimental constraint on the cutoﬀscale Λ [TeV] of the eﬀective operators from LFV leptonic charged meson decays. The second column indicates the relevant experimental constraint. The third and fourth columns give the constraint in TeV. The index of Λ denotes the relevant JHEP02(2016)176 Table 4. Experimental constraint on the cutoﬀscale Λ [TeV] of the eﬀective operators from LF Table 4. Experimental constraint on the cutoﬀscale Λ [TeV] of the eﬀective operators from LFV leptonic charged meson decays. The second column indicates the relevant experimental constraint. The third and fourth columns give the constraint in TeV. The index of Λ denotes the relevant leptons of the operator. The ﬁnal state charged lepton in each process is right-handed and thus corresponds to the second index of the Wilson coeﬃcient. Measured branching ratios are imposed at the 2σ level unless otherwise speciﬁed. The check marks [✓] indicate the constrained operator. 4.4 Leptonic charged meson decays In particular the decays of π+ and K+ have been measured to high precision, and can be used to indirectly constrain the operators in eq. (2.6). In particular the decays of π+ and K+ have been measured to high precision, Rπ = Br(π+ →e+ν) Br(π+ →µ+ν) =(1.230 ± 0.004)×10−4, Br(π+ →µ+ν)=0.9998770 ± 0.0000004, RK = Br(K+ →e+ν) Br(K+ →µ+ν) =(2.489 ± 0.011)×10−5, Br(K+ →µ+ν)=(63.55 ± 0.11)×10−2. (4.14) K+ →µ+ν)=(63.55 ± 0.11)×10−2. (4.14) However we expect our calculation to be precise at the level of 5% and thus our theoretical precision does not match the experimental precision. A precise treatment would require the inclusion of higher order corrections in chiral perturbation theory, which has been done for the SM contribution in ref. [47]. As there are interference terms between the SM and the new physics contribution, it is not possible to use the precise SM result directly. We do not attempt to include higher-order corrections to pion and kaon decays, but conservatively require that the predicted value taking the operator and the SM contribution into account is within 5% of the experimental value. Given that the precision of these measurements is 0.3% (0.4%) for Rπ and RK as well as 4 × 10−5% (0.17%) for pion (kaon) decay to a muon and a neutrino, we naively (neglecting cancellations) expect that it is possible to increase the limit on the cutoﬀscale from Rπ, RK and Br(K+ →µ+ν) by a factor of two. Similarly taking the experimental precision into account, it might be possible to improve the limit from Br(π+ →µ+ν) by up to a factor 20. 4.4 Leptonic charged meson decays The third and fourth column list the obtained lower limit on the cutoﬀ scale Λij, where the indices indicate the two leptons of the operator. Check marks [✓] in the ﬁfth to ninth column indicate the operators, which are constrained by the considered process. The charged lepton in the ﬁnal state of the diﬀerent processes is right-handed, i.e. the one with the index j of the Wilson coeﬃcient. Despite our crude calculation the strongest constraints on the cutoﬀscale Λ are extracted from the ratios Rπ and RK, which could be improved with a more precise calculation. However they are outperformed by µ-e conversion in nuclei. Kaons. We require the new physics contribution to deviate from the experimental result by less than 2σ. The third and fourth column list the obtained lower limit on the cutoﬀ scale Λij, where the indices indicate the two leptons of the operator. Check marks [✓] in the ﬁfth to ninth column indicate the operators, which are constrained by the considered process. The charged lepton in the ﬁnal state of the diﬀerent processes is right-handed, i.e. the one with the index j of the Wilson coeﬃcient. Despite our crude calculation the strongest constraints on the cutoﬀscale Λ are extracted from the ratios Rπ and RK, which could be improved with a more precise calculation. However they are outperformed by µ-e conversion in nuclei. 1The other four fermion interaction with a neutrino will lead to the signature of a mono-lepton with missing energy, which has a large SM background from W-boson production and it will thus not lead to competitive limits. Hence we do not consider it for the LHC study. 4.4 Leptonic charged meson decays The decay width for charged meson decay M+ kl = (uk ¯dl) in the limit of massless neu- trinos is given by The decay width for charged meson decay M+ kl = (uk ¯dl) in the limit of massless neu- trinos is given by Γ(M+ kl →ℓ+ i ν) (4.15) = kℓ 8πm2 M m2 M −m2 ℓi  2G2 F f2 Mm2 ℓi \|Vkl\|2 + m2 M ¯f2 M 4 X j ΞCu ij,kl −ΞCd ij,kl + yℓi (yuk + ydl) m2 W U ∗ ijV ∗ kl 2 − √ 2GF mℓimM ¯fMfM  yℓi (yuk + ydl) m2 W \|Vkl\|2 + Re  X j ΞCu ij,kl −ΞCd ij,kl UijVkl       (4.15)  + m2 M ¯f2 M 4 X j ΞCu ij,kl −ΞCd ij,kl + yℓi (yuk + ydl) m2 W U ∗ ijV ∗ kl 2 − √ 2GF mℓimM ¯fMfM  yℓi (yuk + ydl) m2 W \|Vkl\|2 + Re  X j ΞCu ij,kl −ΞCd ij,kl UijVkl       with the 3-momentum kℓdeﬁned in eq. (4.13). The Yukawa couplings of the charged fermions are deﬁned as yuk ≡muk/v, ydl ≡mdl/v and yℓi ≡mℓi/v with the vacuum expectation value v = 174 GeV. Finally the meson decay constant fM and the scale- dependent scalar meson decay constant ¯fM are both given in appendix A. All results are summarised in table 4. The ﬁrst column lists the observable, like the ratio Rπ,K and the branching ratios. The second column indicates the used experimental constraint. Note that the calculation is limited by the theory error in case of pions and – 11 – q q ℓi ℓj (a) q q ℓi ℓj g (b) g q q ℓi ℓj q (c) g q q ℓi ℓj q (d) Figure 3. Signatures at hadron colliders. g q q ℓi ℓj q (c) g q q ℓi ℓj q (d) g q q ℓi ℓj q (c) g q q ℓi ℓj q (d) q q ℓi ℓj g (b) q q ℓi ℓj (a) (c) (d) (b) (a) Figure 3. Signatures at hadron colliders. JHEP02(2016)176 Kaons. We require the new physics contribution to deviate from the experimental result by less than 2σ. 5 LHC search At colliders, the four fermion interaction ¯ℓLiℓRj ¯qkqk can lead to the charged lepton ﬂavour violating processes,1 pp →ℓiℓj + jets . (5.1) (5.1) We show the Feynman diagrams contributing to this process up to one jet in ﬁgure 3, including the leading order contribution in ﬁgure 3a and the next-to-leading order contri- butions in ﬁgures 3b–3d. CDF and D0 reported on their search for eµ ﬁnal states from s-channel heavy resonance decays in refs. [48, 49]. There are also rich studies about charged lepton ﬂavour violating processes at the LHC. ATLAS has searched for Z →eµ in ref. [50]. Similarly LFV Higgs decay has also been studied in refs. [19, 20]. Both ATLAS and CMS have performed search for heavy resonances decay to eµ in refs. [51, 52]. ATLAS has also expanded their search to include eµ, eτ and µτ in [53]. These analyses examined the eµ, eτ or µτ invariant mass spectrum for the presence of a heavy particle. They found no evidence of new physics and gave model-dependent limits on the mass of the heavy resonances for given couplings. All these searches looked for LFV processes inclusively, i.e. including extra jets. In ref. [54] ATLAS searched exclusively for ﬁnal states with a LFV eµ pair and zero jet for t-channel ˜t exchange. Note that in most analyses well-deﬁned and properly reconstructed jets have pT ≳30 GeV. – 12 – 0 500 1000 1500 2000 M(eµ) [GeV] 0 1000 2000 3000 4000 5000 Arbitrary units u d s c b Figure 4. The distribution of the eµ invariant mass at parton level at √s = 7 TeV. The production cross section of each operator has been normalised to the same value. JHEP02(2016)176 Figure 4. The distribution of the eµ invariant mass at parton level at √s = 7 TeV. The production cross section of each operator has been normalised to the same value. We will take the most up-to-date inclusive and exclusive analyses for a pair of oppo- sitely charged ﬂavour oﬀ-diagonal leptons, i.e. the 8 TeV search for eµ, eτ and µτ with 20.3 fb−1 integrated luminosity in ref. [53] and the 7 TeV search for eµ with 2.08 fb−1 inte- grated luminosity in ref. [54]. The searches have quite distinctive SM background because of the requirement on jets, which will be elaborated in section 5.1. 5 LHC search With Monte Carlo simulation and the aid of hepdata, we will recast both searches and extract the LHC limits for the eﬀective operators chosen in this work. Before we move on to the details of the simulation, we want to stress that the LHC limits depend on the quark ﬂavour in a not-so-trivial manner. Because of the parton distribution functions, the pT distribution and the invariant mass distribution of the lepton pairs in the ﬁnal states are also diﬀerent for operators with diﬀerent quark ﬂavours even if the total production cross sections at the LHC were the same. As an example, we plot invariant mass distribution of eµ ﬁnal states in a pp collider at √s = 7 TeV in ﬁgure 4. It is easy to see that the distributions are quite similar for the two lighter ﬂavours u and d, plotted with a black solid and blue dashed line respectively, and for the heavier ones s, c and b shown with a gray solid, green dashed, and red dot-dashed line respectively. 5.1 Signal and background The signals for the 7 TeV eµ exclusive analysis dominantly come from the tree-level process in ﬁgure 3a. Since the exclusive search rejects any events with a well-ﬁned jet, we neglect all next-to-leading order contributions for the signals. The relevant operators are implemented in FeynRules 2.0 [55] to generate output model ﬁles in UFO format. The signal events are generated in MadGraph 5 [56] at leading order with parton distribution function nn23lo1. The parton level events are subsequently piped to PYTHIA 8.2 [57] for showering and hadronization. The detector eﬀects are simulated using Delphes 3 [58]. For operators with eτ and µτ, the τ-lepton could also decay leptonically and gives an eµ ﬁnal state. However, – 13 – these processes are suppressed by the leptonic branching ratios of τ and lead to really poor limits. Thus we will only consider operators with eµ for the 7 TeV analysis. Similar to the 7 TeV search, the signal of the inclusive search at 8 TeV also comes mainly from the tree level diagram in ﬁgure 3a. The next-to-leading order contribution can result in a K-factor. Assuming a uniform K-factor, the lower limit on the UV cutoﬀ will be scaled up by K 1 4 , which only improves the limits by a few percent. So for the 8 TeV analysis in this work, we will take the leading order contribution from the tree level diagram and assume a unity K-factor for simplicity. The signal samples are generated with the same tool chain. The major SM processes that can lead to eµ ﬁnal states include t¯t, WW, and Z/γ∗→ ττ. The t¯t pair decays to eµ via W bosons and is always accompanied with two hard b-jets. The other two channels, WW and ττ, give rise to a LFV lepton pair through leptonic W and τ decay, which usually have large missing transverse energy, Emiss T , because of the neutrinos in the ﬁnal states. So in the 7 TeV analysis, these background events are quite eﬃciently eliminated by the selection requirements for zero jet and small Emiss T . Because jets can be misidentiﬁed as leptons, W/Z plus jets and multi-jets also contribute. This type of background is denoted as fake background and estimated from data at the ATLAS search. Other subdominant background includes WZ/ZZ, single top and W/Z + γ. 5.1 Signal and background For both 7 TeV and 8 TeV analysis, WW, Z/γ∗→ττ and t¯t make up around 90% of the background. Because of the strict selection rules, the background for the 7 TeV analysis is much cleaner than the 8 TeV one. This ensures a good limit even with a much lower integrated luminosity at 7 TeV. For the eτ and µτ search at the 8 TeV, the background is dominated by the Drell-Yan process Z/γ∗→µµ/ττ and the fake background. The contribution from the fake background can be as much as 50%. JHEP02(2016)176 We use MadGraph 5 at NLO to generate the background sample for WW, Z/γ∗→ ττ and t¯t, where showering and hadronization is handled with Herwig 6 [59]. Detector eﬀects are simulated with Delphes 3. Our simulated background samples agree with the experimental analysis. However, the simulation and the estimation of the fake background requires the analysis on the actual experimental data, which is way beyond the scope of this work. Therefore, we will use the experimental measurements to extract the limits at both 7 TeV and 8 TeV. For the 14 TeV LHC run, we will only try to project the limit on the operators with eµ because of the non-negligible fake background for other ﬁnal states. Of the two search- ing strategies, we will choose the one in the 7 TeV analysis, which gives a much cleaner background and thus a better limit for the same dataset. So we will make use of the tool chain described here for the WW and Z/γ∗→ττ background estimate. We assume the contribution from the fake background in the selected sample at 14 TeV will be slightly less than that from Z/γ∗→ττ as in the 7 TeV analysis. In our simulation we will consider an integrated luminosity of 300 fb−1. 5.2 Event selection For the 7 TeV and 8 TeV search, we take the same selection requirements as in the ATLAS analysis. The event selection requires a pair of oppositely charged leptons. Electrons – 14 – should have ET > 25 GeV and satisfy a set of stringent identiﬁcation requirements referred as tight. We implement the tight identiﬁcation through the electron eﬃciency in Delphes 3 as in ref. [60]. Electrons are rejected if they lie outside the pseudorapidity regions \|η\| < 1.37 or 1.52 < \|η\| < 2.47. Similarly muons are required to have pT > 25 GeV and \|η\| < 2.4. Tau candidates should also have ET > 25 GeV and lie in the proper pseudo-rapidity range \|η\| < 2.47 and \|η\| > 0.03. In addition, we implement the lepton isolation requirements: the scalar sum of the track pT within a cone of ∆R = 0.2(0.4) around the lepton is less than 10% (6%) of the lepton’s pT for the 7 (8) TeV search; similarly the sum of ET within the cone of ∆R = 0.2 is less than 15% (6%) of the lepton’s ET for the 7 (8) TeV search. Jets are reconstructed using the anti-kt algorithm with a radius parameter of 0.4. In the 7 TeV search any events with jets that have pT > 30 GeV or Emiss T < 25 GeV are rejected. Additionally the invariant mass of the lepton pair should be bigger than 100 (200) GeV and the azimuthal angle diﬀerence between them should be bigger than 3 (2.7) for the 7 (8) TeV search. JHEP02(2016)176 For the 14 TeV projection, we will impose the following cuts on lepton transverse momentum, pT , and the total missing transverse energy: pT (ℓ) > 300 GeV and Emiss T < 20 GeV, in addition to the same cuts on the azimuthal angle ∆φ(e, µ) > 3.0 and the lepton identiﬁcation and isolation requirements. After the selection, the only SM background is from WW, while ττ contribution drops much faster with increasing dilepton invariant mass. With the assumption that the contribution from fake background is less than that from ττ, we can also neglect the fake background in the 14 TeV projection. 5.3 Limit setting and results We use maximum likelihood estimator for limit setting at the 7 and 8 TeV searches. The observed invariant mass distributions of the eµ pair as well as the SM background for the 7 and 8 TeV analyses are taken from hepdata. The likelihood function for each bin is deﬁned as Li(µ, ˜θi\|ni) = P(ni\|µ si + bi)G(˜θi, 0, 1), (5.2) (5.2) where P and G are Poisson and Gaussian functions. si, bi and ni are the predicted signal, SM background and the observed events in the i-th bin. The parameter µ is the signal strength and ˜θi is the nuisance parameter. The total likelihood function is the product of Li in each bin. This limit setting method is tested with the hypothesis as described in the 7 and 8 TeV ATLAS analyses and the results agree within percent level. For the 14 TeV projection, we will perform the same limit setting procedure with the binned invariant eµ mass spectrum from 600 GeV to 1 TeV with bin width of 50 GeV as well as an over-ﬂow bin. For the 14 TeV projection, we will estimate the experimental reach simply with Signiﬁcance = S q S + (∆S)2 + (∆B)2 , (5.3) (5.3) where S and B denote the number of signal and background events. ∆S and ∆B param- eterize the systematic uncertainties, ∆S = 10%S and ∆B = 10%B. where S and B denote the number of signal and background events. ∆S and ∆B param- eterize the systematic uncertainties, ∆S = 10%S and ∆B = 10%B. – 15 – HHHHHH ¯qq ¯ℓiℓj ¯eµ ¯eτ ¯µτ 7 TeV 8 TeV 14 TeV 8 TeV 8 TeV ¯uu 2.6 2.9 8.9 2.4 2.2 ¯dd 2.3 2.3 8.0 2.1 1.9 ¯ss 1.1 1.4 4.0 0.95 0.88 ¯cc 0.97 1.3 3.6 0.82 0.78 ¯bb 0.74 1.0 2.7 0.63 0.61 Table 5. Constraints from the LHC searches on the cutoﬀscale Λ [TeV] at the 7 and 8 TeV search. The 14 TeV projection is also listed for the eµ ﬁnal state. Table 5. Constraints from the LHC searches on the cutoﬀscale Λ [TeV] at the 7 and 8 TeV search. The 14 TeV projection is also listed for the eµ ﬁnal state. JHEP02(2016)176 We list the current limits and future projection at the 14 TeV run in table 5. 5.3 Limit setting and results Note that the 8 TeV search does not result in much better limits even with a higher beam energy and 10 times more data than the 7 TeV one, simply due to the large background. The limits for eτ and µτ are both weaker than the eµ channel at 8 TeV as a result of the low τ-tagging rate and higher fake background. 6 Discussion In ﬁgure 5 we compare the most stringent constraints from precision experiments and the LHC. If the constraint depends on a free parameter like the phase of the Wilson coeﬃcient or a mixing angle, we show the possible constraints in a band and include the second- most stringent constraint as well. For completeness, we kept the constraints from Rπ and RK, since they suﬀer from their theoretical uncertainty and can be further improved with a more detailed calculation by a factor of a few. Operators with diﬀerent lepton combinations are separated by a gray vertical line. The ﬁgure shows the limits on operators with the quarks u, d, s, c, b ordered from left to right in each of the blocks. The current 8 TeV LHC constraints are denoted by a solid red line and the future (14 TeV) sensitivity by a dashed orange line. The constraints from µ-e conversion are shown in green indicating the range between direct nuclear mediation and meson exchange mediation. Blue lines indicate limits from τ-decays to a charged lepton and a neutral pseudoscalar mesons besides f0, which is shown in purple. It depends on the undetermined mixing angle ϕm between the diﬀerent quark compositions. Finally, constraints from leptonic charged meson decays are shown in gray. The limits from leptonic neutral meson decays were generally weaker than the presented limits and are thus not included in the ﬁgure. For operators with eµ across all quark ﬂavours, the limit from µ-e conversion in nuclei clearly outperforms any other limit. Even its current limit on Λ assuming direct nuclear mediation is two orders of magnitude higher than that from the 14 TeV projection of the LHC. The limits certainly will be further improved by the two proposed experiments, Mu2E [12, 13] at FNAL and COMET [14, 15] at J-PARC, which aim to improve the sensitivity of µ-e conversion in 48Ti down to 10−16, and possibly even to 10−18 in a future proposed experiment PRISM/PRIME [14, 15]. For eτ and µτ, pion and Kaon decays and semi-leptonic τ decays places the strongest constraints for the light quark ﬂavours. The – 16 – Figure 5. Summary plots of most stringent limits from precision experiments and the LHC. See the text for a detailed explanation. Figure 5. Summary plots of most stringent limits from precision experiments and the LHC. See the text for a detailed explanation. JHEP02(2016)176 Figure 5. 6 Discussion Summary plots of most stringent limits from precision experiments and the LHC. See the text for a detailed explanation. limits from τ decays will be further improved at the Belle-II experiment [61]: Belle-II aims to increase the sensitivity on the branching fraction by two orders of magnitude. Note that constraints from precision measurements for ¯uu and ¯dd are quite similar, which can be easily explained with the isospin symmetry. However, the constraints on the operators with heavy quark ﬂavours are generally weaker. That is exactly where the LHC comes into play. With the 8 TeV LHC search, the collider limit is competitive with constraints from τ-decays and charged meson decays for operators with ¯cc and ¯bb and a right-handed τ-lepton. In particular it does not depend on the phase of the Wilson coeﬃcient. The limits from charged meson decays feature an interference with the SM contribution and thus depend on the phase of the Wilson coeﬃcient. Using the 8 TeV LHC data, we set limits of 600–800 GeV on the cutoﬀscale Λ for LFV operators with right-handed τ leptons and we expect that those limits can be further improved with more integrated luminosity, similar to projected sensitivity for the eµ channel at 14 TeV with 300 fb−1. In case of the eµ channel we ﬁnd that the sensitivity of the LHC can be improved by a factor of 2.7–3.5. Finally we want to address the validity of the eﬀective operator descriptions at the LHC. When the momentum transfer Qtr in the interaction is comparable to the heavy mediator of mass M in the UV completion of the eﬀective theory, the eﬀective operator description is no longer a good approximation. The UV cutoﬀscale Λ is related to the mass of the heavy mediator with (6.1) M = Λ√gqgl, (6.1) where gq (gl) denote the couplings between the heavy mediator and the quarks (leptons). To properly preserve the validity of the eﬀective operators, a procedure referred as truncation can be conducted when Qtr > M, i.e. the event is discarded [62]. So if we take the optimistic limit gq = gl = 4π, the heavy mediator mass should be at least ≳7.7 TeV for the 8 TeV search and the results are surely valid. 7 Conclusions From the comprehensive case study in this work, we see that precision measurements and the LHC study are indeed complementary. Which experiment gives the best reach depends on both the quark ﬂavour and the lepton pair in the operator. For light quarks u, d and s, precision measurements clearly outperform the LHC irrespective of the charged lepton ﬂavour. However, the LHC becomes competitive for heavier quarks, c and b, and there is an interesting interplay between the two approaches to obtain limits on LFV operators with two quarks and two leptons. Operators with eµ are still highly constrained by precision measurements, particularly µ-e conversion in nuclei, but the LHC competes for LFV operators with right-handed τ leptons and can set limits independent of the phase of the Wilson coeﬃcient. We set a lower limit of 600–800 GeV on the cutoﬀscale of all these operators. JHEP02(2016)176 In this study we restricted ourselves to scalar operators and did not consider operators with top quarks. For other Lorentz structures we expect similar limits from the LHC, but the limits from the precision experiments have to be reevaluated. In case of top quarks, there are no direct limits from precision experiments, although operator mixing will lead to some constraint. We expect that a similar analysis of the collider phenomenology can set new interesting limits in addition to constraints from ﬂavour violating top decays. Finally we only considered non-resonant searches and did not consider possible underlying UV completions. A complementary study of simpliﬁed models, where the operators are opened up, might lead to more stringent, although model-dependent, limits. Acknowledgments We thank Martin Holthausen for collaboration during the initial stages of this project and Tong Li, Fei Gao, Lei Wu, Aldo Saavedra and Bruce Yabsley for useful discussions. This work was supported in part by the Australian Research Council. We acknowledge the use of matplotlib [63] and ipython [64]. 6 Discussion Even with relatively conservative – 17 – option gq = gl = 1, the LHC limit is still quite sound, because the LHC analyses we use in this work rely mostly on events with smaller momentum transfer. Moreover, if very small values of gq and gl are chosen, we are bound to return to the UV completions, which is a completely diﬀerent type of study not meant to be contained in this work. A Mesons The quark bilinear in the operator we choose to study determines that the only mesons involved in the LFV processes are either neutral scalars or pseudoscalars. The decay constants fM for scalar (S) and pseudo-scalar (P) mesons are deﬁned as 0\|¯qiγµqj\|S(p) = fSpµ , 0\|¯qiqj\|S(p) = mS ¯fS , (A.1a) 0\|¯qiγµγ5qj\|P(p) = fP pµ , 0\|¯qiγ5qj\|P(p) = mP ¯fP = mP fP mP miq + mj q . (A.1b) 0\|¯qiqj\|S(p) = mS ¯fS , (A.1a) 0\|¯qiγ5qj\|P(p) = mP ¯fP = mP fP mP miq + mj q . (A.1b) (A.1a) – 18 – Meson mM/MeV τM/s ΓM/MeV fM/MeV ¯fM/MeV π0 134.9766 8.52 × 10−17 7.725 × 10−6 130.41 2500 η 547.862 1.31 ⋆ ⋆ η′ 957.78 0.198 ⋆ ⋆ K0 L 497.614 5.116 × 10−8 1.287 × 10−14 156.2 790 K0 S 497.614 8.954 × 10−11 7.351 × 10−12 156.2 790 D0 1864.84 4.101 × 10−13 1.605 × 10−9 204.6 300 B0 5279.58 1519 × 10−15 4.333 × 10−10 190.6 240 π+ 139.57018 2.6033 × 10−8 2.5284 × 10−14 130.41 2600 K+ 493.677 1.2380 × 10−8 5.3167 × 10−14 156.2 780 D+ 1869.61 1.040 × 10−12 6.329 × 10−10 204.6 300 D+ s 1968.30 5.00 × 10−13 1.32 × 10−9 257.5 370 B+ 5279.26 1638 × 10−15 4.018 × 10−10 190.6 240 f0(980) 990 40–100 ⋆ ⋆ JHEP02(2016)176 Table 6. Relevant data for the scalar and pseudoscalar mesons studied in this work. Besides the last meson f0(980) which has JP C = 0++, all mesons are pseudoscalar mesons with JP C = 0−+ according to the quark model assignment. We list the decay constants for most mesons assuming isospin symmetry to relate the decay constants of charged mesons with the corresponding neutral meson. ⋆Please refer to the text for the decay constants of η(′) and f0(980). The scale-dependent scalar decay constants ¯fM are related to the decay constants fM via the equations of motion. We use the experimental values (where available) for the pseudoscalar decay constants and the quark masses in ref. [40] ¯m = mu + md 2 = 3.5+0.7 −0.2 MeV , mc = (1.275 ± 0.025) GeV , (A.2) ms = (95 ± 5) MeV , mb = (4.18 ± 0.03) GeV (A.2) to obtain the scale-dependent scalar decay constants. All decay constants are listen in table 6 except for the states η(′) and f0(980), where the decay constants depend on a mixing angle. A Mesons The pseudo-scalars η and η′ mix with each other and are a mixture of \|s¯s⟩and the isospin singlet \|q¯q⟩≡ \|u¯u⟩+ \|d ¯d⟩ / √ 2 and their decay constants can be parameterised in terms of two decay constants fq,s and two mixing angles φq,s fq η fs η fq η′ fs η′ ! ≡ fq cos φq −fs sin φs fq sin φq fs cos φs ! . (A.3) (A.3) In the FKS formalism [65–67], the mixing angles coincide φs = φq ≡φ and glueball admixtures are neglected. The vector decay constants fq,s and the mixing angle φ are – 19 – given by [65, 67] given by [65, 67] fq = (1.07 ± 0.02)fπ , fs = (1.34 ± 0.06)fπ , φ = (39.3 ± 1.0)◦. (A.4) (A.4) The corresponding vector decay constant for the η and η′ meson are The corresponding vector decay constant for the η and η′ meson are ¯fq η′ = fq sin φ ≃88 MeV , (A.5) ¯fs η′ = fs cos φ ≃130 MeV . ¯fq η = fq cos φ ≃110 MeV , ¯fq η′ = fq sin φ ≃88 MeV , (A.5) ¯fs η = −fs sin φ ≃−110 MeV , ¯fs η′ = fs cos φ ≃130 MeV . (A.5) The meson masses given in table 6 the scalar decay constants are thus JHEP02(2016)176 ¯fq η = fq cos φmη 2 ¯m ≃8400 MeV , ¯fq η′ = fq sin φmη′ 2 ¯m ≃12000 MeV , (A.6) ¯fs η = −fs sin φ mη 2ms ≃−320 MeV , ¯fs η′ = fs cos φ mη′ 2ms ≃680 MeV . (A.7) ¯fq η′ = fq sin φmη′ 2 ¯m ≃12000 MeV , (A.6) ¯fs η′ = fs cos φ mη′ 2ms ≃680 MeV . (A.7) (A.6) (A.7) Finally, in the case of f0(980) with mass mf0(980) = 990 ± 20 MeV [40] the scale-dependent decay constant ¯fM is given by [68] ¯ff0(980) = 370 ± 20 MeV . (A.8) (A.8) In the simple quark picture f0(980) together with f0(500) are a mixture of \|s¯s⟩and the isospin singlet \|q¯q⟩. The exact mixing angle ϕm between the f0(500) and the f0(980) meson is not known yet. See ref. [69] for a list of experimental results. Note however that it is unclear whether the description in the simple quark picture is actually correct or whether the f0(980) is a multi-quark state [40]. A Mesons We will assume the simple quark model and parameterize our result in terms of the mixing angle ϕm between \|s¯s⟩and the isospin singlet \|q¯q⟩ \|f0(980)⟩ \|f0(500)⟩ ! = cos ϕm sin ϕm −sin ϕm cos ϕm ! \|s¯s⟩ \|q¯q⟩ ! . (A.9) (A.9) Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References [1] ATLAS collaboration, Observation of a new particle in the search for the Standard Model Higgs boson with the ATLAS detector at the LHC, Phys. Lett. B 716 (2012) 1 [arXiv:1207.7214] [INSPIRE]. [2] CMS collaboration, Observation of a new boson at a mass of 125 GeV with the CMS experiment at the LHC, Phys. Lett. B 716 (2012) 30 [arXiv:1207.7235] [INSPIRE]. [3] Super-Kamiokande collaboration, Y. Fukuda et al., Evidence for oscillation of atmospheric neutrinos, Phys. Rev. Lett. 81 (1998) 1562 [hep-ex/9807003] [INSPIRE]. [4] P. Minkowski, µ →eγ at a Rate of One Out of 109 Muon Decays?, Phys. Lett. B 67 (1977) 421 [INSPIRE]. [1] ATLAS collaboration, Observation of a new particle in the search for the Standard Model Higgs boson with the ATLAS detector at the LHC, Phys. Lett. B 716 (2012) 1 [arXiv:1207.7214] [INSPIRE]. [2] CMS collaboration, Observation of a new boson at a mass of 125 GeV with the CMS experiment at the LHC, Phys. Lett. B 716 (2012) 30 [arXiv:1207.7235] [INSPIRE]. [3] Super-Kamiokande collaboration, Y. Fukuda et al., Evidence for oscillation of atmospheric neutrinos, Phys. Rev. Lett. 81 (1998) 1562 [hep-ex/9807003] [INSPIRE]. [4] P. Minkowski, µ →eγ at a Rate of One Out of 109 Muon Decays?, Phys. Lett. B 67 (1977) 421 [INSPIRE]. – 20 – [5] T.P. Cheng and L.-F. Li, Neutrino Masses, Mixings and Oscillations in SU(2) × U(1) Models of Electroweak Interactions, Phys. Rev. D 22 (1980) 2860 [INSPIRE]. [6] R. Foot, H. Lew, X.G. He and G.C. Joshi, Seesaw Neutrino Masses Induced by a Triplet of Leptons, Z. Phys. C 44 (1989) 441 [INSPIRE]. [7] A. Zee, A Theory of Lepton Number Violation, Neutrino Majorana Mass and Oscillation, Phys. Lett. B 93 (1980) 389 [Erratum ibid. B 95 (1980) 461] [INSPIRE]. [8] R. Barbier et al., R-parity violating supersymmetry, Phys. Rept. 420 (2005) 1 [hep-ph/0406039] [INSPIRE]. [9] P. Langacker, The Physics of Heavy Z′ Gauge Bosons, Rev. Mod. Phys. 81 (2009) 1199 [arXiv:0801.1345] [INSPIRE]. JHEP02(2016)176 JHEP02(2016)176 [10] MEG collaboration, J. Adam et al., New constraint on the existence of the µ+ →e+γ decay, Phys. Rev. Lett. 110 (2013) 201801 [arXiv:1303.0754] [INSPIRE]. [11] A. Blondel et al., Research Proposal for an Experiment to Search for the Decay µ →eee, arXiv:1301.6113 [INSPIRE]. [12] Mu2e collaboration, R. Carey et al., Proposal to search for µ−N →e−N with a single event sensitivity below 10−16, FERMILAB-PROPOSAL-0973 [INSPIRE]. [13] R.K. References Kutschke, The Mu2e Experiment at Fermilab, arXiv:1112.0242 [INSPIRE]. [14] COMET collaboration, E.V. Hungerford, COMET/PRISM muon to electron conversion at J-PARC, AIP Conf.Proc. 1182 (2009) 694 [INSPIRE]. [15] COMET collaboration, Y.G. Cui et al., Conceptual design report for experimental search for lepton ﬂavor violating µ−−e−conversion at sensitivity of 10−16 with a slow-extracted bunched proton beam (COMET), [INSPIRE]. [16] SINDRUM II collaboration, C. Dohmen et al., Test of lepton ﬂavor conservation in µ →e conversion on titanium, Phys. Lett. B 317 (1993) 631 [INSPIRE]. [17] BaBar collaboration, B. Aubert et al., The BaBar detector, Nucl. Instrum. Meth. A 479 (2002) 1 [hep-ex/0105044] [INSPIRE]. [18] A. Abashian et al., The Belle Detector, Nucl. Instrum. Meth. A 479 (2002) 117 [INSPIRE]. [19] CMS collaboration, Search for Lepton-Flavour-Violating Decays of the Higgs Boson, Phys. Lett. B 749 (2015) 337 [arXiv:1502.07400] [INSPIRE]. [20] ATLAS collaboration, Search for lepton-ﬂavour-violating H →µτ decays of the Higgs boson with the ATLAS detector, JHEP 11 (2015) 211 [arXiv:1508.03372] [INSPIRE]. [21] W. Buchm¨uller and D. Wyler, Eﬀective Lagrangian Analysis of New Interactions and Flavor Conservation, Nucl. Phys. B 268 (1986) 621 [INSPIRE]. [22] B. Grzadkowski, M. Iskrzynski, M. Misiak and J. Rosiek, Dimension-Six Terms in the Standard Model Lagrangian, JHEP 10 (2010) 085 [arXiv:1008.4884] [INSPIRE]. [23] M. Carpentier and S. Davidson, Constraints on two-lepton, two quark operators, Eur. Phys. J. C 70 (2010) 1071 [arXiv:1008.0280] [INSPIRE]. [24] A.A. Petrov and D.V. Zhuridov, Lepton ﬂavor-violating transitions in eﬀective ﬁeld theory and gluonic operators, Phys. Rev. D 89 (2014) 033005 [arXiv:1308.6561] [INSPIRE]. [25] J.D. Bjorken and S. Weinberg, A Mechanism for Nonconservation of Muon Number, Phys. Rev. Lett. 38 (1977) 622 [INSPIRE]. – 21 – [26] G.C. Branco, W. Grimus and L. Lavoura, Relating the scalar ﬂavor changing neutral couplings to the CKM matrix, Phys. Lett. B 380 (1996) 119 [hep-ph/9601383] [INSPIRE]. [27] F.J. Botella, G.C. Branco, A. Carmona, M. Nebot, L. Pedro and M.N. Rebelo, Physical Constraints on a Class of Two-Higgs Doublet Models with FCNC at tree level, JHEP 07 (2014) 078 [arXiv:1401.6147] [INSPIRE]. [28] G.C. Branco, P.M. Ferreira, L. Lavoura, M.N. Rebelo, M. Sher and J.P. Silva, Theory and phenomenology of two-Higgs-doublet models, Phys. Rept. 516 (2012) 1 [arXiv:1106.0034] [INSPIRE]. [29] UTfit collaboration, M. Bona et al., The Unitarity Triangle Fit in the Standard Model and Hadronic Parameters from Lattice QCD: A Reappraisal after the Measurements of ∆ms and BR(B →τντ), JHEP 10 (2006) 081 [hep-ph/0606167] [INSPIRE]. References Honecker et al., Improved limit on the branching ratio of µ →e conversion on lead, Phys. Rev. Lett. 76 (1996) 200 [INSPIRE]. [44] SINDRUM II collaboration, W. Honecker et al., Improved limit on the branching ratio of µ →e conversion on lead, Phys. Rev. Lett. 76 (1996) 200 [INSPIRE]. [44] SINDRUM II collaboration, W. Honecker et al., Improved limit on the branching ratio of µ →e conversion on lead, Phys. Rev. Lett. 76 (1996) 200 [INSPIRE]. [45] SINDRUM II collaboration, W.H. Bertl et al., A Search for muon to electron conversion in muonic gold, Eur. Phys. J. C 47 (2006) 337 [INSPIRE]. [45] SINDRUM II collaboration, W.H. Bertl et al., A Search for muon to electron conversion in muonic gold, Eur. Phys. J. C 47 (2006) 337 [INSPIRE]. [45] SINDRUM II collaboration, W.H. Bertl et al., A Search for muon to electron conversion in muonic gold, Eur. Phys. J. C 47 (2006) 337 [INSPIRE]. [46] LHCb collaboration, Analysis of the resonant components in ¯B0 →J/ψπ+π−, Phys. Rev. D 87 (2013) 052001 [arXiv:1301.5347] [INSPIRE]. JHEP02(2016)176 [47] V. Cirigliano and I. Rosell, Two-loop eﬀective theory analysis of π(K) →e¯νe[γ] branching ratios, Phys. Rev. Lett. 99 (2007) 231801 [arXiv:0707.3439] [INSPIRE]. [48] CDF collaboration, A. Abulencia et al., Search for high-mass resonances decaying to e mu in p¯p collisions at √s = 1.96-TeV., Phys. Rev. Lett. 96 (2006) 211802 [hep-ex/0603006] [INSPIRE]. [49] D0 collaboration, V.M. Abazov et al., Search for sneutrino production in emu ﬁnal states in 5.3 fb−1 of p¯p collisions at √s = 1.96 TeV, Phys. Rev. Lett. 105 (2010) 191802 [arXiv:1007.4835] [INSPIRE]. [50] ATLAS collaboration, Search for the lepton ﬂavor violating decay Z →eµ in pp collisions at √s TeV with the ATLAS detector, Phys. Rev. D 90 (2014) 072010 [arXiv:1408.5774] [INSPIRE]. [51] ATLAS collaboration, Search for a heavy particle decaying into an electron and a muon with the ATLAS detector in √s = 7 TeV pp collisions at the LHC, Phys. Rev. Lett. 106 (2011) 251801 [arXiv:1103.5559] [INSPIRE]. [52] CMS Collaboration, Search for Lepton Flavour Violating Decays of Heavy Resonances and Quantum Black Holes to electron/muon Pairs in pp Collisions at a centre of mass energy of 8 TeV, CMS-PAS-EXO-13-002 (2015). [53] ATLAS collaboration, Search for a Heavy Neutral Particle Decaying to eµ, eτ, or µτ in pp Collisions at √s = 8 TeV with the ATLAS Detector, Phys. Rev. Lett. 115 (2015) 031801 [arXiv:1503.04430] [INSPIRE]. References JHEP02(2016)176 JHEP02(2016)176 [30] M.C. Gonzalez-Garcia, M. Maltoni and T. Schwetz, Updated ﬁt to three neutrino mixing: status of leptonic CP-violation, JHEP 11 (2014) 052 [arXiv:1409.5439] [INSPIRE]. [31] G. Buchalla, A.J. Buras and M.E. Lautenbacher, Weak decays beyond leading logarithms, Rev. Mod. Phys. 68 (1996) 1125 [hep-ph/9512380] [INSPIRE]. [32] K.G. Chetyrkin, J.H. Kuhn and M. Steinhauser, RunDec: A Mathematica package for running and decoupling of the strong coupling and quark masses, Comput. Phys. Commun. 133 (2000) 43 [hep-ph/0004189] [INSPIRE]. [33] LHCb collaboration, Diﬀerential branching fraction and angular analysis of the decay B0 s →φµ+µ−, JHEP 07 (2013) 084 [arXiv:1305.2168] [INSPIRE]. [34] LHCb collaboration, Measurement of Form-Factor-Independent Observables in the Decay B0 →K∗0µ+µ−, Phys. Rev. Lett. 111 (2013) 191801 [arXiv:1308.1707] [INSPIRE]. [35] LHCb collaboration, Test of lepton universality using B+ →K+ℓ+ℓ−decays, Phys. Rev. Lett. 113 (2014) 151601 [arXiv:1406.6482] [INSPIRE]. [36] BaBar collaboration, J.P. Lees et al., Evidence for an excess of ¯B →D(∗)τ −¯ντ decays, Phys. Rev. Lett. 109 (2012) 101802 [arXiv:1205.5442] [INSPIRE]. [37] Belle collaboration, M. Huschle et al., Measurement of the branching ratio of ¯B →D(∗)τ −¯ντ relative to ¯B →D(∗)ℓ−¯νℓdecays with hadronic tagging at Belle, Phys. Rev. D 92 (2015) 072014 [arXiv:1507.03233] [INSPIRE]. [38] LHCb collaboration, Measurement of the ratio of branching fractions B( ¯B0 →D∗+τ −¯ντ)/B( ¯B0 →D∗+µ−¯νµ), Phys. Rev. Lett. 115 (2015) 111803 [arXiv:1506.08614] [INSPIRE]. [39] L. Calibbi, A. Crivellin and T. Ota, Eﬀective Field Theory Approach to b →sℓℓ(′), B →K(∗)ν¯ν and B →D(∗)τν with Third Generation Couplings, Phys. Rev. Lett. 115 (2015) 181801 [arXiv:1506.02661] [INSPIRE]. [40] Particle Data Group collaboration, K.A. Olive et al., Review of Particle Physics, Chin. Phys. C 38 (2014) 090001 [INSPIRE]. [41] M. Gonzalez, T. Gutsche, J.C. Helo, S. Kovalenko, V.E. Lyubovitskij and I. Schmidt, Limits on lepton ﬂavor violation from µ−−e−conversion, Phys. Rev. D 87 (2013) 096020 [arXiv:1303.0596] [INSPIRE]. [42] A. Faessler, T. Gutsche, S. Kovalenko, V.E. Lyubovitskij and I. Schmidt, Scalar meson mediated nuclear µ−−e−conversion, Phys. Rev. D 72 (2005) 075006 [hep-ph/0507033] [INSPIRE]. – 22 – [43] T.S. Kosmas, S. Kovalenko and I. Schmidt, B quark mediated neutrinoless µ−−e− conversion in presence of R parity violation, Phys. Lett. B 519 (2001) 78 [hep-ph/0107292] [INSPIRE]. [43] T.S. Kosmas, S. Kovalenko and I. Schmidt, B quark mediated neutrinoless µ−−e− conversion in presence of R parity violation, Phys. Lett. B 519 (2001) 78 [hep-ph/0107292] [INSPIRE]. [44] SINDRUM II collaboration, W. References [54] ATLAS collaboration, Search for lepton ﬂavour violation in the emu continuum with the ATLAS detector in √s = 7 TeV pp collisions at the LHC, Eur. Phys. J. C 72 (2012) 2040 [arXiv:1205.0725] [INSPIRE]. [55] A. Alloul, N.D. Christensen, C. Degrande, C. Duhr and B. Fuks, FeynRules 2.0 — A complete toolbox for tree-level phenomenology, Comput. Phys. Commun. 185 (2014) 2250 [arXiv:1310.1921] [INSPIRE]. [56] J. Alwall, M. Herquet, F. Maltoni, O. Mattelaer and T. Stelzer, MadGraph 5: Going Beyond, JHEP 06 (2011) 128 [arXiv:1106.0522] [INSPIRE]. [57] T. Sj¨ostrand, S. Mrenna and P.Z. Skands, A Brief Introduction to PYTHIA 8.1, Comput. Phys. Commun. 178 (2008) 852 [arXiv:0710.3820] [INSPIRE]. [58] DELPHES 3 collaboration, J. de Favereau et al., DELPHES 3, A modular framework for fast simulation of a generic collider experiment, JHEP 02 (2014) 057 [arXiv:1307.6346] [INSPIRE]. – 23 – [59] G. Corcella et al., HERWIG 6: An event generator for hadron emission reactions with interfering gluons (including supersymmetric processes), JHEP 01 (2001) 010 [hep-ph/0011363] [INSPIRE]. [60] M. Drees, H. Dreiner, D. Schmeier, J. Tattersall and J.S. Kim, CheckMATE: Confronting your Favourite New Physics Model with LHC Data, Comput. Phys. Commun. 187 (2014) 227 [arXiv:1312.2591] [INSPIRE]. [61] T. Aushev et al., Physics at Super B Factory, arXiv:1002.5012 [INSPIRE]. [62] G. Busoni, A. De Simone, E. Morgante and A. Riotto, On the Validity of the Eﬀective Field Theory for Dark Matter Searches at the LHC, Phys. Lett. B 728 (2014) 412 [arXiv:1307.2253] [INSPIRE]. JHEP02(2016)176 JHEP02(2016)176 [63] J.D. Hunter, Matplotlib: A 2d graphics environment, Comput. Sci. Eng. 9 (2007) 90. [64] F. P´erez and B.E. Granger, IPython: a system for interactive scientiﬁc computing, Comput. Sci. Eng. 9 (2007) 21. [65] T. Feldmann, P. Kroll and B. Stech, Mixing and decay constants of pseudoscalar mesons, Phys. Rev. D 58 (1998) 114006 [hep-ph/9802409] [INSPIRE]. [66] T. Feldmann, P. Kroll and B. Stech, Mixing and decay constants of pseudoscalar mesons: The sequel, Phys. Lett. B 449 (1999) 339 [hep-ph/9812269] [INSPIRE]. [67] T. Feldmann, Quark structure of pseudoscalar mesons, Int. J. Mod. Phys. A 15 (2000) 159 [hep-ph/9907491] [INSPIRE]. [68] H.-Y. Cheng, C.-K. Chua, K.-C. Yang and Z.-Q. Zhang, Revisiting charmless hadronic B decays to scalar mesons, Phys. Rev. D 87 (2013) 114001 [arXiv:1303.4403] [INSPIRE]. [69] R. Fleischer, R. Knegjens and G. Ricciardi, Anatomy of B0 s,d →J/ψf0(980), Eur. Phys. J. C 71 (2011) 1832 [arXiv:1109.1112] [INSPIRE]. – 24 –
https://openalex.org/W2135526915	https://dro.deakin.edu.au/articles/journal_contribution/Fine-scale_movements_of_the_broadnose_sevengill_shark_and_its_main_prey_the_gummy_shark/20977318/1/files/37272331.pdf	English	null	Fine-Scale Movements of the Broadnose Sevengill Shark and Its Main Prey, the Gummy Shark	PloS one	2,010	cc-by	9,621	Fine-scale movements of the broadnose sevengill shark and its main prey, the gummy shark AUTHOR(S) Adam Barnett, K Abrantes, J Stevens, B Bruce, J Semmens PUBLICATION DATE 01-01-2010 HANDLE 10536/DRO/DU:30048759 Downloaded from Deakin University’s Figshare repository Deakin University CRICOS Provider Code: 00113B Fine-scale movements of the broadnose sevengill shark and its main prey, the gummy shark AUTHOR(S) Adam Barnett, K Abrantes, J Stevens, B Bruce, J Semmens DOI: 10.1371/journal.pone.0015464 DOI: 10.1371/journal.pone.0015464 This is the published version. Reproduced by Deakin University under the terms of the Creative Commons Attribution Licence uced by Deakin University under the terms of the Creative Commons Attribution Licenc Reproduced by Deakin University under the terms of the Creative Commons Attribution Licence 10536/DRO/DU:30048759 Downloaded from Deakin University’s Figshare repository Deakin University CRICOS Provider Code: 00113B Barnett, Adam, Abrantes, Katya G., Stevens, John D., Bruce, Barry D. and Semmens, Jayson M. 2010, Fine- scale movements of the broadnose sevengill shark and its main prey, the gummy shark, PLoS one, vol. 5, no. 12, Article number: e15464, pp. 1-9. Barnett, Adam, Abrantes, Katya G., Stevens, John D., Bruce, Barry D. and Semmens, Jayson M. 2010, Fine- scale movements of the broadnose sevengill shark and its main prey, the gummy shark, PLoS one, vol. 5, no. 12, Article number: e15464, pp. 1-9. Fine-Scale Movements of the Broadnose Sevengill Shark and Its Main Prey, the Gummy Shark Adam Barnett1,2, Ka´tya G. Abrantes1,3, John D. Stevens2, Barry D. Bruce2, Jayson M. Semmens1 1 Marine Research Laboratories, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Hobart, Tasmania, Australia, 2 CSIRO Marine and Atmospheric Research, Hobart, Tasmania, Australia, 3 Coastal and Estuary Ecosystems, School of Marine and Tropical Biology, James Cook University, Townsville, Queensland, Australia 1 Marine Research Laboratories, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Hobart, Tasmania, Australia, 2 CSIRO Marine and Atmospheric Research, Hobart, Tasmania, Australia, 3 Coastal and Estuary Ecosystems, School of Marine and Tropical Biology, James Cook University, Townsville, Queensland, Australia Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2010 Barnett et al. This is an open-access article distributed under the term unrestricted use, distribution, and reproduction in any medium, provided the original author a Funding: This study was supported by grants from the Save Our Seas Foundation (www.saveourseas.com), Winifred Violet Scott Foundation (no website available) and the Holsworth Wildlife Research Endowment (http://www.anz.com/personal/private-bank-trustees/trustees/apply-grant/named-charitable-trusts/). No grant numbers are available at present due to finance department restructuring. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. E-mail: adam.barnett@utas.edu.au Available from Deakin Research Online: http://hdl.handle.net/10536/DRO/DU:30048759 Abstract Information on the fine-scale movement of predators and their prey is important to interpret foraging behaviours and activity patterns. An understanding of these behaviours will help determine predator-prey relationships and their effects on community dynamics. For instance understanding a predator’s movement behaviour may alter pre determined expectations of prey behaviour, as almost any aspect of the prey’s decisions from foraging to mating can be influenced by the risk of predation. Acoustic telemetry was used to study the fine-scale movement patterns of the Broadnose Sevengill shark Notorynchus cepedianus and its main prey, the Gummy shark Mustelus antarcticus, in a coastal bay of southeast Tasmania. Notorynchus cepedianus displayed distinct diel differences in activity patterns. During the day they stayed close to the substrate (sea floor) and were frequently inactive. At night, however, their swimming behaviour continually oscillated through the water column from the substrate to near surface. In contrast, M. antarcticus remained close to the substrate for the entire diel cycle, and showed similar movement patterns for day and night. For both species, the possibility that movement is related to foraging behaviour is discussed. For M. antarcticus, movement may possibly be linked to a diet of predominantly slow benthic prey. On several occasions, N. cepedianus carried out a sequence of burst speed events (increased rates of movement) that could be related to chasing prey. All burst speed events during the day were across the substrate, while at night these occurred in the water column. Overall, diel differences in water column use, along with the presence of oscillatory behaviour and burst speed events suggest that N. cepedianus are nocturnal foragers, but may opportunistically attack prey they happen to encounter during the day. Citation: Barnett A, Abrantes KG, Stevens JD, Bruce BD, Semmens JM (2010) Fine-Scale Movements of the Broadnose Sevengill Shark and Its Main Prey, the Gummy Shark. PLoS ONE 5(12): e15464. doi:10.1371/journal.pone.0015464 Citation: Barnett A, Abrantes KG, Stevens JD, Bruce BD, Semmens JM (2010) Fine-Scale Movements of the Broadnose Sevengill Shark and Its Main Prey, the Gummy Shark. PLoS ONE 5(12): e15464. doi:10.1371/journal.pone.0015464 Editor: Richard K F Unsworth University of Glamorgan United Kingdom Editor: Richard K. F. Unsworth, University of Glamorgan, United Kingdom Received August 3, 2010; Accepted September 29, 2010; Published December 3, 2010 Received August 3, 2010; Accepted September 29, 2010; Published December 3, 2010 Copyright: 2010 Barnett et al. PLoS ONE \| www.plosone.org December 2010 \| Volume 5 \| Issue 12 \| e15464 Introduction difficult [9]. The advent of remote electronic data collection and monitoring techniques in recent decades has allowed for some of the complexities associated with studying large marine predators to be overcome. For example, active acoustic tracking, radio-acoustic positioning and animal-borne video, audio and data collection systems have been used to study fine-scale movements of a relatively small number of large shark species [9–12]. Information on the foraging behaviour of large mobile predators provides additional information to methods such as dietary analysis to better understand predator-prey relationships and their effects on community dynamics [1,2]. The foraging behaviour of a predator can determine anti-predatory behaviour of its prey such as increased vigilance, or it can influence habitat selection [3–5]. Increased vigilance or foregoing foraging oppor- tunities in risky habitats can decrease the prey’s foraging ability, consequently affecting its fitness [4–6]. Conversely, increased anti- predator behaviour may mean that the predator needs to be continually moving between foraging locations to keep the element of surprise in the predator-prey game [1,5,7,8] and, as a result, the predator’s fitness may also be affected. Determining fine-scale movement patterns of predators and their prey is an important component of studying predator-prey interactions and evaluating the likely consequences of these interactions for the predator, prey and overall community [1]. Active tracking can give high spatial resolution, and the pattern of a movement path can be measured and used to predict foraging behaviour. For example, the tortuosity of the path was used to estimate foraging strategy and patch use of blacktip reef sharks Carcharhinus melanopterus among various tropical reef habitats [12]. Animal-borne video, audio and data collection system (Crittercam) was used to provide information on foraging behaviour and fine- scale habitat preferences of tiger sharks Galeocerdo cuvier in shallow sea grass habitats in Shark Bay, Western Australia [9,13]. In California, a radio-acoustic positioning system was used to describe the foraging behaviour and interactions between white sharks Carcharodon carcharias while hunting seals [10]. To date, this is the only shark species studied using this system. Large mobile marine predators are often elusive, have large home ranges and low population densities [9]. Obtaining information on fine-scale movement patterns and foraging behaviours can be Broadnose sevengill sharks N. cepedianus are large coastal predators with a wide temperate distribution [14]. VRAP: animal capture and transmitter attachment VRAP: animal capture and transmitter attachment The VRAP was deployed from the 25th January to the 22nd April 2008. Sharks were tagged with coded transmitters (VEMCO Ltd., Halifax, Canada) during the period from the 26th January to 7th March 2008 (Table S1). For each species, half of the transmitters (nine for N. cepedianus and five for M. antarcticus) also recorded depth via a calibrated pressure sensor. Tags for N. cepedianus were V16 6H .3 year battery life and V16P 5H .2 year battery life programmed to transmit randomly every 50 to 130 seconds, while those for M. antarcticus were V13 1L .3 year battery life n = 1, V13P 1H .1 year battery life n = 5 and V9 2H ,180 day battery life n = 4 programmed to transmit every 80 to 160 seconds. Transmitter sizes and program times were chosen to accommo- date the different sizes of sharks. Sharks were caught on bottom-set longlines and brought on board the boat and restrained by two people holding them down on a foam mattress while a third person implanted the transmitter into the body cavity via a 1–2 cm incision in the abdominal wall. The incision was closed with a surgical suture and the shark released. Total length was measured in a straight line from snout to tip of tail to the nearest cm with the shark on its side and the caudal fin flexed, so the individual was as straight as possible (i.e. the longest longitudinal axis). The entire procedure was normally accomplished in 3–5 minutes during which time running water was pumped over the shark’s gills. Due to the large size of the sharks and the rapid completion of surgery, the animals were not sedated. The large sizes (150–290 cm) of N. cepedianus consistently caught in coastal areas of Tasmania and the absence of neonates in the catches indicates that these areas are not used for shelter or as nursery areas and pupping grounds for this species [15,18]. Furthermore, the large number of mature and immature individuals, and the low incidence of mature females containing mating scars (16%) also suggests that these areas do not have any specific reproductive relevance [15]. Therefore, foraging is probably the primary reason that N. cepedianus use these coastal areas. The fact that the peak in N. VRAP: animal capture and transmitter attachment cepedianus abundance in summer coincides with the seasonal occurrence of its main elasmobranch prey [15,18,20–23] further indicates that N. cepedianus may be moving into coastal areas following prey. The high abundances of N. cepedianus in coastal systems suggest that they have the potential to significantly influence community dynamics through both direct and indirect interactions, but to date no studies have addressed the movement patterns and possible foraging behaviour of N. cepedianus. The aim of this study was to use acoustic telemetry methods to examine the fine-scale movements of N. cepedianus and its primary prey species, M. antarcticus, in a coastal bay in southeastern Tasmania. In particular, we aimed to compare activity patterns over the diel cycle and describe potential foraging behaviours. Study area y A VEMCO radio-acoustic positioning system (VRAP) was deployed in Norfolk Bay in southeast Tasmania, Australia (Fig. 1) to track 18 N. cepedianus and 10 M. antarcticus (see Table S1 for sex and size ranges) that were fitted with coded acoustic transmitters. Norfolk Bay is a relatively shallow (average depth 15 m, max depth 20 m), semi-enclosed bay with an area of approximately 180 km2. The substrate is mostly silt and silty sand with algae (predominately species from the family Caulerpaceae) and seagrass around the edges. The VRAP was positioned in the southwest corner of Norfolk Bay, in a depth of ,16 m. This site was chosen due to previous records of high catch rates of N. cepedianus [15,23]. VRAP (VEMCO radio-acoustic positioning system) (VEMCO Ltd., Halifax, Canada) The VRAP consisted of three buoys aligned in an equilateral triangle array. Each buoy had a unidirectional hydrophone and receiver for detecting ultrasonic signals. The buoys triangulate the x, y and time coordinates of an animal fitted with an acoustic transmitter. This information was sent to a base station computer in near real time. For transmitters configured with a pressure sensor, the depth (the z coordinate) was also retrieved [10,25]. The VRAP records two types of data: resolved and unresolved. Resolved data are available when all three buoys triangulate the coordinates of an individual, giving a precise location. Previous studies have indicated that the accuracy of such coordinates can be within two metres [10,25]. The precision of calculated positions in this study was high, with the positions of each sentinel tag consistently being within 1 m of each other. Unresolved data are obtained when only one or two of the buoys detect a tagged animal, giving information only on whether the animal is in range, and its depth, if it has been fitted with a pressure tag, but no information on its precise location is available. In this study, the VRAP buoys were moored 400 m apart, giving the triangle an area of 0.069 km2. Resolved detections were recorded up to 1200 m outside the triangle. Ethics Statement All research was conducted with approval from the University of Tasmania Animal Ethics Committee (#A0009120) and the Department of Primary Industries and Water (Permit # 8028). Introduction Within their PLoS ONE \| www.plosone.org 1 December 2010 \| Volume 5 \| Issue 12 \| e15464 December 2010 \| Volume 5 \| Issue 12 \| e15464 Fine-Scale Movement of Sevengill Sharks Two individuals were also manually tracked using a VEMCO VR100 acoustic receiver coupled with a directional hydrophone, one in Norfolk Bay and the other in the Derwent Estuary (Fig. 1). The Derwent Estuary runs through the City of Hobart, before opening into Storm Bay (Fig. 1) and consistently reaches depths of 20–30 m, with a maximum depth of 44 m. distributional range, they can be one of the most abundant apex predators in shallow coastal areas over summer [15–17]. N. cepedianus has similar dietary patterns globally, consuming a variety of prey including sharks, batoids, teleosts and marine mammals [16–20]. In particular, sharks of the genus Mustelus are one of the most common prey in all parts of their range [16–21]. In southeast Tasmania, high abundances of elasmobranchs including the Gummy shark, M. antarcticus, occur in coastal regions over summer [15,18,22,23], with this shark being the main prey species of N. cepedianus [21]. The use of these coastal areas by juvenile M. antarcticus (mature at ,95–110 cm TL) during summer implies these areas are beneficial for protection or that they contain abundant food to accelerate growth [24]. Although there might be other explanations, such as protection from exposure or socializing, it is more likely that Juvenile sharks use these habitats based on trade-offs between predation risk and food availability [24]. As such, the high abundance of N. cepedianus and the common occurrence of M. antarcticus in their diets [15,18] suggests that M. antarcticus is exposed to high predation risk and therefore that these coastal areas may have little benefit for protection. So, foraging may be the primary reason for the continued use of these areas. Data analysis For each species, the Rao’s spacing test (Oriana v.3 software) was used to test if depth use was uniformly distributed over the 24 h period. Additionally, a three-dimensional contingency table was used to test for mutual independence between depth use, species and time of day (day vs. night). Here, depth was divided in three groups: 0–5 m (surface), 5–10 m (mid) and .10 m (bottom). This was followed by a three-dimensional chi-square partial independence test to determine if each of the three variables is independent of the other two. Rao’s spacing tests were also used to test for uniformity in time of use of each depth range. Frequency of occurrence. Detections from the day of tagging were excluded from all VRAP data analysis to eliminate any bias resulting from the tagging event. Both resolved and unresolved VRAP data was used to investigate differences in occurrence between night and day for each species. Chi-square (x2) analysis was used to test for a departure from the expected 14.5:9.5 ratio (daylight vs. night hours) in the number of hits between day and night hours for each individual shark. A t-test was also used to test for differences in number of hits between the day and night periods for each species. This was done by comparing the proportion of hits detected during the day to 60.4%, the proportion of time corresponding to the 14.5 h of daylight. Each individual was considered as a replicate. Active tracks of N. cepedianus (VR100 data) were overlayed on bathymetric maps of Norfolk Bay and the Derwent Estuary to observe the distance and area covered by the two sharks. To further examine diel depth profiles, the altitude of the shark in relation to the bottom was determined for the animal tracked in the Derwent Estuary using Eonfusion software (Myriax, Hobart, Australia) and segments representative of the day and night tracks presented graphically. p Diel depth profiles. For each species, detection data from all individuals were pooled before analyses. This was done only after detecting no interaction between individual and depth (surface (0– 5 m), mid (5–10 m), bottom (.10 m)) (F16, 589 = 0.682, p = 0.8130 for N. cepedianus; F6,143 = 0.468, p = 0.8310 for M. antarcticus); or individual and period of day (day vs. night) (F8, 598 = 1.5333, p = 0.1621 for N. cepedianus; F3 147 = 1.390, p = 0.2481 for M. Active tracking Two N. cepedianus were equipped with a continuous acoustic transmitter with a pressure sensor that was configured to transmit every second at a set frequency. The tags were externally attached with a stainless steel tag head inserted into the muscle region near PLoS ONE \| www.plosone.org December 2010 \| Volume 5 \| Issue 12 \| e15464 2 Fine-Scale Movement of Sevengill Sharks Figure 1. Study area, showing Norfolk Bay and the Derwent Estuary in southeast Tasmania. Triangle represents the VRAP location. The two thick bold black lines (one in the Derwent Estuary and one in Norfolk Bay) are the movement paths of the two actively tracked N. cepedianus. doi:10.1371/journal.pone.0015464.g001 Figure 1. Study area, showing Norfolk Bay and the Derwent Estuary in southeast Tasmania. Triangle represents the VRAP location. The two thick bold black lines (one in the Derwent Estuary and one in Norfolk Bay) are the movement paths of the two actively tracked N. cepedianus. doi:10.1371/journal.pone.0015464.g001 the base of the dorsal fin. In both cases, the procedure took less than a minute (see Table S1 for shark details). The sharks were tracked from a 6 m vessel using a VEMCO VR100 acoustic receiver and a directional hydrophone. and the area covered by the VRAP (,1 km2), any individual could enter and exit the area many times in a day, and detections from each individual shark in any day can be considered as independent from detections in any other day. This means that data can be pooled for all individual sharks of each species. Diel depth profiles – VRAP Rao’s spacing test indicated that both N. cepedianus (U = 142.365, p,0.0001, n = 6241 depth records) and M. antarcticus (U = 149.442, p,0.0001, n = 1349) did not use the different depths evenly throughout the diel cycle. During night-time, N. cepedianus were consistently detected at all depths from the bottom to near surface, whereas during the day most detections where close to the substrate (Fig. 2). During the night, M. antarcticus was also detected in the water column, particularly during the first half of the night. However, most detections at night (79%) and all detections during the day were close to the substrate (Fig. 2). The two species used various depths differently, and the depths used also differed between time of day (day vs. night). Depth use (surface, mid, bottom), time (day vs. night) and species (N. cepedianus vs. M. antarcticus) were not all mutually independent for the animals sampled (x2 0.05,7 = 185.4721, p,0.0001). Partial independence tests also indicate that each of the three variables was not independent from the other two (species: x2 0.05,5 = 140.5915, p,0.0001; depth use: x2 0.05,6 = 4088.726, p,0.0001; and time: x2 0.05,5 = 2133.895, p,0.0001), indicating an interaction between the three factors. Path analysis. Circular statistics (Oriana v.3 software) were used to calculate angular changes in the movement paths of M. antarcticus. VRAP Data were pooled for night and day period and the angular changes were used to compare the linearity of movement and path structure between day and night. To enable the analysis of angular change, a shark must have been recorded by the VRAP for at least three consecutive resolved detections, when the detections were not separated by more than the maximum tag off time (i.e. ,3 minutes for M. antarcticus). Watson- Williams F-test was used to compare angular changes between day and night. Rayleigh’s Uniformity Test was used to determine if movements were uniformly distributed over the 360u or if they show some directionality. Due to the low number of three consecutive positional fixes for N. cepedianus, angular analysis could not be performed. Movement paths of M. antarcticus were also analysed in Fractal 5.0 software (V. O. Nams, Nova Scotia Agricultural College, Nova Scotia, Canada) to measure the fractal dimension. A fractal dimension is a measure of tortuosity of a movement path. Data analysis Movement rates were pooled into day and night periods and examined for each species separately using a one-way ANOVA. times between points indicates that the shark left the detection range of the VRAP and later re-entered to be recorded again. Thus, the shark would be swimming in a non linear path. The accuracy of movement rates may also be influenced by variation in depth between the two points (i.e. up and down movement in the water column) since changes in depth would imply faster swimming rates than those calculated by considering exclusively horizontal distances. However, this confounding effect is probably minimal as the depth range in the study area is less than 15 m [10]. Considering these possible sources for bias, movement results should be considered as conservative estimates. Movement rates were pooled into day and night periods and examined for each species separately using a one-way ANOVA. Frequency of occurrence N. cepedianus were detected in the VRAP on 52% of days and M. antarcticus 75% of days. Daily detections ranged from 0–4 individuals (x = 0.961.1 (6SD)) for N. cepedianus and 0–5 individuals (x = 1.861.4 (6SD)) for M. antarcticus. Multiple detections (individuals detected at the same time) of M. antarcticus occurred 40 times during the day compared to 11 times at night. Resolved detections constituted only 14% of the total number of detections for N. cepedianus and 9% for M. antarcticus. Resolved detections for N. cepedianus individuals ranged from 0 to 63, with a mean of 15, and unresolved ranged from 1 to 502, with a mean of 118. Resolved detections for M. antarcticus individuals ranged from 2 to 415 (mean = 60) and unresolved 52 to 3804 (mean = 730). On several occasions an individual N. cepedianus was continually detected on or near the substrate for extended periods of time. The majority of these detections were unresolved, so an exact location could not be obtained. However, the constant recording of a particular depth for periods that ranged from 30 min to 4 hours indicates that the shark would have to be either stationary or milling (limited or slow movements in a centralised area) around the location. These periods of inactivity were only observed during the day. Diel depth profiles – VRAP The fractal mean function was used to estimate the overall fractal D value for movement paths at night and day. Fractal D movement paths range from 1 to 2, where 1 is a straight line and 2 is a path so tortuous that it completely covers a plane [26,27]. For N. cepedianus, Rao’s spacing test indicated that the use of the three depth ranges was not uniformly distributed over time (p,0.01 in all cases). The top 0–5 m was mostly used during the night period, although there were some detections at this depth during the crepuscular periods (Fig. 3). Intermediate depths of 5– 10 m were used throughout the 24 h period, but mostly during the night-time, while the bottom (.10 m depth) was mostly used during the day (Fig. 3). As with N. cepedianus, Rao’s test indicates that depth use for M. antarcticus was not uniformly distributed in time for any of the three depth ranges (p,0.01 in all cases). They occurred in the top 5 m during dusk (,19:00–20:00 h), and again from around midnight to 04:00, but there were no detections at this depth during the day hours (Fig. 3). Intermediate depths were mainly used during the first few hours after sunset. Bottom depths were used throughout the diel cycle, although a greater proportion of detections was in the afternoons (Fig. 3). However, only 6% of the total detections for this species were in the top 0–5 m and 4% between 5 and 10 m, while the great majority (90%) was close to the bottom. Therefore, M. antarcticus is highly substrate associated throughout the diel cycle, although it occasionally moves up into the water column at night. Data analysis antarcticus) with a two-way ANOVA. Here, the number of detections per day for each individual was considered as independent in the following analysis. Hence, data from each individual in each day can be considered as independent. Moreover, given the size of the animals, their movement rates p Diel depth profiles. For each species, detection data from all individuals were pooled before analyses. This was done only after detecting no interaction between individual and depth (surface (0– 5 m), mid (5–10 m), bottom (.10 m)) (F16, 589 = 0.682, p = 0.8130 for N. cepedianus; F6,143 = 0.468, p = 0.8310 for M. antarcticus); or individual and period of day (day vs. night) (F8, 598 = 1.5333, p = 0.1621 for N. cepedianus; F3 147 = 1.390, p = 0.2481 for M. antarcticus) with a two-way ANOVA. Here, the number of detections per day for each individual was considered as independent in the following analysis. Hence, data from each individual in each day can be considered as independent. Moreover, given the size of the animals, their movement rates Movement rates. Arcview 3.2 Animal Extension v.2 program was used to determine the distances sharks moved between two successive points in the VRAP. This distance was then divided by the elapsed time to gain the minimum estimate of movement rate. To increase accuracy, only tracks where the time between the two detection points was less than the maximum transmission interval of the tags were used. This gives the best chance that the shark was swimming in a direct path, as longer PLoS ONE \| www.plosone.org PLoS ONE December 2010 \| Volume 5 \| Issue 12 \| e15464 PLoS ONE \| www.plosone.org 3 Fine-Scale Movement of Sevengill Sharks times between points indicates that the shark left the detection range of the VRAP and later re-entered to be recorded again. Thus, the shark would be swimming in a non linear path. The accuracy of movement rates may also be influenced by variation in depth between the two points (i.e. up and down movement in the water column) since changes in depth would imply faster swimming rates than those calculated by considering exclusively horizontal distances. However, this confounding effect is probably minimal as the depth range in the study area is less than 15 m [10]. Considering these possible sources for bias, movement results should be considered as conservative estimates. Movement rates Eleven N. cepedianus individuals recorded movement events that met the criteria of being less than 130 s between detection points (maximum tag transmit period). In total, there were 65 movement events that could be used to analyse rate of movement. The average swimming rate was 0.9960.16 m.s21 (6 SE) (range: 0.05 m.s21 to 6.1 m. s21). To gain a more accurate estimate of the animals’ cruising speed, burst speed events were removed from the analysis. As the vast majority of movement rates were less than 1 ms21 (82%), any movement rate greater than 1 ms21 was considered to be faster than a possible cruising speed. The adjusted average movement rate was considerably lower, with a value of 0.4860.03. There were no significant differences in movement There was no evidence of a diel pattern in detections between day and night period for N. cepedianus (n = 16) or M. antarcticus (n = 9), with some individuals being detected more during day and others more during the night (see Table S1 for x2 results). Therefore, there was no significant effect of time of day on occurrence of N. cepedianus (t = 20.8799, df = 16, p = 0.3919) or M. antarcticus (t = 1.4855, df = 8, p = 0.1809). However, for four M. antarcticus, there were more detections during the day, with three of these individuals showing considerably higher number of detec- tions (71–83%) during this period (Table S1). PLoS ONE \| www.plosone.org December 2010 \| Volume 5 \| Issue 12 \| e15464 December 2010 \| Volume 5 \| Issue 12 \| e15464 4 Fine-Scale Movement of Sevengill Sharks Fine-Scale Movement of Sevengill Sharks Figure 2. Diel pattern in depth use for N. cepedianus and M. antarcticus in southeast Tasmania. Box plots show the median (line within the boxes), interquartile ranges (boxes), 10th and 90th percentiles (whiskers) and outliers (z) of depths detected during each 1 h interval. For each species, data from the whole sampling period was pooled. Dashed area indicates nocturnal period. doi:10.1371/journal.pone.0015464.g002 Figure 2. Diel pattern in depth use for N. cepedianus and M. antarcticus in southeast Tasmania. Box plots show the median (line within the boxes), interquartile ranges (boxes), 10th and 90th percentiles (whiskers) and outliers (z) of depths detected during each 1 h interval. For each species, data from the whole sampling period was pooled. Dashed area indicates nocturnal period. Movement rates doi:10.1371/journal.pone.0015464.g002 rates between day (0.5060.04 m.s21) and night (0.436 0.04 m.s21) (ANOVA: F (1, 52) = 1.2039, p = 0.2343). between night and day (Watson-Williams F-test: F = 0.213, df = 1, p = 0.6450). For both periods, there were relatively small angular changes (Rayleigh test, p,0.0001 for both day and night), represented by the mean bearings: mean day: 350u69u; night: 345u624u (6SD) (Fig. 4). Fractal D showed that M. antarcticus did not move in a tortuous path for day or night (D = 1.0960.02 in both cases). Twelve burst speed events (5 individuals) were recognised for N. cepedianus. These events ranged from 1.4 m.s21 up to 6.1 m. s21. The seven burst speed events during the day were all close to the substrate (shallowest depth 11.3 m). In contrast, the five nocturnal burst speed events were closer to the surface, ranging from 2.5 to 7 m depth. Nocturnal burst speed events were associated with the shark entering the VRAP area with high speed. In contrast, day burst speed events were associated with long periods of inactivity, where the shark may have been stationary or milling around a restricted area, as the depth did not change during this period. On two occasions, burst speed events were followed by an extremely slow track (0.1 ms21 and 0.3 ms21). Active tracking The Norfolk Bay individual (female N. cepedianus) was tracked for 20 daylight hours over 3 days. Due to bad weather, tracking was only conducted during the day. The shark only moved within an area of 9,722 m2 (Fig. 1) and remained milling or stationary on the substrate for the entire tracking period. Five M. antarcticus individuals recorded movement events that met the criteria of being less than 180 seconds between detections (maximum tag transmit period). In total, 245 events were recorded. M. antarcticus showed no difference in movement speed between day (0.3360.01 m.s21) and night (0.3260.02 m.s21). Movement rates ranged from 0.07 ms21 to 1.02 ms21. The Derwent Estuary individual was tracked continuously for 22 hours, from 1100 h to 0900 h the following morning. The shark moved constantly and when tracking ceased it had moved over 37 km (straight line measurement) (Fig. 1). During the day, the shark swam at constant depths and was often moving just above the substrate. In contrast, at night, it continually moved up and down in the water column (Fig. 5). This oscillating behaviour was consistent throughout the nocturnal period (Fig. 5) and was characterised by slow ascents (x = 0.07 m.s21) off the bottom followed by faster descents (x = 0.16 m.s21) back to the substrate. December 2010 \| Volume 5 \| Issue 12 \| e15464 Discussion The distinct difference in depth use between day and night periods detected for N. cepedianus in the VRAP indicates different activity patterns for day and night. The use of multiple depths during the night suggests similar movements to that observed in the active track, where nocturnal movement was characterised by an oscillatory, or yo-yo, swimming motion, in which they repeatedly ascended into the water column and dived back to the substrate. Figure 4. Rose diagrams showing the angular changes of N. cepedianus for both day and night periods. Note differences in scale between the two periods. doi:10.1371/journal.pone.0015464.g004 Oscillatory swimming motion has previously been reported for other shark species in offshore or deeper waters [28–32], but has not been commonly observed in shallow coastal areas. However, tiger sharks G. cuvier also display similar oscillating movements to N. cepedianus in shallow inshore habitats (,10 m depth) in Shark Bay, Western Australia [9]. Both N. cepedianus and G. cuvier move slower on the ascent stage (x = 0.07 m.s21 and x = 0.10 m.s21 respectively) than on descents (x = 0.16 m.s21 for both species). However, N. cepedianus is more bottom oriented and initiates the oscillations from the substrate, ascending into the water column before returning to the substrate. G. cuvier appears to be more of a surface swimmer, initiating the oscillations from the surface and descending before returning to near surface waters [9]. In contrast to N. cepedianus, the yo-yo behaviour in G. cuvier was observed during the day [9]. g y Although Heithaus et al. [9] suggested a number of possible explanations for the oscillatory movement of G. cuvier, they believed that foraging behaviour was the most likely cause. They proposed that an oscillating foraging strategy allowed G. cuvier to ambush benthic prey from above, and air breathing prey such as mammals and turtles from below. For instance, Crittercam showed that G. cuvier descending from the surface were able to get close to benthic prey before evoking a flight response [9]. Since bottom associated prey such as M. antarcticus, skates and urolophids are the most common prey in the locations of the present study [18], N. cepedianus may similarly use the yo-yo behaviour to attack benthic prey from above. This could also explain the faster decent rates. Path analysis Mustelus antarcticus showed similar movement paths for day and night (Fig. 4). Turning angles were not significantly different PLoS ONE \| www.plosone.org December 2010 \| Volume 5 \| Issue 12 \| e15464 December 2010 \| Volume 5 \| Issue 12 \| e15464 5 Fine-Scale Movement of Sevengill Sharks PLoS ONE \| www.plosone.org 6 December 2010 \| Volume 5 \| Issue 12 \| e1546 PLoS ONE \| www.plosone.org December 2010 \| Volume 5 \| Issue 12 \| e15464 PLoS ONE \| www.plosone.org December 2010 \| Volume 5 \| Issue 12 \| e15464 PLoS ONE \| www.plosone.org Fine-Scale Movement of Sevengill Sharks Figure 3. Diel variations in depth use for N. cepedianus (in % time) for the top (0–5 m depth), middle (5–10 m depth) and bottom (.10 m depth) depth ranges. doi:10 1371/journal pone 0015464 g003 Figure 3. Diel variations in depth use for N. cepedianus (in % time) for the top (0–5 m depth), middle (5–10 m depth) and bottom (.10 m depth) depth ranges. doi:10.1371/journal.pone.0015464.g003 The depth to which the shark ascended to varied, but almost all descents concluded with the shark swimming along the substrate (Fig. 5). Crepuscular periods showed marked changes between bottom or constant depth swimming and oscillating behaviour (dusk: ,20:00 to 20:30; dawn: ,05:30 to 06:00) (Fig. 5). Discussion doi:10.1371/journal.pone.0015464.g005 Isurus oxyrinchus sharks [28,47,48] and it may take hours [13,48] to days [47,49–51] for sharks to return to their natural behaviour. However, as both the VRAP and active tracking produced similar results, the tagging event appeared to have minimal influence on behaviour. cepedianus during the day could also be linked to hunting marine mammals, an important part of their diet [18], using vision as their primary sense. If this hypothesis is the case, then N. cepedianus may remain close to the substrate during the day so that they can attack marine mammals at the surface from below. This stalking approach is also characteristic of white sharks hunting pinnipeds, where vision is thought to be the primary sense used to detect prey [45,46]. Overall, the regular occurrence of both benthic prey and marine mammals in N. cepedianus diets [18] suggests that the foraging strategy proposed by Heithaus et al. [9], where G. cuvier attacks benthic prey from above and surface prey from below may also be applicable to N. cepedianus. In addition, if this hypothesis is correct, N. cepedianus may be using different senses to hunt at different diel periods. Movement rates and path analysis of M. antarcticus suggest similar activity patterns for day and night periods. The lack of directional changes or burst speed events may be connected with a diet of predominately slow or stationary prey such as crabs, sipunculids and polychaete worms. However, as most of the water column detections were at night, it is possible that this was a result of M. antarcticus chasing nocturnally active prey such as cephalopods off the substrate. The diet of M. antarcticus in coastal Tasmania supports this hypothesis. Crabs and worms dominate their diets with teleosts and cephalopods less frequent [23,52]. Due to the short duration of the two active tracks in this study, the observed behaviours could be associated with adverse reactions to the tagging event. For instance, the long period of inactivity observed in the shark tracked in Norfolk Bay could have been a result of stress or injury caused by its capture and tagging. The individual actively tracked in the Derwent Estuary travelled more than 37 km in 22 hours, and this could have been a flight reaction after tagging. Discussion Alternative hypotheses for this yo-yo behaviour include searching through the water column for olfactory cues [33], or minimization of energy consumption by swimming on the ascent and gliding (resting) on the descent. Gliding behavior on descents has been noted in pinnipeds and is possibly used to gain energetic benefits during foraging dives [34,35]. Diel differences in depth use have been observed for a number of shark species, but have normally been associated with nocturnal migrations from deep water to forage in shallow waters [36–39]. For example, vertical oscillations at night by pacific sleeper sharks Somniosus pacificus have been related to foraging [32]. This behaviour is believed to be consistent with predators that use olfactory cues to search through the water column for prey [28,32]. Both N. cepedianus and S. pacificus are large sluggish looking predators that consume fast moving animals such as marine mammals and teleosts [40–44]. The similarities in movement patterns, body size and diets suggest that the two species employ similar hunting strategies, where they use vertical oscillations and possibly olfactory cues, during no or low light conditions to ambush fast moving prey. Figure 4. Rose diagrams showing the angular changes of N. cepedianus for both day and night periods. Note differences in scale between the two periods. doi:10.1371/journal.pone.0015464.g004 In contrast to night-time behaviour, N. cepedianus appears to be less active during the day, and may have periods of resting and reduced foraging. The bottom associated movements of N. December 2010 \| Volume 5 \| Issue 12 \| e15464 December 2010 \| Volume 5 \| Issue 12 \| e15464 PLoS ONE \| www.plosone.org 7 Figure 5. Typical depth profiles for each of the different periods of the diel cycle. Data illustrates the profile over representative 1 h periods during the day (16:00–17:00 h), dusk (20:00–21:00 h), night (01:00–02:00 h) and dawn (05:00–06:00 h) periods for the N. cepedianus individual tracked in the Derwent Estuary on the 16th March 2007. doi:10.1371/journal.pone.0015464.g005 Fine-Scale Movement of Sevengill Sharks Fine-Scale Movement of Sevengill Sharks Figure 5. Typical depth profiles for each of the different periods of the diel cycle. Data illustrates the profile over representative 1 h periods during the day (16:00–17:00 h), dusk (20:00–21:00 h), night (01:00–02:00 h) and dawn (05:00–06:00 h) periods for the N. cepedianus individual tracked in the Derwent Estuary on the 16th March 2007. PLoS ONE \| www.plosone.org December 2010 \| Volume 5 \| Issue 12 \| e15464 Discussion Increased swimming rates, heightened activity levels, deep dives and depth holding behaviour (little or no movement to other depths) in relatively shallow water have all been observed immediately after release in blue Prionace glauca and shortfin mako Given the likely high rate of natural mortality inflicted by N. cepedianus on M. antarcticus in Norfolk Bay [21], movement into the water column at night could also be anti-predator behaviour, as fine-scale movement may mitigate predation risk [53–56]. Additionally, the higher occurrence of M. antarcticus individuals detected together in the VRAP during the day could be a result of temporary group formation. Group formation by juvenile shark species during the day and dispersal at night has been previously described, and associated to anti-predator behaviour [57,58]. As Norfolk Bay does not have much structure to provide shelter, December 2010 \| Volume 5 \| Issue 12 \| e15464 8 Fine-Scale Movement of Sevengill Sharks increased movement (including vertical) at night, when N. cepedianus is more active, and group formation during the day may be a tactic to avoid predation in a relatively featureless landscape. Landscape features are important factors influencing foraging locations and escape tactics for a range of prey and predator species [2,59]. However, despite only covering a small spatial scale, radio- acoustic positioning systems provide the triangulated position of tracked animals, which can elucidate fine-scale behaviours, compared to the presence/absence data provided over larger spatial scales by passive receivers [60]. Regardless, for large mobile predators such as sharks, this system will be more useful for species that have a focal point, i.e. a discreet area of intense activity to centre the study around. For example, white sharks congregating seasonally around seal colonies to hunt [10]. However, despite the inherent difficulties of obtaining fine-scale movement behaviour of large predators, empirical data is needed to complement other data sources such as dietary information and prey abundance, and to supplement predator-prey modelling studies [61]. The reason for the burst speed events recorded for N. cepedianus is unclear. However, we speculate that they were predation attempts. Due to the size of these animals and the absence of likely predators, it is unlikely that these burst speed events were escape behaviour. Although these could be a result of social interaction, there is a lack of evidence that these areas are used for mating or any other reproductive purpose [15]. Conclusion Although Norfolk Bay supports large seasonal aggregations of both N. cepedianus and M. antarcticus, the bay is large and the radio- acoustic positioning system only detects animals over a limited spatial range (,1 km2 diameter [10]). Therefore, the low percentage of resolved detections and the low number of individuals detected per day in the current study suggests that movements recorded in the VRAP area are only a small representation of the total movement in the Norfolk Bay. Supporting Information Table S1 N. cepedianus and M. antarcticus. Details of sharks tagged; date - date of tagging, TL - total length in cm, Days- number of days detected in VRAP, DP- detection period representing the time in days between first detection until last detection. P-values in bold are significant and % ratio indicates if they occurred more during the day (above 60%) or the night (below 60%). The 60% expected is based on 14.5 hours of daylight being 60% of the hours in the day. * denotes animals omitted from Chi-square x2 and t-test because they were not detected on more than one day. (DOCX) Burst speeds for N. cepedianus during the day took place on the substrate and were associated with periods of inactivity. Con- versely, burst speeds at night were all in the water column and had no association with inactivity periods. These results further suggest different foraging behaviours for day and night. One hypothesis is that during the day N. cepedianus cruise about on the substrate and may opportunistically attack prey they happen to encounter, while during the night they move throughout the water column actively searching for prey. Alternatively, N. cepedianus may hunt close to the substrate during periods of maximum light using mainly visual cues, and forage throughout the water column during periods of low light using mainly olfactory senses. Discussion If we consider these burst speed events to be predation attempts, for the two occasions that these events were followed by an extremely slow track (0.1 ms21 and 0.3 ms21), we could speculate that the shark was either resting after exerting energy on an unsuccessful chase, or that it may have secured the prey and was eating it. White sharks showed similar behaviour in which speeds of 7 m s21 were followed by a period of limited movement indicated that the shark may have caught prey [10]. References 10. Klimley AP, Le Boeuf BJ, Cantara KM, Richert JE, Davis SF, et al. (2001) Radio acoustic positioning as a tool for studying site-specific behavior of the white shark and other large marine species. Mar Biol 138: 429–446. 1. Lima SL (2002) Putting predators back into behavioural predator-prey interactions. Trends Ecol Evol 17: 70–75. 1. Lima SL (2002) Putting predators back into behavioural predator-prey interactions. Trends Ecol Evol 17: 70–75. 2. Heithaus MR, Wirsing AJ, Burkholder D, Thomson J, Dill LM (2009) Towards a predictive framework for predator risk effects: the interaction of landscape features and prey escape tactics. J Anim Ecol 78: 556–562. 2. Heithaus MR, Wirsing AJ, Burkholder D, Thomson J, Dill LM (2009) Towards a predictive framework for predator risk effects: the interaction of landscape features and prey escape tactics. J Anim Ecol 78: 556–562. 11. Klimley AP, Le Boeuf BJ, Cantara KM, Richert JE, Davis SF, et al. (2001) The hunting strategy of white sharks (Carcharodon carcharias) near a seal colony. Mar Biol 138: 617–636. 3. Sih A (1980) Optimal behavior - can foragers balance two conflicting demands. Science 210: 1041–1043. 3. Sih A (1980) Optimal behavior - can foragers balance two conflicting demands. Science 210: 1041–1043. 12. Papastamatiou YP, Lowe CG, Caselle JE, Friedlander AM (2009) Scale- dependent effects of habitat on movements and path structure of reef sharks at a predator-dominated atoll. Ecology 90: 996–1008. 4. Lima SL, Dill LM (1990) Behavioral decisions made under the risk of predation - a review and prospectus. Can J Zool 68: 619–640. 13. Heithaus MR, Marshall GJ, Buhleier B, Dill LM (2001) Employing crittercam to study habitat use and behaviour of large sharks. Mar Ecol Prog Ser 209: 307–310. 5. Brown JS, Laundre JW, Gurung M (1999) The ecology of fear: Optimal foraging, game theory, and trophic interactions. J Mammal 80: 385–399. 6. Wirsing AJ, Heithaus MR, Dill LM (2007) Living on the edge: dugongs prefer to forage in microhabitats that allow escape from rather than avoidance of predators. Anim Behav 74: 93–101. 14. Last PR, Stevens JD (2009) Sharks and rays of Australia 2nd edn. Melbourne: CSIRO Publishing. 656 p. p 7. Mitchell WA, Lima SL (2002) Predator-prey shell games: large-scale movement and its implications for decision-making by prey. Oikos 99: 249–259. 15. Author Contributions Conceived and designed the experiments: AB JMS. Performed the experiments: AB JMS KGA JDS BDB. Analyzed the data: AB KGB. Contributed reagents/materials/analysis tools: AB BDB JMS. Wrote the paper: JMS KGA JDS BDB. Acknowledgments We would like to thank E. Forbes and D. Jones for field and technical assistance with the VRAP, the active tracking crew J. Hulls, E. Butal, T. Alexander, J. Yick and A. Pender, the TAFI habitat mapping section for supplying mapping data, H. Pederson for plotting the active track in Eonfusion and Dr. J. Seymour for statistical advice. We would also like to thank V. Namms and his wife for designing the Fractal analysis package and Dr. C. Simpfendorfer for advice for improving the manuscript. Fine-Scale Movement of Sevengill Sharks Klimley PA, Beavers SC, Curtis TH, Jorgensen SJ (2002) Movements and swimming behavior of three species of sharks in La Jolla Canyon, California. Environ Biol Fishes 63: 117–135. 54. Johnson CJ, Parker KL, Heard DC, Gillingham MP (2002) Movement parameters of ungulates and scale-specific responses to the environment. J Anim Ecol 71: 225–235. 32. Hulbert LB, Sigler MF, Lunsford CR (2006) Depth and movement behaviour of the Pacific sleeper shark in the north-east Pacific Ocean. J Fish Biol 69: 406–425. 55. Anderson DP, Turner MG, Forester JD, Zhu J, Boyce MS, et al. (2005) Scale- dependent summer resource selection by reintroduced elk in Wisconsin, USA. J Wildlife Manage 60: 298–310. 33. Gardiner JM, Atema J (2010) The function of bilateral odor arrival time differences in olfactory orientation of sharks. Curr Biol 20: 1187–1191. y 34. Crocker GE, Gales NJ, Costa DP (2001) Swimming speed and foraging strategies of New Zealand sea lions (Phocarctos hookeri). J Zool Lond 254: 267–277. 34. Crocker GE, Gales NJ, Costa DP (2001) Swimming speed and foraging strategies of New Zealand sea lions (Phocarctos hookeri). J Zool Lond 254: 267–277. 35. Davis RW, Weihs D (2007) Locomotion in diving elephant seals: physical and h i l i l t i t Phil T R S B 362 2141 2150 56. Hebblewhite M, Merrill EH (2007) Multiscale wolf predation risk for elk: does migration reduce risk? Oecologia 152: 377–387. strategies of New Zealand sea lions (Phocarctos hookeri). J Zool Lond 254: 267–277. 35. Davis RW, Weihs D (2007) Locomotion in diving elephant seals: physical and physiological constraints. Phil Trans R Soc B 362: 2141–2150. 35. Davis RW, Weihs D (2007) Locomotion in diving elephant seals: physical and physiological constraints. Phil Trans R Soc B 362: 2141–2150. 57. Holland KN, Wetherbee BM, Peterson JD, Lowe CG (1993) Movements and distribution of hammerhead shark pups on their natal grounds. Copeia 2: 495–502. 36. West GJ, Stevens JD (2001) Archival tagging of school shark, Galeorhinus galeus, in Australia: Initial results. Environ Biol Fish 60: 283–298. 58. Heupel MR, Simpfendorfer CA (2005) Quantitative analysis of aggregation behaviour in juvenile blacktip sharks. Mar Biol 147: 1239–1249. 37. Nakano H, Matsunaga H, Okamoto H, Okazaki M (2003) Acoustic tracking of bigeye thresher shark Alopias supercilius in the eastern Pacific Ocean. Mar Ecol Prog Ser 265: 255–261. 59. Fine-Scale Movement of Sevengill Sharks 76 p. 47. Campana SE, Joyce W, Manning MJ (2009) Bycatch and discard mortality in commercially caught blue sharks Prionace glauca assessed using archival satellite pop-up tags. Mar Ecol Prog Ser 387: 241–253. p j p p 24. Heupel MR, Carlson JK, Simpfendorfer CA (2007) Shark nursery areas: concepts, definition, characterization and assumptions. Mar Ecol Prog Ser 337: 287–297. p p p g g 48. Holts DB, Bedford DW (1993) Horizontal and vertical movements of the shortfin Mako shark, Isurus oxyrinchus, in the southern California Bight. Aust J Mar Freshw Res 44: 901–909. 25. Jadot C, Donnay A, Acolas ML, Cornet Y, Anras MLB (2006) Activity patterns, home-range size, and habitat utilization of Sarpa salpa (Teleostei: Sparidae) in the Mediterranean Sea. ICES J Mar Sci 63: 128–139. 49. Tricas TC, Taylor LR, Naftel G (1981) Diel behaviour of the tiger shark, Galeocerdo Cuvier, at French Frigate Shoals, Hawaiian Islands. Copeia 4: 904–908. J 26. Nams VO (1996) The VFractal: A new estimator for fractal dimension of animal movement paths. Landscape Ecol 11: 289–297. 50. Sundstrom LF, Gruber SH, Clermont SM, Correia JPS, de Marignac JRC, et al. (2001) Review of elasmobranch behavioral studies using ultrasonic telemetry with special reference to the lemon shark, Negaprion brevirostris, around Bimini Islands, Bahamas. Environ Biol Fish 60: 225–250. 27. Nams VO (2005) Using animal movement paths to measure response to spatial scale. Oecologia 143: 179–188. g 28. Carey FG, Scharold JV (1990) Movements of blue sharks (Prionace glauca) in depth and course. Mar Biol 106: 329–342. 51. Sundstrom LF, Gruber SH (2002) Effects of capture and transmitter attachments on the swimming speed of large juvenile lemon sharks in the wild. J Fish Biol 61: 834–838. 29. Klimley AP (1993) Highly directional swimming by scalloped hammerhead sharks, Sphyrna lewini, and subsurface irradiance, temperature, bathymetry, and geomagnetic field. Mar Biol 117: 1–22. 52. Yick J The foraging and feeding ecology of Tasmanian coastal water elasmobranchs, Honours thesis, University of Tasmania. g g 30. Gunn JS, Stevens JD, Davis TLO, Norman BM (1999) Observations on the short-term movements and behaviour of whale sharks (Rhincodon typus) at Ningaloo Reef, Western Australia. Mar Biol 135: 553–559. 53. Johnson CJ, Parker KL, Heard DC, Gillingham MP (2002) A multiscale behavioral approach to understanding the movements of woodland caribou. Ecol Appl 12: 1840–1860. ( Ningaloo Reef, Western Australia. Mar Biol 135: 553–559. 31. Fine-Scale Movement of Sevengill Sharks 41. Yang MS, Page BN (1999) Diet of Pacific sleeper shark, Somniosus pacificus, in the Gulf of Alaska. Fish Bull 97: 406–409. 18. Barnett A, Abrantes K, Stevens JD, Yick J, Frusher SD, Semmens JM (2010) Predator-prey relationships and foraging ecology of a marine apex predator with a wide temperate distribution. Mar Ecol Prog Ser 416: 189–200. p y p g g gy p a wide temperate distribution. Mar Ecol Prog Ser 416: 189–200. 42. Ebert DA (2002) Ontogenetic changes in the diet of the sevengill shark (N. cepedianus). Mar Freshwat Res 53: 517–52. p g 19. Ebert DA (1991) Diet of the sevengill shark N. cepedianus in the temperate coastal waters of Southern Africa. S Afr J Mar Sci 11: 565–572. waters of Southern Africa. S Afr J Mar Sci 11: 565–572 43. Sigler MF, Hulbert LB, Lunsford CR, Thompson NH, Burek K, et al. (2006) Diet of Pacific sleeper shark, a potential Steller sea lion predator, in the north- east Pacific Ocean. J Fish Biol 69: 392–405. 20. Braccini JM (2008) Feeding ecology of two high-order predators from south- eastern Australia: the coastal broadnose and the deepwater sharpnose sevengill sharks. Mar Ecol Prog Ser 371: 273–284. 44. Yano K, Stevens JD, Compagno LJV (2007) Distribution, reproduction and feeding of the Greenland shark Somniosus (Somniosus) microcephalus, with notes on two other sleeper sharks, Somniosus (Somniosus) pacificus and Somniosus (Somniosus) antarcticus. J Fish Biol 70: 374–390. g 21. Barnett A, Redd KS, Frusher SD, Stevens JD, Semmens JM (2010) Non-lethal method to obtain stomach samples from a large marine predator and the use of DNA analysis to improve dietary information. J Exp Mar Biol Ecol 393: 188–192. 45. Strong WR (1996) Shape discrimination and visual predatory tactics in white sharks. In: Klimley PA, Ainley DG, eds. Great white sharks the biology of Carcharodon carcharias. San Diego: Academic press. pp 229–249. 22. Williams H, Schaap AH (1992) Preliminary results of a study into the incidental mortality of sharks in gill-nets in two Tasmanian shark nursery areas. Aust J Mar Freshwat Res 43: 237–250. 46. Laroche RK, Kock AA, Dill LM, Oosthuizen WH (2008) Running the gauntlet: a predator-prey game between sharks and two age classes of seals. Anim Behav 76: 1901–1917. 23. Stevens JD, West GJ (1997) Investigation of school and Gummy shark nursery areas in southeastern Australia. Australia: Fisheries Research and Development Corporation Project 93/061 final report, CSIRO Marine Research. References Barnett A, Stevens JD, Frusher SD, Semmens JM (2010) Seasonal occurrence and population structure of the broadnose sevengill shark (Notorynchus cepedianus) in coastal habitats of south east Tasmania. J Fish Biol 77(7): 1688–1701. 8. Meyer CG, Clark TB, Papastamatiou YP, Whitney NM, Holland KN (2009) Long-term movement patterns of tiger sharks Galeocerdo cuvier in Hawaii. Mar Ecol Prog Ser 381: 223–235. 16. Ebert DA (1989) Life History of the Sevengill Shark, N. cepedianus (Peron 1807), in two Northern California Bays. Calif Fish and Game 75: 102–112. in two Northern California Bays. Calif Fish and Game 75: 102 17. Lucifora LO, Menni RC, Escalante AH (2005) Reproduction, abundance and feeding habits of the broadnose sevengill shark N. cepedianus in north Patagonia, Argentina. Mar Ecol Prog Ser 289: 237–244. 9. Heithaus MR, Dill LM, Marshall GJ, Buhleier B (2002) Habitat use and foraging behaviour of tiger sharks (Galeocerdo cuvier) in a seagrass ecosystem. Mar Biol 140: 37–248. PLoS ONE \| www.plosone.org December 2010 \| Volume 5 \| Issue 12 \| e15464 PLoS ONE \| www.plosone.org 9 Fine-Scale Movement of Sevengill Sharks Macia A, Abrantes KGS, Paula J (2003) Thorn fish Terapon jarbua (Forska˚l) predation on juvenile white shrimp Penaeus indicus H. Milne Edwards and brown shrimp Metapenaeus monoceros (Fabricius): the effect of turbidity, prey density, substrate type and pneumatophore density. J Exp Mar Biol Ecol 291: 29–56. 38. Stokesbury MJW, Harvey-Clark C, Gallant J, Block BA, Myers RA (2005) Movement and environmental preferences of Greenland sharks (Somniosus microcephalus) electronically tagged in the St. Lawrence Estuary, Canada. Mar Biol 148: 159–165. 60. Heupel MR, Semmens JM, Hobday AJ (2006) Automated acoustic tracking of aquatic animals: scales, design and deployment of listening station arrays. Mar Freshwat Res 57: 1–13. 39. Andrews KS, Williams GD, Farrer D, Tolimieri N, Harvey CJ, et al. (2009) Diel activity patterns of sixgill sharks, Hexanchus griseus: the ups and downs of an apex predator. Anim Behav 78: 525–536. 61. Frid A, Burns J, Baker GG, Thorne RE (2009) Predicting synergistic effects of resources and predators on foraging decisions by juvenile Steller sea lions. Oecologia 158: 775–786. 40. Ebert DA (1991) Observations on the predatory behaviour of the sevengill sharks N. cepedianus. S Afr J Mar Sci 11: 455–465. PLoS ONE \| www.plosone.org December 2010 \| Volume 5 \| Issue 12 \| e15464 10
https://openalex.org/W2580085487	https://link.springer.com/content/pdf/10.1007%2Fs00330-016-4720-9.pdf	English	null	Diagnostic performance of imaging modalities in chronic pancreatitis: a systematic review and meta-analysis	European radiology	2,017	cc-by	17,300	Abstract Objectives Obtain summary estimates of sensitivity and spec- ificity for imaging modalities for chronic pancreatitis (CP) assessment. • EUS, ERCP, MRI and CT have high diagnostic sensitivity for chronic pancreatitis Methods A systematic search was performed in Cochrane Library, MEDLINE, Embase and CINAHL databases for studies evaluating imaging modalities for the diagnosis of CP up to September 2016. A bivariate random-effects model- ing was used to obtain summary estimates of sensitivity and specificity. • Diagnostic specificity is comparable for all imaging modalities • EUS and ERCP are outperformers and US has the lowest accuracy • The choice of imaging can be made based on clinical considerations • The choice of imaging can be made based on clinical considerations Results We included 43 studies evaluating 3460 patients. Sensitivity of endoscopic retrograde cholangiopancreatography (ERCP) (82%; 95%CI: 76%-87%) was significant higher than that of abdominal ultrasonography (US) (67%; 95%CI: 53%- 78%; P=0.018). The sensitivity estimates of endoscopic ultraso- nography (EUS), magnetic resonance imaging (MRI), and com- puted tomography (CT) were 81% (95%CI: 70%-89%), 78% (95%CI: 69%-85%), and 75% (95%CI: 66%-83%), respective- ly, and did not differ significantly from each other. Estimates of specificity were comparable for EUS (90%; 95%CI: 82%-95%), ERCP (94%; 95%CI: 87%-98%), CT (91%; 95% CI: 81%- 96%), MRI (96%; 95%CI: 90%-98%), and US (98%; 95%CI: 89%-100%). Results We included 43 studies evaluating 3460 patients. Sensitivity of endoscopic retrograde cholangiopancreatography (ERCP) (82%; 95%CI: 76%-87%) was significant higher than that of abdominal ultrasonography (US) (67%; 95%CI: 53%- 78%; P=0.018). The sensitivity estimates of endoscopic ultraso- nography (EUS), magnetic resonance imaging (MRI), and com- puted tomography (CT) were 81% (95%CI: 70%-89%), 78% (95%CI: 69%-85%), and 75% (95%CI: 66%-83%), respective- Keywords Chronic pancreatitis . Diagnostic imaging . Diagnostic accuracy . Meta-analysis Diagnostic accuracy . Meta-analysis Eur Radiol (2017) 27:3820–3844 DOI 10.1007/s00330-016-4720-9 GASTROINTESTINAL Diagnostic performance of imaging modalities in chronic pancreatitis: a systematic review and meta-analysis Y. Issa1 & M. A. Kempeneers1 & H. C. van Santvoort1 & T. L. Bollen2 & S. Bipat3 & M. A. Boermeester1 Y. Issa1 & M. A. Kempeneers1 & H. C. van Santvoort1 & T. L. Bollen2 & S. Bipat3 & 1 Received: 3 May 2016 /Revised: 20 September 2016 /Accepted: 16 December 2016 /Published online: 27 January 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com and ERCP are outperformers and US has the lowest accuracy. The choice of imaging modality can therefore be made based on invasiveness, local availability, experience and costs. Key Points and ERCP are outperformers and US has the lowest accuracy. The choice of imaging modality can therefore be made based on invasiveness, local availability, experience and costs. Key Points 1 Department of Surgery, Academic Medical Centre, Meibergdreef 9, 1100DD Amsterdam, The Netherlands * M. A. Boermeester m.a.boermeester@amc.uva.nl 2 Department of Radiology, St Antonius Ziekenhuis, Koekoekslaan 1, 3430EM Nieuwegein, The Netherlands 3 Department of Radiology, Academic Medical Centre, Meibergdreef 9, 1100DD Amsterdam, The Netherlands Introduction Chronic pancreatitis (CP) is a disabling inflammatory disease of the pancreas characterized by severe recurrent or continu- ous abdominal pain and considerable impact on the quality of life [1–4]. Patients with CP usually develop endocrine and exocrine insufficiency during the course of the disease as a result of the progressive loss of pancreatic parenchyma. Conclusions EUS, ERCP, MRI and CT all have comparable high diagnostic accuracy in the initial diagnosis of CP. EUS There is lack of international consensus regarding the ini- tial diagnosis of CP, particularly at its early stages. The diag- nosis is often made by a combination of clinical symptoms (e.g. abdominal pain, malabsorption, diabetes mellitus), pan- creatic function tests (e.g. fecal elastase-1) and morphological abnormalities seen on imaging (e.g. calcifications, ductal le- sions, pseudocysts) [5, 6]. Imaging plays a key role in the diagnosis and therapeutic management of patients with CP. The most frequently used imaging modalities for CP are en- doscopic ultrasonography (EUS), endoscopic retrograde Eur Radiol (2017) 27:3820–3844 3821 cholangiopancreatography (ERCP), magnetic resonance im- aging (MRI), computed tomography (CT) and ultrasonogra- phy (US). Data was extracted regarding the imaging characteristics: type of imaging modality, scoring criteria, technical features for each modality, and reported observer experience. Also data on the reference standard was extracted, such as clinical fol- low-up, surgery and histology. cholangiopancreatography (ERCP), magnetic resonance im- aging (MRI), computed tomography (CT) and ultrasonogra- phy (US). The aim of this meta-analysis was to determine the diag- nostic accuracy of imaging modalities for the initial diagnostic assessment of CP. The methodological quality of the included articles was assessed by the Quality Assessment of Diagnostic Accuracy Studies version 2 (QUADAS-2) tool [7]. The QUADAS-2 tool evaluates the risk of bias in four do- mains (patient selection, index test, reference standard, flow and timing) and the clinical applicability in the first three domains. Signaling questions were used to help assess the risk of bias and applicability. Possible answers were ‘yes’, ‘no’ or ‘unclear’ in which ‘yes’ indicates no risk of bias. In addition the GRADE scor- ing system for diagnostic tests was used, which assesses the quality of evidence for each imaging modality [8, 9]. Although the criteria are applicable to diagnostic test accuracy, the methods are less well established com- pared to interventional studies [10]. Search A search was performed in Cochrane Library, MEDLINE, EMBASE and CINAHL databases, without restrictions for publication date or language up to September 2016. The search included terms for chronic pancreatitis, EUS, ERCP, MR imaging, CT and US. For detailed search details, see Appendix Table 5. Selection of studies All search hits were screened on title and abstract and eligible articles on full text by two reviewers independently (YI and MAK). Disagreements were solved through discussion with a third reviewer (MAB). Studies were eligible when EUS, ERCP, MR imaging, CT or US was evaluated in patients with suspected CP. Duplicates, reviews, letters, case reports and book chapters were excluded. The remaining studies were potentially eligible and their full text was retrieved. To identify additional relevant studies, the reference lists of the included studies were checked manually. Studies were included if they met the following criteria: (1) sufficient data was reported to construct 2 × 2 tables (true positive, false positive, true nega- tive and false negative); (2) the imaging technique was com- pared with a reference standard (e.g. surgery, histology, fol- low-up). Exclusion criteria were: (1) evaluation of imaging techniques other than the aforementioned (e.g. PET-CT, EUS-FNA, EUS-elastography); (2) imaging techniques used for treatment of patients with CP (e.g. therapeutic ERCP, EUS-guided pseudocyst drainage); (3) in vitro studies; (4) studies that included less than five patients with CP; (5) stud- ies where no separate analysis were done for patients with CP; and (6) full-text articles that were not available or retrievable. MEDLINE N=4243 EMBASE N=6376 CINAHL N=300 COCHRANE N=192 Combined N=8123 Duplicates N=2988 Eligible studies N=277 Not eligible based on title and abstract N=7846 Included studies N=43 Excluded on full-text N=234 Reason for exclusion: - Article not available (n=5) - Different patient group (n=37) - In vitro (n=1) - Insufficient data (n=92) - Lack of reference standard (n=15) - Less than 5 patients (n=4) - Only data on sensitivity (n=33) - Other disease (n=14) - Other imaging modality (n=11) - Other type of article (n=22) Fig. 1 Flow chart Not eligible based on title and abstract N=7846 Introduction Two reviewers in- dependently (YI and MAK) assessed the QUADAS-2 and the GRADE scoring system and all disagreements were resolved by reaching consensus. Data analysis calculated. Sensitivity and specificity estimates, the positive predictive value and negative predictive values, and the accu- racy were calculated from the reconstructed contingency ta- bles. We used the I2 test with 95% confidence interval (95% CI) to quantify heterogeneity [11]. Mean logit sensitivity and specificity were acquired, and the anti-logit transformation was then obtained to calculate summary estimates of Overall diagnostic accuracy For each included study we constructed a 2 × 2 contingency table for each imaging modality. If diagnostic accuracy was compared between different observers, mean values were Table 1 Study characteristics of included studies Study Year Country P/R OE Modality Reference standard for CP diagnosis Adamek et al 2000 Germany P No MRCP/ERCP Histology (NA), FU (NA) Albashir et al 2010 USA R Yes EUS Histology (all) Alcaraz et al 2000 Spain P Yes MRCP Surgery (4), ERCP (70), PTC (7) Balci et al 2006 USA and Germany R No MRCP ePFT (all) Bolog et al 2004 Romania R No MRCP Surgery (NA), ERCP (NA), FU (NA) Brand et al 2000 Germany P No EUS Histology (all) Buscail et al 1995 France P No US/CT/ERCP /EUS Histology (7), morphological changes (i.e. Data extraction and critical appraisal Data was extracted systematically from the included studies by using a structured study record form. The following study design and patient characteristics were extracted: name of the first author, country of origin, year of publication, name of journal, study design, total number of patients included, num- ber of included patients with CP, median or mean age, the proportion of male patients, and the patient inclusion criteria. 3822 Eur Radiol (2017) 27:3820–3844 P prospective, R retrospective, OE observer experience reported, PTC percutaneous transhepatic cholangiogram, ePFT endoscopic pancreatic function test, FU follow-up, NA not available Data analysis In cases where a negative covariance between the logit sensitivity and logit specificity was obtained, summary receiver operating characteristic curve (sROC) were generated for each separate imaging modality. We used the z test to sensitivity and specificity with 95% CIs. Forest plots were made to visualize the sensitivity and specificity with the 95% CIs. Data analysis calcifications) and exocrine insufficiency (42) + FU (all) Catalano et al 1998 USA P No EUS ERCP + ePFT (all) Chong et al 2007 USA R Yes EUS Surgery (all) Conwell et al 2007 USA R Yes EUS ePFT (all) Dramaix et al 1980 France P No US/CT Surgery (NA), ERCP (NA) Fusari et al 2010 Italy P Yes CT/MRCP Biopsy (33), histology (7) Gebel et al 1985 Germany P No US/ERP Obduction (NA), Surgery (NA), FU (NA) Giovannini et al 1994 France P No EUS ERCP (all) Glasbrenner et al 2000 Germany P Yes EUS/ERCP Surgery (all) Gmelin et al 1981 Germany P No US/CT/ERCP Surgery (NA)+FU (NA) Hellerhoff et al 2002 Germany P Yes MRCP/sMRCP ERCP (35), surgery (4), FU (56) Imdahl et al 1999 Germany P Yes CT Histology (42), FU (6) Kremer et al 1977 Germany R No US Clinical diagnosis (338), ERCP, surgery, ePFT, angiography (NA) Lammer et al 1980 Germany R No ERCP/CT Surgery (31), angiography (16), clinical diagnosis (60) Lawson et al 1978 USA R Yes ERCP/US Surgery (25), FU (50) Lees et al 1979 UK P No US Surgery (36), ERCP (46) Lin et al 1989 Taiwan R No US/EUS Histology (26), CT (4), surgery+ERCP (3) Nattermann et al 1993 Germany P No EUS ERCP (94), FU (20) Pamos et al 1998 Spain P Yes MRCP ERCP (all) Parsi et al 2008 USA R Yes ERCP FU (all) Pistolesi et al 1981 Italy P No CT Surgery (all) Pungpapong et al 2007 USA P Yes EUS Clinical history, lab data, ERCP/CT/MRI and/or surgical pathology (all) Pungpapong et al 2007 USA P Yes MRCP/EUS ERCP (48), surgery (9), FU (57) Rudowicz-Pietruszewska et al 2002 Poland P No MRCP ERCP (all) Sai et al 2008 Japan P Yes sMRCP ERCP (all) Savarino et al 1980 Italy R No CT Surgery (NA), calcifications (NA), clinical and lab data (NA) Scarabino et al 1989 Italy R No ERCP, US, CT Combination of CT, US and ERCP (all) Schlaudraff et al 2008 USA and Germany P Yes MRCP/sMRCP Clinical history, laboratory, radiology (≥2 methods) (all) Stevens et al 2009 USA P Yes EUS ePFT (all) Sverko et al 2011 Croatia R No MRCP Histology (all) Swobodnik et al 1983 Germany P No US/CT/ERCP FU (59), surgery (22) Tox et al 2007 Germany R Yes EUS Surgery (79), FU (92) Trikudanathan et al 2016 USA R YES EUS Histology (all) Triller et al 1975 Switzerland P No ERCP Surgery (14), autopsy (1), FU (9) Wiersema et al 1993 USA P No EUS/ERCP FU (51), ePFT (16) Zhang et al 2003 USA R No MRCP US (12), CT (11), ERCP (6) Zuccaro et al 2009 USA R No MRCP/sMRCP ePFT (all) P prospective, R retrospective, OE observer experience reported, PTC percutaneous transhepatic cholangiogram, ePFT endoscopic pancreatic function t t FU f ll NA t il bl Table 1 Study characteristics of included studies 3823 Eur Radiol (2017) 27:3820–3844 model [12]. Study and patient characteristics Study characteristics, including the reference standard for the diagnosis of CP for each included study, are listed in Table 1. The 43 included studies were published between 1975 and 2016; 26 studies were prospective and 23 studies were pub- lished after the year 2000. A total of 3460 patients were eval- uated, of which 1242 patients were diagnosed with CP [14–56]. The age of the patients ranged from 36 to 65 years, with a median of 50% male. Criteria for selection of patients were those with suspected pancreatic disease or patients with suspected CP. Patient characteristics are depicted in Table 2. Heterogeneity exploration Heterogeneity exploration The following factors were incorporated in the bivariate mod- el and we evaluated the effect on the sensitivity and specific- ity, and cause of heterogeneity for all imaging modalities ac- cording to the QUADAS-2 tool: clear description of criteria for bias (low bias versus high bias or unclear) for (a) patient selection, (b) criteria for the index test used, (c) sufficient description and verification with the reference standard, and (d) the flow and timing. Data analysis 2 Summary of study quality (QUADAS-2) 0% 20% 40% 60% 80% 100% PATIENT SELECTION INDEX TEST REFERENCE STANDARD FLOW AND TIMING Proportion of studies with low, high or unclear RISK of BIAS 0% 20% 40% 60% 80% 100% Proportion of studies with low, high, or unclear CONCERNS regarding APPLICABILITY Low High Unclear Fig. 2 Summary of study quality (QUADAS-2) Fig. 2 Summary of study quality (QUADAS-2) 0% 20% 40% 60% 80% 100% Proportion of studies with low, high or unclear RISK of BIAS 0% 20% 40% 60% 80% 100% Proportion of studies with low, high, or unclear CONCERNS regarding APPLICABILITY evaluate differences in sensitivity and specificity between the five imaging modalities. A p value of less than 0.05 indicated a statistically significant difference. Study selection The initial search resulted in 11,111 hits, of which 2988 du- plicates were removed, resulting in a total of 8123 titles and abstracts that were screened for eligibility. The full text of 277 articles was retrieved; 43 of these articles fulfilled the inclu- sion criteria. See Appendix Table 6 for the excluded articles. Figure 1 shows the flow chart of the search. Data analysis Summary estimates of sensitivity and specificity, including 95% CI, were obtained by using a random-effects Table 2 Patient characteristics of included studies Table 2 Patient characteristics of included studies Study Nr pts Age Male (%) Nr pts CP Patient selection Adamek et al 124 55 61% 57 Suspected pancreatic mass (clinical presentation, lab, US) Albashir et al 23 43* 57% 19 Suspected chronic pancreatitis (clinical presentation) Alcaraz et al 81 65** 31% 8 Suspected pancreatobiliary disease (clinical presentation, US) Balci et al 30 48* 17% 11 Suspected early CP (clinical presentation) Bolog et al 103 57* 43% 15 Suspected pancreatobiliary disease (US/CT or clinical presentatio Brand et al 115 61* 59% 24 Suspected focal pancreatic lesion (US/CT/ERCP or lab/tumour markers) Buscail et al 62 50* 79% 44 Suspected chronic pancreatitis (clinical presentation, lab, imaging) Catalano et al 80 51* 40% 38 Non-alcoholic recurrent acute pancreatitis (3–11 episodes) Chong et al 71 45* 46% 64 Suspected chronic pancreatitis (clinical presentation) Conwell et al 56 44* 45% 38 Suspected chronic pancreatitis (clinical presentation) Dramaix et al 50 52* 66% 18 Suspected pancreatic disease (clinical presentation) Fusari et al 40 62* 55% 8 Suspected pancreatic mass (clinical presentation and US) Gebel et al US: 56, ERP: 45 NA NA US: 22, ERP: 16 Suspected pancreatic disease (clinical presentation) Giovannini et al 26 NA NA 17 Suspected pancreatobiliary disease (clinical presentation, imaging/lab) Glasbrenner et al 85 NA NA 41 Suspected pancreatic mass (clinical presentation, US/CT) Gmelin et al 41 54* 68% 19 Suspected pancreatic disease (clinical presentation) Hellerhoff et al 95 NA NA 26 Suspected pancreatic disease (clinical presentation) Imdahl et al 48 58* 60% 12 Suspected pancreatic disease (clinical presentation) Kremer et al 446 NA NA 61 Suspected pancreatic disease (clinical presentation) Lammer et al 107 NA NA 39 Suspected pancreatic disease (clinical presentation) Lawson et al 75 NA NA 26 Suspected pancreatic disease (clinical presentation) Lees et al 98 NA NA 20 Suspected pancreatic disease (clinical presentation) Lin et al 33 47* 58% 7 Suspected pancreatic disease (clinical presentation) Nattermann et al 114 53* 67% 51 Suspected pancreatic disease (clinical presentation) Pamos et al 41 64* 59% 5 Suspected pancreatobiliary disease (clinical presentation) Parsi et al 35 46 46% 24 Suspected chronic pancreatitis (clinical presentation) Pistolesi et al 100 NA NA 31 Suspected pancreatic disease (clinical presentation) Pungpapong et al 79 50 35% 38 Suspected chronic pancreatitis (clinical presentation) Pungpapong et al 99 55** 41% 40 Suspected chronic pancreatitis (clinical presentation) Rudowicz-Pietruszewska et al 88 52* 64% 9 Suspected pancreatobiliary disease (clinical presentation, lab, US/CT) Sai et al 28 36* NA 16 Mild chronic pancreatitis (ERCP) Savarino et al 108 47 67% 59 Suspected pancreatic disease (clinical presentation) Scarabino et al 63 44 63% 12 Suspected of biliopancreatic disease (clinical presentation) Schlaudraff et al 62 NA NA 9 Suspected chronic pancreatitis (clinical presentation) Stevens et al 100 NA 38% 41 Suspected chronic pancreatitis (clinical presentation) Sverko et al 29 44** 52% 14 Suspected pancreatic disease (clinical presentation) Swobodnik et al 81 49* 52% 27 Suspected pancreatic disease (clinical presentation) Tox et al 171 61* NA 65 Suspected pancreatic disease (clinical presentation) Trikudanathan et al 68 39* 18% 56 Total pancreatectomy for non-calcific chronic pancreatitis Triller et al 24 52* 83% 11 Suspected pancreatobiliary disease (clinical presentation) Wiersema et al 67 45* 20% 30 Suspected pancreatobiliary disease (clinical presentation) Zhang et al 44 50* 30% 24 Suspected early or mild chronic pancreatitis (clinical presentation, US/CT/ERCP) Zuccaro et al 69 43* 35% 28 Suspected chronic pancreatitis (clinical presentation) NA not available 3824 Eur Radiol (2017) 27:3820–3844 0% 20% 40% 60% 80% 100% PATIENT SELECTION INDEX TEST REFERENCE STANDARD FLOW AND TIMING Proportion of studies with low, high or unclear RISK of BIAS 0% 20% 40% 60% 80% 100% Proportion of studies with low, high, or unclear CONCERNS regarding APPLICABILITY Low High Unclear Fig. Fig. 3 Forest plot for sensitivity and specificity Head to head comparison A head to head comparison was performed in studies that compared the diagnostic accuracy of two or more imaging modalities. Heterogeneity was quantified by I2 test, with 95% CI. The random-effects (I2 > 25%) and fixed effects (I2 ≤25%) models were used to obtain summary estimates of sensitivity and specificity, and compared with one another by a paired z test. The risk of bias, assessed by QUADAS-2, was low in 28% of the studies and high in 19% of the studies. The concerns about applicability were low in 30% of the studies and high in 40% of the studies. The QUADAS-2 characteristics for each domain are depicted in Fig. 2 and outlined for each study in Appendix Table 7. The quality of evidence for all five imaging modalities according to the GRADE scoring system was very For data analysis, Review Manager (RevMan, version 5.3. Copenhagen: The Cochrane Collaboration, 2014) and SAS (version 9.3; SAS Institute, Cary, NC) were used. We adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [13]. Table 3 Estimated overall sensitivity, specificity and heterogeneity according to imaging modality Modality N studies N patients Sensitivity (95% CI) Specificity (95% CI) Heterogeneity (I2) EUS 16 1249 81% (70–89%) 90% (82–95%) 82%/73% ERCP 11 742 82% (76–87%) 94% (87–98%) 39%/67% MRCP 14 933 78% (69–85%) 96% (90–98%) 59%/65% CT 10 700 75% (66–83%) 91% (81–96%) 50%/71% US 10 1005 67% (53–78%) 98% (89–100%) 40%/93% Random effects model Modality N studies N patients Sensitivity (95% CI) Specificity (95% CI) Heterogeneity (I2) EUS 16 1249 81% (70–89%) 90% (82–95%) 82%/73% ERCP 11 742 82% (76–87%) 94% (87–98%) 39%/67% MRCP 14 933 78% (69–85%) 96% (90–98%) 59%/65% CT 10 700 75% (66–83%) 91% (81–96%) 50%/71% US 10 1005 67% (53–78%) 98% (89–100%) 40%/93% Random effects model 3825 Eur Radiol (2017) 27:3820–3844 Forest plot: EUS Forest plot: ERCP Forest plot: MRCP Forest plot CT Forest plot: US for sensitivity and specificity 3826 Eur Radiol (2017) 27:3820–3844 low. The GRADE scores for each imaging modality and char- acteristics for each study are outlined in Appendix Tables 8 and 9. Heterogeneity exploration The bivariate model for heterogeneity exploration showed that the factor ‘flow and timing’ was significantly associated with a higher sensitivity of US (p = 0.01). ‘Description and verifi- cation with the reference standard’ was significantly associat- ed with a higher specificity for MRCP (p = 0.0002). Head to head comparison The head to head comparison of US versus ERCP compar- ison yields a sensitivity of 57% (49–65%) versus 78% (71– 85%) (p < 0.001); and a specificity of 94% (74–99%) versus EUS was the most frequently evaluated imaging modality; 16 studies including 1249 patients [15, 19–23, 27, 28, 36, 37, 41, 42, 48, 51, 53, 56]. ERCP was studied in 11 studies in- cluding 742 patients [14, 20, 26, 28, 29, 33, 34, 39, 46, 50, 52]; MRCP, including secretin-enhanced MRCP, was evaluat- ed in 14 studies including 933 patients [14, 16–18, 25, 30, 38, 42–44, 47, 49, 54, 55]; CT in 10 studies including 700 patients [20, 24, 25, 29, 31, 33, 40, 45, 46, 50] and abdominal US in 10 studies which included 1005 patients [20, 24, 26, 29, 32, 34–36, 46, 50]. The imaging characteristics for each study and modality in an individual study are listed in Appendix Table 11. Three of the 43 articles reported about complications of the imaging modality used; these were complications relat- ed to ERCP (being post-ERCP pancreatitis) with a mean com- plication rate of 4% [14, 20, 28]. EUS ERCP EUS ERCP CT Fig. 4 Receiver operator curves (ROC) Overall diagnostic accuracy Analyses for summary estimates of sensitivity and specificity were done for EUS, ERCP, MRI, CT and US (Table 3). Figures 3 and 4 show sensitivity and specificity of individual studies in forest plots and in receiver operator curves (ROC), respectively. A negative covariance between the logit sensi- tivity and logit specificity was not obtained; therefore, no sROC for MRI and US could be drawn. The summary esti- mate of sensitivity for EUS, ERCP, MRCP, CT and US was 81%, 82%, 78%, 75% and 67%, respectively. The summary estimate of specificity for EUS, ERCP, MRCP, CT and US was 90%, 94%, 96%, 91% and 98%, respectively. Sensitivity of ERCP was significant higher than sensitivity of US (p = 0.018). Other pairwise comparisons of sensitivity between imaging modalities revealed no significant diffe- rence. Specificity did not differ significantly among all modalities (Table 3). Sensitivity and specificity values for each study are listed in Appendix Table 10. CT CT CT Fig. 4 Receiver operator curves (ROC) Head to head comparison Six head to head comparisons were performed (Table 4). The specificity of ERCP and EUS, and the sensitivity of ERCP, EUS and CT in the summary estimates of the head to head studies were significantly higher as compared with US. Fig. 4 Receiver operator curves (ROC) Eur Radiol (2017) 27:3820–3844 3827 98% (89–100%) (p = 0.003), respectively [20, 26, 29, 34, 46, 50]. The comparison between US and CT yields a sensitivity of 58% (49–66%) and 77% (68–83%) (p = 0.002), respective- ly [20, 24, 29, 46, 50]. And finally, the comparison of EUS versus US comparison yields a sensitivity of 90% (82–98%) versus 63% (49–76%) (p = 0.001); and a specificity of 100% versus 91% (82–99%) (p = 0.04), respectively [20, 36]. There were no significant differences in the sensitivity and specific- ity estimates between ERCP and EUS [20, 28, 53], MRCP and sMRCP [30, 47, 55] or ERCP and CT [20, 29, 33, 46, 50]. The heterogeneity (I2) between US and ERCP (>25%) was higher (>25%) than in the other comparisons (I2 ≤25%). and 78%, respectively, in the present meta-analyses. The European Society of Radiology (ESR) is developing the ESR iGuide, a clinical decision support system for European imaging referral guidelines, covering various clinical scenari- os, indications and recommendations (www.esriguide.org) [61–63]. The results from the present systematic review may be useful to incorporate in that system. We excluded three studies where sensitivity and specificity data were provided, but it was not possible to extract sufficient data to produce 2 × 2 tables and calculate the diagnostic ac- curacy values, because only the sensitivity and specificity es- timates were given [64–66]. In the study by Wang et al., esti- mates of sensitivity and specificity for EUS, ERCP and US were in line with the present results; the sensitivity of MR imaging and CT, however, were much lower (66% and 61%) [66]. The studies by Clave et al. and Orti et al. showed a lower sensitivity of ERCP (62% and 70%, respectively) compared to present results (82%) [64, 65]. Discussion EUS, ERCP, MRI and CTall have comparable high diagnostic accuracy in the initial diagnosis of chronic pancreatitis. EUS and ERCP are outperformers and US has the lowest accuracy. The choice of imaging modality can therefore be made on the basis of invasiveness, local availability, experience and costs. Several recent guidelines [57–59] advocate the use of EUS, MRCP or CT for the diagnosis of CP, although summary estimates of their accuracy, thus far, were lacking. There is one guideline from Germany on CP that has reported sensi- tivity and specificity regarding EUS, ERCP, MRCP and US, although not for CT [60]. In this guideline 14 studies were selected, reporting ranges rather than pooling the data on sen- sitivity and specificity estimates. This method resulted in re- sults slightly different from those in the present meta-analyses. For example the guideline reports a sensitivity of 70–80% for ERCP and 88% for MRI versus summary estimates of 82% EUS, ERCP, MRI and CTall have comparable high diagnostic accuracy in the initial diagnosis of chronic pancreatitis. EUS and ERCP are outperformers and US has the lowest accuracy. The choice of imaging modality can therefore be made on the basis of invasiveness, local availability, experience and costs. The risk of missing important studies was minimized by performing a search in four major databases by two reviewers independently, without setting any restrictions for language and publication date. However, this systematic review has some limitations. The heterogeneity of the pooled studies was moderate to high in all analyses (between 39% and 93%). However, in the head to head comparison analyses, the heterogeneity was low in most comparisons (<25%). Furthermore, the heterogeneity of the reference standards used in the studies could have influenced individual study results. Surgery, histology and long-term follow-up of patients are reliable methods. Some reference standards, such as the use of endoscopic pancreatic function test (ePFT) for establishing the diagnosis of CP, could have resulted in under- or Several recent guidelines [57–59] advocate the use of EUS, MRCP or CT for the diagnosis of CP, although summary estimates of their accuracy, thus far, were lacking. There is one guideline from Germany on CP that has reported sensi- tivity and specificity regarding EUS, ERCP, MRCP and US, although not for CT [60]. In this guideline 14 studies were selected, reporting ranges rather than pooling the data on sen- sitivity and specificity estimates. Table 4 Head to head comparison Sensitivity: US vs ERCP (p < 0.001), US vs CT (p = 0.002), EUS vs US (p = 0.001) Discussion This method resulted in re- sults slightly different from those in the present meta-analyses. For example the guideline reports a sensitivity of 70–80% for ERCP and 88% for MRI versus summary estimates of 82% Table 4 Head to head comparison Comparison N studies N patients Modality Sensitivity (95% CI) Specificity (95% CI) US vs ERCPa 6 423 US 57% (49–65%) 94% (74–99%) ERCP 78% (71–85%) 98% (89–100%) US vs CTb 5 297 US 58% (49–66%) 77% (71–83%) CT 77% (68–83%) 82% (74–88%) CT vs ERCPb 5 354 CT 75% (67–82%) 86% (81–90%) ERCP 84% (77–89%) 90% (85–93%) EUS vs ERCPb 3 214 EUS 88% (80–93%) 85% (76–91%) ERCP 86% (78–91%) 92% (85–96%) MRCP vs sMRCPb 3 226 MRCP 62% (49–73%) 94% (89–97%) sMRCP 68% (56–79%) 91% (85–94%) EUS vs USb 2 95 EUS 90% (82–98%) 100% US 63% (49–76%) 91% (82–99%) Sensitivity: US vs ERCP (p < 0.001), US vs CT (p = 0.002), EUS vs US (p = 0.001) Specificity: US vs ERCP (p = 0.003), EUS vs US (p = 0.04) a Random effects model b Fixed effects model 3828 Eur Radiol (2017) 27:3820–3844 overestimation of the sensitivity and specificity. In addition, the diagnosis of CP and the criteria used are different in dif- ferent stages of the disease (e.g. absence of calcifications in the early phase of the disease). Another limitation was that our analyses included imaging studies and imaging protocols per- formed over the last 40 years in different centres with inherent variations in techniques and equipment. Especially in the last decade the quality of some imaging modalities (e.g. MRCP and CT) has improved considerably. Also there were concerns about the quality of the available evidence, as assessed by QUADAS-2 and the GRADE scoring system. (e.g. PD dilatation and strictures) and slight changes of the pancreatic parenchyma and side branches, which can be at- tributed to early signs CP (i.e. atrophy, side branch ectasia) compared to CT [74]. Early diagnosis can also lead to a timely start of treatment, which has been associated with improved long-term outcome [75]. Nevertheless, for very early CP this association needs to be established in further research, such as the ESCAPE trial, evaluating the effect of early intervention in patients with CP [76]. Discussion As diagnostic sensitivity of CT and MRCP is not significantly lower than that of ERCP and EUS, and specificity is comparable, non-invasive modalities except for US are a likely first choice in patients with suspected pancreatic disease including chronic pancreatitis. The highest scores for accuracy in the diagnosis of CP were found for EUS and ERCP, but these are invasive techniques. ERCP has a relatively high risk of complications, such as post- ERCP pancreatitis (1.6–15.7%, mean complication rate of 4%) and is nowadays only used for therapeutic purposes (e.g. stenting of pancreatic duct) [67–69]. To date, diagnostic ERCP is largely replaced by EUS and the cross-sectional im- aging modalities CT and MRCP. Acknowledgements The scientific guarantor of this publication is M.A. Boermeester. The authors of this manuscript declare relationships with the following companies: Baxter, Acelity/KCI, Ipsen, Mylan, Johnson & Johnson, Bard. The authors of this manuscript declare no relationships with any companies whose products or services may be related to the subject matter of the article. The authors state that this work has not received any funding. Two of the authors have significant statis- tical expertise (SB, MAB). Institutional review board approval was not required because of the nature of the study (a systematic review). Acknowledgements The scientific guarantor of this publication is M.A. Boermeester. The authors of this manuscript declare relationships with the following companies: Baxter, Acelity/KCI, Ipsen, Mylan, Johnson & Johnson, Bard. The authors of this manuscript declare no relationships with any companies whose products or services may be related to the subject matter of the article. The authors state that this work has not received any funding. Two of the authors have significant statis- tical expertise (SB, MAB). Institutional review board approval was not required because of the nature of the study (a systematic review). It has been suggested that CT is better in detecting paren- chymal calcifications and intraductal calcifications compared to MRCP [70–73]. On the other hand, MRCP is more often able to detect significant abnormalities of the pancreatic duct MeSH Medical Subject Headings APPENDIX Table 5 Search terms Table 5 Search terms MeSH terms All Fields Chronic pancreatitis Pancreatitis, chronic [MeSH] Chronic pancreatitis [All Fields] AND EUS Endosonography [MeSH] EUS [All Fields] OR ERCP Cholangiopancreatography, Endoscopic Retrograde [MeSH] Endoscopic Retrograde Cholangiopancreatograp* [All Fields] OR ERCP [All Fields] OR MRCP Magnetic Resonance Imaging [MeSH] OR Cholangiopancreatography, Magnetic Resonance [MeSH] Magnetic resonance imaging [All Fields] OR MRI [All Fields] OR MRCP [All Fields] OR Magnetic Resonance Cholangio* [All Fields] OR sMRCP Magnetic Resonance Imaging [All Fields] AND secretin [All Fields] OR sMRI [All Fields] OR CT Tomography, X-Ray Computed [MeSH] (Tomography [All Fields] AND x-ray [All Fields] AND computed [All Fields]) OR Computed Tomography [All Fields]) OR CT scan* [All Fields] OR US Ultrasonography [MeSH] Ultrasonogra* [All Fields] OR ultrasound [All Fields] MeSH Medical Subject Headings EUS [All Fields] 3829 Eur Radiol (2017) 27:3820–3844 Author Year Journal Borsukov et al 2001 Ross Gastroenterol Zh Diad'kin et al 2013 Vestnik rentgenologii i radiologii Dotsenko et al 1985 Vrach Delo Rosch et al 1989 Z Gastroenterologie Suzdalev et al 1992 Likars'ka sprava Agarwal et al 2008 GIE Brailski et al 1989 Vutr Boles Brailski et al 1984 Vutr Boles Brimiene et al 2011 Medicina Carlucci et al 1989 HPB Surgery Chowdhury et al 2005 Pancreas Cotton et al 1980 Radiology DelMaschio et al 1991 Radiology Erturk et al 2006 Am J Gastroenterol Frick et al 1982 Gastrointest Rad Gheonea et al 2013 BMC Gastroenterology Goodale et al 1981 Ann Surg Hanninen et al 2002 Radiology Hatano et al 1998 Nippon rinsho J Hocke et al 2008 Dtsch Med Wochenschr Hocke et al 2006 WJG Hocke et al 2012 Z Gastroenterologie Huang et al 2011 J Dig dis Imbriaco et al 2006 Radiol Med Kawai et al 2012 Eur J Rad Kim et al 2007 J MRI Kursawa et al 1991 Radiol Diagn Lu et al 2013 Acad J Sec Mil Med University Lutz et al 1975 Klin Wschr Morris-Stiff et al 2009 J Pancreas Papp et al 1978 Wiener klin Wchnschrft Pomerri et al 1991 Radiologia Med Rosch et al 2000 Am J Gastroenterol Sandrasegaran et al 2013 AJR Sendler et al 2000 World J Surg Sugumar et al 2011 Gut Testoni et al 1981 Acta Endoscopica Tiushin et al 2003 Voprosy onkologii Varadarajulu et al 2007 GIE Viceconte et al 1980 Ann ital chir Yamada et al 2010 Abdom Imaging Zhu et al 2013 PLOS one Bhutani et al 2009 Pancreas Akisik et al 2013 AJR Alempijević et al 2005 Vojnosanit Pregl Alpern et al 1985 Radiology Ardelean et al 2014 Med Ultrason Ardengh et al 2011 GIE Ascunce et al 2010 Surg End Baert et al 1977 Radiologe Balci et al 2010 J MRI Beliao et al 2012 Eur J Rad Bender et al 1999 Invest Rad Bhatt et al 2005 Indian J Rad Imag Ass Bonanno et al 1994 Giorn Ital End Dig Bruhlmann et al 1976 RoFo Caletti et al 1982 British j Surgery Cao 1989 Zhonghua yi xue za zhi Cappeliez et al 2000 Radiology Chang et al 2010 GIE Author Year Journal Reason for exclusion Borsukov et al 2001 Ross Gastroenterol Zh Article not available Diad'kin et al 2013 Vestnik rentgenologii i radiologii Article not available Dotsenko et al 1985 Vrach Delo Article not available Rosch et al 1989 Z Gastroenterologie Article not available Suzdalev et al 1992 Likars'ka sprava Article not available Agarwal et al 2008 GIE Exclusive patient group Brailski et al 1989 Vutr Boles Exclusive patient group Brailski et al 1984 Vutr Boles Exclusive patient group Brimiene et al 2011 Medicina Exclusive patient group Carlucci et al 1989 HPB Surgery Exclusive patient group Chowdhury et al 2005 Pancreas Exclusive patient group Cotton et al 1980 Radiology Exclusive patient group DelMaschio et al 1991 Radiology Exclusive patient group Erturk et al 2006 Am J Gastroenterol Exclusive patient group Frick et al 1982 Gastrointest Rad Exclusive patient group Gheonea et al 2013 BMC Gastroenterology Exclusive patient group Goodale et al 1981 Ann Surg Exclusive patient group Hanninen et al 2002 Radiology Exclusive patient group Hatano et al 1998 Nippon rinsho J Exclusive patient group Hocke et al 2008 Dtsch Med Wochenschr Exclusive patient group Hocke et al 2006 WJG Exclusive patient group Hocke et al 2012 Z Gastroenterologie Exclusive patient group Huang et al 2011 J Dig dis Exclusive patient group Imbriaco et al 2006 Radiol Med Exclusive patient group Kawai et al 2012 Eur J Rad Exclusive patient group Kim et al 2007 J MRI Exclusive patient group Kursawa et al 1991 Radiol Diagn Exclusive patient group Lu et al 2013 Acad J Sec Mil Med University Exclusive patient group Lutz et al 1975 Klin Wschr Exclusive patient group Morris-Stiff et al 2009 J Pancreas Exclusive patient group Papp et al 1978 Wiener klin Wchnschrft Exclusive patient group Pomerri et al 1991 Radiologia Med Exclusive patient group Rosch et al 2000 Am J Gastroenterol Exclusive patient group Sandrasegaran et al 2013 AJR Exclusive patient group Sendler et al 2000 World J Surg Exclusive patient group Sugumar et al 2011 Gut Exclusive patient group Testoni et al 1981 Acta Endoscopica Exclusive patient group Tiushin et al 2003 Voprosy onkologii Exclusive patient group Varadarajulu et al 2007 GIE Exclusive patient group Viceconte et al 1980 Ann ital chir Exclusive patient group Yamada et al 2010 Abdom Imaging Exclusive patient group Zhu et al 2013 PLOS one Exclusive patient group Bhutani et al 2009 Pancreas In vitro Akisik et al 2013 AJR No diagnostic values for CP Alempijević et al 2005 Vojnosanit Pregl No diagnostic values for CP Alpern et al 1985 Radiology No diagnostic values for CP Ardelean et al 2014 Med Ultrason No diagnostic values for CP Ardengh et al 2011 GIE No diagnostic values for CP Ascunce et al 2010 Surg End No diagnostic values for CP Baert et al 1977 Radiologe No diagnostic values for CP Balci et al 2010 J MRI No diagnostic values for CP Beliao et al 2012 Eur J Rad No diagnostic values for CP Bender et al 1999 Invest Rad No diagnostic values for CP Bhatt et al 2005 Indian J Rad Imag Ass No diagnostic values for CP Bonanno et al 1994 Giorn Ital End Dig No diagnostic values for CP Bruhlmann et al 1976 RoFo No diagnostic values for CP Caletti et al 1982 British j Surgery No diagnostic values for CP Cao 1989 Zhonghua yi xue za zhi No diagnostic values for CP Cappeliez et al 2000 Radiology No diagnostic values for CP Chang et al 2010 GIE No diagnostic values for CP Table 6 Excluded articles based on full text Eur Radiol (2017) 27:3820–3844 3830 Author Year Journal Cohen et al 2014 Dig Dis Sci Concia et al 2014 Invest Rad Dale et al 1979 Electromedica Das et al 2008 GIE Delbeke et al 1999 J Nucl Med Dite et al 1982 Vnitrni Lekarstvi Dronamraju et al 2016 Ann Gastroenterol D’Souza et al 2015 Dig Dis Sci Eitner et al 1979 Dtsch Zeitschr Verdauungs- und Stoffwechselkrankheiten Eloubeidi et al 2013 Pancreas Ergul et al 2014 Rev Esp Med Nucl Im Mol Ferrucci et al 1979 Radiology Foley et al 1980 Gastrointest Rad Fontana et al 1976 Gut Foster et al 1984 BMJ Gardner et al 2014 Pancreas Gincul et al 2014 Endoscopy Gowland et al 1981 Lancet Grant et al 1981 J Am Osteopathic Ass Harada et al 1977 Gastroenterologica Jap He et al 2014 Pancreas Hoki et al 2009 J Gastroenterol Hollerbach et al 1994 Med Klinik Horii et al 1982 Jap J Gastroenterol Johnson et al 1999 Radiology Jones et al 1988 Clin Radiol Kamisawa et al 2007 J Gastroenterol Kersting et al 2009 Gastroenterology Kitano et al 2004 Gut Laghi et al 1998 Chirurgia Leblanc et al 2014 Pancreas Leblanc et al 2014 Pancreas Li et al 2001 Zhongguo yi xue ke xue Loginov et al 1976 Sovetskaya Meditsina Lopez et al 2002 Radiology Manfredi 2000 Radiology Modder et al 1979 RoFo Montori et al 1979 Min Diet Gastroent Napoleon et al 2010 Endoscopy Novis et al 1976 S Afr Med J Ohtsubo et al 2008 Gastroenterolog Endoscopy Orlikov et al 2007 Ter Arkh Park et al 2008 The Korean J Gastroenter Petersein et al 2002 RoFo Pezzelli et al 2013 Pancreas Pomerri et al 1987 Radiologia Med Rickes et al 2002 Scand J Gastroenterol Rosenberger et al 1979 MMW Russell et al 1978 Gut Sahai et al 1998 GIE Sainani et al 2009 AJG Sica et al 2002 J MRI Sica et al 1999 Radiology Songur et al 2000 Digest Endoscopy Stevens et al 2010 WJG Struve et al 1982 Diagnostik & Intensivtherapie Sun et al 2010 Acad J Sec Mil Med University Tamura et al 2006 Radiology Tellez-Avila et al 2014 WJG Author Year Journal Reason for exclusion Cohen et al 2014 Dig Dis Sci No diagnostic values for CP Concia et al 2014 Invest Rad No diagnostic values for CP Dale et al 1979 Electromedica No diagnostic values for CP Das et al 2008 GIE No diagnostic values for CP Delbeke et al 1999 J Nucl Med No diagnostic values for CP Dite et al 1982 Vnitrni Lekarstvi No diagnostic values for CP Dronamraju et al 2016 Ann Gastroenterol No diagnostic values for CP D’Souza et al 2015 Dig Dis Sci No diagnostic values for CP Eitner et al 1979 Dtsch Zeitschr Verdauungs- und Stoffwechselkrankheiten No diagnostic values for CP Eloubeidi et al 2013 Pancreas No diagnostic values for CP Ergul et al 2014 Rev Esp Med Nucl Im Mol No diagnostic values for CP Ferrucci et al 1979 Radiology No diagnostic values for CP Foley et al 1980 Gastrointest Rad No diagnostic values for CP Fontana et al 1976 Gut No diagnostic values for CP Foster et al 1984 BMJ No diagnostic values for CP Gardner et al 2014 Pancreas No diagnostic values for CP Gincul et al 2014 Endoscopy No diagnostic values for CP Gowland et al 1981 Lancet No diagnostic values for CP Grant et al 1981 J Am Osteopathic Ass No diagnostic values for CP Harada et al 1977 Gastroenterologica Jap No diagnostic values for CP He et al 2014 Pancreas No diagnostic values for CP Hoki et al 2009 J Gastroenterol No diagnostic values for CP Hollerbach et al 1994 Med Klinik No diagnostic values for CP Horii et al 1982 Jap J Gastroenterol No diagnostic values for CP Johnson et al 1999 Radiology No diagnostic values for CP Jones et al 1988 Clin Radiol No diagnostic values for CP Kamisawa et al 2007 J Gastroenterol No diagnostic values for CP Kersting et al 2009 Gastroenterology No diagnostic values for CP Kitano et al 2004 Gut No diagnostic values for CP Laghi et al 1998 Chirurgia No diagnostic values for CP Leblanc et al 2014 Pancreas No diagnostic values for CP Leblanc et al 2014 Pancreas No diagnostic values for CP Li et al 2001 Zhongguo yi xue ke xue No diagnostic values for CP Loginov et al 1976 Sovetskaya Meditsina No diagnostic values for CP Lopez et al 2002 Radiology No diagnostic values for CP Manfredi 2000 Radiology No diagnostic values for CP Modder et al 1979 RoFo No diagnostic values for CP Montori et al 1979 Min Diet Gastroent No diagnostic values for CP Napoleon et al 2010 Endoscopy No diagnostic values for CP Novis et al 1976 S Afr Med J No diagnostic values for CP Ohtsubo et al 2008 Gastroenterolog Endoscopy No diagnostic values for CP Orlikov et al 2007 Ter Arkh No diagnostic values for CP Park et al 2008 The Korean J Gastroenter No diagnostic values for CP Petersein et al 2002 RoFo No diagnostic values for CP Pezzelli et al 2013 Pancreas No diagnostic values for CP Pomerri et al 1987 Radiologia Med No diagnostic values for CP Rickes et al 2002 Scand J Gastroenterol No diagnostic values for CP Rosenberger et al 1979 MMW No diagnostic values for CP Russell et al 1978 Gut No diagnostic values for CP Sahai et al 1998 GIE No diagnostic values for CP Sainani et al 2009 AJG No diagnostic values for CP Sica et al 2002 J MRI No diagnostic values for CP Sica et al 1999 Radiology No diagnostic values for CP Songur et al 2000 Digest Endoscopy No diagnostic values for CP Stevens et al 2010 WJG No diagnostic values for CP Struve et al 1982 Diagnostik & Intensivtherapie No diagnostic values for CP Sun et al 2010 Acad J Sec Mil Med University No diagnostic values for CP Tamura et al 2006 Radiology No diagnostic values for CP Tellez-Avila et al 2014 WJG No diagnostic values for CP Table 6 (continued) No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP No diagnostic values for CP Author Year Journal Tirkes et al 2016 J MRI Trikudanathan et al 2015 Am J Gastroenterol Tripathi et al 2002 Indian J Gastroenterol Tympner et al 1979 Leber Magen Darm Tympner et al 1977 Verhand Dtschen Gesellschaft fur I Medizin Uskudar et al 2009 Pancreas Valentini et al 1981 Endoscopy Varghese et al 2002 Clin Radiol Wang et al 2013 WJG Wierzbicka-Paczos et al 1998 Gastroenterologia Polska Wierzbicka-Paczos et al 1999 Polski Merk Lek Will et al 2010 Ultraschall Med Zaheer et al 2014 Eur J Rad Bian et al 2014 Chin J Radiol Braganza et al 1978 Clin Radiol Gillams et al 2007 Eur J Rad Helmberger et al 2000 RoFo Hernandez Garces et al 2004 J Pancreas Ho et al 2006 Clin Gastroenterol Hep Kalmar et al 1984 Southern Medical J Kalmin et al 2011 Can J Gastroenterol Kaufman et al 1989 GIE Kumon et al 2012 GIE Manfredi et al 1998 La Rad Medica Novotny et al 2000 Bratisl Lek Listy Ponette et al 1976 Acta Gastro-Enterol Belgica Sanyal et al 2012 AJR Yoshimoto et al 1980 Jap J Gastroenterol Grossjohann et al 2010 Scand J Gastroenterol Sood et al 1992 Indian J Gastroenterol Zhi et al 2002 Chin J Digestive Dis Zhong et al 2003 WJG Ainsworth et al 2003 Endoscopy Bastid et al 1995 J d'Echographie et de Med par Ultr Campisi et al 2009 Clin Radiol Dancygier et al 1986 Scand J Gastroenterol Giday et al 2011 J Gastr Hep Guarita et al 1982 AMB Guo et al 2003 Chin J Digestive Dis Kahl et al 2002 GIE Kim et al 2001 AJR Kolmannskog et al 1981 Acta Radiologica Lackner et al 1980 RoFo Lawson 1978 Radiology Manfredi 2002 Radiology Mao et al 2011 WCJD Nakashio 1992 Acta medica Noguchi et al 1985 Gastroenterolog Endoscopy Propp 2011 Vestnik khirurgii imeni Rossi et al 1996 Giorn Ital End Dig Sahel et al 1976 Acta Endoscopica Seicean et al 2010 Ultraschall Med Sildiroglu 1985 Rontgenpraxis Singh et al 1993 Indian J Rad Imag Sivak et al 1986 Scand J Gastroenterol Stabile Ianora et al 2013 Recenti Prog Med Stevens et al 2008 Dig Dis Sci 3831 Eur Radiol (2017) 27:3820–3844 Author Year Journal Reason for exclusion Tirkes et al 2016 J MRI No diagnostic values for CP Trikudanathan et al 2015 Am J Gastroenterol No diagnostic values for CP Tripathi et al 2002 Indian J Gastroenterol No diagnostic values for CP Tympner et al 1979 Leber Magen Darm No diagnostic values for CP Tympner et al 1977 Verhand Dtschen Gesellschaft fur Innere Medizin No diagnostic values for CP Uskudar et al 2009 Pancreas No diagnostic values for CP Valentini et al 1981 Endoscopy No diagnostic values for CP Varghese et al 2002 Clin Radiol No diagnostic values for CP Wang et al 2013 WJG No diagnostic values for CP Wierzbicka-Paczos et al 1998 Gastroenterologia Polska No diagnostic values for CP Wierzbicka-Paczos et al 1999 Polski Merk Lek No diagnostic values for CP Will et al 2010 Ultraschall Med No diagnostic values for CP Zaheer et al 2014 Eur J Rad No diagnostic values for CP Bian et al 2014 Chin J Radiol No reference standard Braganza et al 1978 Clin Radiol No reference standard Gillams et al 2007 Eur J Rad No reference standard Helmberger et al 2000 RoFo No reference standard Hernandez Garces et al 2004 J Pancreas No reference standard Ho et al 2006 Clin Gastroenterol Hep No reference standard Kalmar et al 1984 Southern Medical J No reference standard Kalmin et al 2011 Can J Gastroenterol No reference standard Kaufman et al 1989 GIE No reference standard Kumon et al 2012 GIE No reference standard Manfredi et al 1998 La Rad Medica No reference standard Novotny et al 2000 Bratisl Lek Listy No reference standard Ponette et al 1976 Acta Gastro-Enterol Belgica No reference standard Sanyal et al 2012 AJR No reference standard Yoshimoto et al 1980 Jap J Gastroenterol No reference standard Grossjohann et al 2010 Scand J Gastroenterol Not enough patients Sood et al 1992 Indian J Gastroenterol Not enough patients Zhi et al 2002 Chin J Digestive Dis Not enough patients Zhong et al 2003 WJG Not enough patients Ainsworth et al 2003 Endoscopy Only sensitivity reported Bastid et al 1995 J d'Echographie et de Med par Ultrasons Only sensitivity reported Campisi et al 2009 Clin Radiol Only sensitivity reported Dancygier et al 1986 Scand J Gastroenterol Only sensitivity reported Giday et al 2011 J Gastr Hep Only sensitivity reported Guarita et al 1982 AMB Only sensitivity reported Guo et al 2003 Chin J Digestive Dis Only sensitivity reported Kahl et al 2002 GIE Only sensitivity reported Kim et al 2001 AJR Only sensitivity reported Kolmannskog et al 1981 Acta Radiologica Only sensitivity reported Lackner et al 1980 RoFo Only sensitivity reported Lawson 1978 Radiology Only sensitivity reported Manfredi 2002 Radiology Only sensitivity reported Mao et al 2011 WCJD Only sensitivity reported Nakashio 1992 Acta medica Only sensitivity reported Noguchi et al 1985 Gastroenterolog Endoscopy Only sensitivity reported Propp 2011 Vestnik khirurgii imeni Only sensitivity reported Rossi et al 1996 Giorn Ital End Dig Only sensitivity reported Sahel et al 1976 Acta Endoscopica Only sensitivity reported Seicean et al 2010 Ultraschall Med Only sensitivity reported Sildiroglu 1985 Rontgenpraxis Only sensitivity reported Singh et al 1993 Indian J Rad Imag Only sensitivity reported Sivak et al 1986 Scand J Gastroenterol Only sensitivity reported Eur Radiol (2017) 27:3820–3844 3832 Author Year Journal Reason for exclusion Stevens et al 2010 Dig Dis Sci Only sensitivity reported Triller et al 1983 Computertomographie Only sensitivity reported Uchida et al 1997 Jap J Clin Radiology Only sensitivity reported Vitale et al 2009 The Am Surgeon Only sensitivity reported Wang et al 2009 J Gastr Hep Only sensitivity reported Wu et al 2006 World Chin J Dig Only sensitivity reported Yanling et al 2001 Chinese J Gastroenterol Only sensitivity reported Zhou et al 1993 Zhonghua nei ke za zhi Only sensitivity reported Aithal et al 2002 GIE Other disease Doust et al 1976 Radiology Other disease Engjom et al 2015 Scan J Gastroenterol Other disease Huang et al 2009 Acad J Sec Mil Med University Other disease Kushnir et al 2011 GIE Other disease Lai et al 2004 Endoscopy Other disease Leblanc et al 2014 Pancreas Other disease Matos et al 2001 GIE Other disease Mosler et al 2012 Dig Dis Sci Other disease Novis et al 2010 Rev Colegio Brasileiro Cirurg Other disease Rana et al 2012 J Gastr Hep Other disease Ranney et al 2012 GIE Other disease Sainani et al 2015 Pancreas Other disease Soto et al 2005 Radiology Other disease Akisik et al 2009 Radiology Other imaging modality Cherian et al 2010 HPB Surgery Other imaging modality Glaser et al 1994 Int J Pancreatology Other imaging modality Glaser et al 1989 Scand J Gastroenterol Other imaging modality Glaser et al 1985 Ultraschall Med Other imaging modality Hocke et al 2007 Pancreas Other imaging modality Kumon et al 2010 GIE Other imaging modality Saftoiu et al 2008 GIE Other imaging modality Sreenarasimhaiah 2008 J Clin Gastroenterol Other imaging modality Tummula et al 2013 Clin Transl Gastroenterol Other imaging modality Uehara et al 2011 J Gastr Hep Other imaging modality Abdalla et al 2012 Gastroenterolgy Other type of article Arsac et al 1981 Med Chirurgie Digest Other type of article Ashida et al 2011 J Gastr Hep Other type of article Chvatalova et al 2012 Pancreatology Other type of article Czako et al 2007 J Gastroenterol Other type of article Gupta et al 2013 JIMSA Other type of article Heverhagen et al 2007 RoFo Other type of article Kasugai et al 1982 Stomach and intestine Other type of article Kent et al 2008 Pancreas Other type of article Markwardt et al 1980 Radiologia Diagn Other type of article Munoz et al 2010 Rev Med de Chile Other type of article Musunuri et al 2015 Ind J Gastroenterol Other type of article Quinn et al 2012 Gut Other type of article Romagnuolo et al 2012 GIE Other type of article Sherman et al 2012 GIE Other type of article Shibukawa et al 2015 Dig Endos Other type of article Stevens et al 2008 Pancreas Other type of article Takahashi et al 2014 AJR Other type of article Trus et al 1998 Probl Gen Surg Other type of article Vadrot et al 1981 Med Chirurgie Digest Other type of article Zaruba et al 2012 Pancreatology Other type of article Zhang et al 2011 J Gastr Hep Other type of article Table 6 (continued) 3833 Eur Radiol (2017) 27:3820–3844 Table 7 QUADAS-2 characteristics for each study Study Bias Applicability Patient selection Index test Reference standard Flow and timing Patient selection Index test Reference stan Adamek et al Low Low Low Low Unclear Unclear Low Albashir et al Low Low Low Low Low Low Low Alcaraz et al Low Low Low Low High Unclear Low Balci et al Low Low Unclear Low Low Low Unclear Bolog et al Low Unclear Low Low High Unclear Low Brand et al Low Low Low High High Low Low Buscail et al Low Unclear Low Low High Unclear Low Catalano et al Unclear Low Unclear Low Low Low Low Chong et al Low Low Low Low Low Low Low Conwell et al Low Low High Low Low Low Unclear Dramaix et al Low Low Low Low Low Unclear Low Fusari et al Unclear Low Low Low High Low Low Gebel et al Low Low Low High Low Unclear Low Giovannini et al Unclear Unclear Low Low High Unclear Unclear Glasbrenner et al Low Low Low Low High Low Low Gmelin et al Low Low Low Low Low High Unclear Hellerhoff et al Low Low Low Low Low Low Low Imdahl et al Low Low Unclear Low Low Unclear Low Kremer et al High Unclear Unclear High High Unclear Low Lammer et al Low Low Unclear Low Low Unclear Unclear Lawson et al Low Low Unclear Low Low Low Unclear Lees et al Low Low Low High Low High Low Lin et al High Unclear Low Low Low Unclear Low Nattermann et al Unclear Low Low Low High Unclear Low Pamos et al Low Low Low Low High Unclear Low Parsi et al Low Low Low Low Low Low Low Pistolesi et al Unclear Low Low Low Low Low Low Pungpapong et al Low Low Low Low Low Low Low Pungpapong et al Low Unclear Unclear Low Low Low Low Rudowicz Pietr-uszewska et al Low Unclear Low Low High Unclear Unclear Sai et al High Low Low Low Low Low Low Savarino et al Unclear Low Low Low Low Low Low Scarabino et al Low Unclear Unclear Low High Unclear Unclear Schlaudraff et al Low Unclear Low Low Low Low Low Stevens et al Low Low Unclear Low Low Low Unclear Sverko et al Unclear Unclear Low Low Low Unclear Low Swobodnik et al Low Low Low Low Low Low Low Tox et al Low Unclear Unclear Low Low Low Low Trikudanathan et al Unclear Low Unclear Low High Low Low Triller et al Unclear Low Unclear Low Unclear Unclear Low Wiersema et al Unclear Low Unclear Low High Low Unclear Zhang et al High Unclear High Low Low Unclear High Zuccaro et al Unclear Low Unclear Low Low Low Unclear Table 7 QUADAS-2 characteristics for each study Eur Radiol (2017) 27:3820–3844 3834 EUS Outcome №of studies (№of patients) Study design Factors that may decrease quality of evidence Effect per 1000 patients tested Quality of evidence Risk of bias Indirectness Inconsistency Imprecision Publication bias Pre-test probability of 47.2% True positives 16 (1249) Cohort & case-control Serious a Serious b Very serious c Very serious d NA 387 (335 to 425) ⨁◯◯◯VERY LOW False negatives 85 (47 to 137) True negatives 16 (1249) Cohort & case-control Serious a Serious b Serious c Serious d NA 480 (438 to 502) ⨁◯◯◯VERY LOW False positives 48 (26 to 90) ERCP Outcome №of studies (№of patients) Study design Factors that may decrease quality of evidence Effect per 1000 patients tested Quality of evidence Risk of bias Indirectness Inconsistency Imprecision Publication bias Pre-test probability of 42.6% True positives 11 (742) Cohort & case-control Not serious e Serious f Serious g Serious h NA 349 (324 to 371) ⨁◯◯◯ Very low False negatives 77 (55 to 102) True negatives 11 (742) Cohort & case-control Not serious e Serious f Serious g Serious h NA 540 (499 to 563) ⨁◯◯◯ Very low False positives 34 (11 to 75) MRCP Outcome №of studies (№of patients) Study design Factors that may decrease quality of evidence Effect per 1000 patients tested Quality of evidence Risk of bias Indirectness Inconsistency Imprecision Publication bias Pre-test probability of 28.9% True positives 14 (933) Cohort & case-control- type studies Serious i Serious j Serious k Very serious l NA 225 (199 to 246) ⨁◯◯◯ Very low False negatives 64 (43 to 90) True negatives 14 (933) Cohort & case-control- type studies Serious i Serious j Serious k Not serious l NA 683 (640 to 697) ⨁◯◯◯ Very low False positives 28 (14 to 71) CT Outcome №of studies (№of patients) Study design Factors that may decrease quality of evidence Effect per 1000 patients tested Quality of evidence Risk of bias Indirectness Inconsistency Imprecision Publication bias Pre-test probability of 38.4% True positives 10 (700) Cohort & case-control Serious m Serious n Serious o Very serious p NA 288 (253 to 319) ⨁◯◯◯ Very low False negatives 96 (65 to 131) True negatives 10 (700) Cohort & case-control Serious m Serious n Serious o Serious p NA 561 (499 to 591) ⨁◯◯◯ Very low False positives 55 (25 to 117) US Outcome №of studies (№of patients) Study design Factors that may decrease quality of evidence Effect per 1000 patients tested Quality of evidence Risk of bias Indirectness Inconsistency Imprecision Publication bias pre-test probability of 25.7% Eur Radiol (2017) 27:3820–3844 3835 nued) 10 (1005) Cohort & case-control Serious q Serious r Serious s Very serious t NA 172 (136 to 200) ⨁◯◯◯ Very low 85 (57 to 121) 10 (1005) Cohort & case-control Serious q Serious r Very serious s Serious t NA 728 (661 to 743) ⨁◯◯◯ Very low 15 (0 to 82) e ased on QUADAS-2 risk of bias; 7 studies not serious, 9 studies serious ased on QUADAS-2 applicability; 7 studies not serious, 9 studies serious based on heterogeneity and visual inspection CIs ased on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) ADAS-2 risk of bias: 8 studies not serious, 3 studies serious ADAS-2 applicability: 6 studies not serious, 5 studies serious rogeneity and visual inspection CIs y numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) ased on QUADAS-2 risk of bias; 7 studies not serious, 5 studies serious, 1 study very serious ased on QUADAS-2 applicability; 6 studies not serious, 8 studies serious based on heterogeneity and visual inspection CIs ased on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) based on QUADAS-2 risk of bias; 5 studies not serious, 5 studies serious ased on QUADAS-2 applicability; 6 studies not serious, 4 studies serious based on heterogeneity and visual inspection CIs ased on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) based on QUADAS-2 risk of bias; 6 studies not serious, 3 studies serious, 1 study very serious ased on QUADAS-2 applicability; 5 studies not serious, 5 studies serious based on heterogeneity and visual inspection CIs ased on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) ur Radiol (2017) 27:3820–3844 383 ued) 10 (1005) Cohort & case-control Serious q Serious r Serious s Very serious t NA 172 (136 to 200) ⨁◯◯◯ Very low 85 (57 to 121) 10 (1005) Cohort & case-control Serious q Serious r Very serious s Serious t NA 728 (661 to 743) ⨁◯◯◯ Very low 15 (0 to 82) ased on QUADAS-2 risk of bias; 7 studies not serious, 9 studies serious ased on QUADAS-2 applicability; 7 studies not serious, 9 studies serious based on heterogeneity and visual inspection CIs sed on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) DAS-2 risk of bias: 8 studies not serious, 3 studies serious DAS-2 applicability: 6 studies not serious, 5 studies serious ogeneity and visual inspection CIs numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) sed on QUADAS-2 risk of bias; 7 studies not serious, 5 studies serious, 1 study very serious sed on QUADAS-2 applicability; 6 studies not serious, 8 studies serious based on heterogeneity and visual inspection CIs sed on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) ased on QUADAS-2 risk of bias; 5 studies not serious, 5 studies serious ased on QUADAS-2 applicability; 6 studies not serious, 4 studies serious based on heterogeneity and visual inspection CIs sed on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) ased on QUADAS-2 risk of bias; 6 studies not serious, 3 studies serious, 1 study very serious sed on QUADAS-2 applicability; 5 studies not serious, 5 studies serious based on heterogeneity and visual inspection CIs sed on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) ( ) Table 8 (continued) True positives 10 (1005) Cohort & case-control Serious q Serious r Serious s Very serious t NA False negatives True negatives 10 (1005) Cohort & case-control Serious q Serious r Very serious s Serious t NA False positives NA not available a Risk of bias: based on QUADAS-2 risk of bias; 7 studies not serious, 9 studies serious b Indirectness: based on QUADAS-2 applicability; 7 studies not serious, 9 studies serious c Inconsistency: based on heterogeneity and visual inspection CIs d Imprecision: based on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) e Based on QUADAS-2 risk of bias: 8 studies not serious, 3 studies serious f Based on QUADAS-2 applicability: 6 studies not serious, 5 studies serious g Based on heterogeneity and visual inspection CIs h Based on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) i Risk of bias: based on QUADAS-2 risk of bias; 7 studies not serious, 5 studies serious, 1 study very serious j Indirectness: based on QUADAS-2 applicability; 6 studies not serious, 8 studies serious k Inconsistency: based on heterogeneity and visual inspection CIs l Imprecision: based on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) m Risk of bias: based on QUADAS-2 risk of bias; 5 studies not serious, 5 studies serious n Indirectness: based on QUADAS-2 applicability; 6 studies not serious, 4 studies serious o Inconsistency: based on heterogeneity and visual inspection CIs p Imprecision: based on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) q Risk of bias: based on QUADAS-2 risk of bias; 6 studies not serious, 3 studies serious, 1 study very serious r Indirectness: based on QUADAS-2 applicability; 5 studies not serious, 5 studies serious s Inconsistency: based on heterogeneity and visual inspection CIs t Imprecision: based on study numbers and CIs of summary estimate (CIs 0–10 = not serious, 11–15 = serious, more than 15 = very serious) Eur Radiol (2017) 27:3820–3844 3836 Table 9 GRADE characteristics for each study Modality Name first author Risk of bias Indirectness Inconsistency Imprecision Publication bias EUS Albashir et al Low Low Sensitivity: very serious Specificity: serious Sensitivity: very serious Specificity: serious Not assessed Brand et al Serious Serious Buscail et al Serious Serious Catalano et al Low Low Chong et al Low Low Conwell et al Serious Serious Giovannini et al Serious Serious Glasbrenner et al Low Low Lin et al Serious Serious Nattermann et al Low Serious Pungpapong et al Low Low Pungpapong et al Low Low Stevens et al Serious Serious Tox et al Serious Low Trikudanathan et al Serious Serious Wiersema et al Serious Serious ERCP Adamek et al Low Low Sensitivity: serious Specificity: serious Sensitivity: serious Specificity: serious Not assessed Buscail et al Low Serious Gebel et al Low Low Glasbrenner et al Low Low Gmelin et al Low Serious Lammer et al Serious Serious Lawson et al Low Low Parsi et al Low Low Scarabino et al Serious Serious Swobodnik et al Low Low Triller et al Serious Serious MRCP Adamek et al Low Low Sensitivity: serious Specificity: serious Sensitivity: very serious Specificity: not serious Not assessed Alcaraz et al Low Serious Balci et al Serious Serious Bolog et al Serious Serious Fusari et al Low Low Hellerhoff et al Low Low Pamos et al Low Serious Pungpapong et al Low Low Rudowicz-Pietruszewska Serious Serious Sai et al Serious Low Schlaudraff et al Low Low Sverko et al Serious Serious Zhang et al Very serious Serious Zuccaro et al Serious Serious CT Buscail et al Low Serious Sensitivity: serious Specificity: serious Sensitivity: very serious Specificity: serious Not assessed Dramaix et al Low Low Fusari et al Low Low Gmelin et al Low Serious Imdahl et al Serious Low Lammer et al Serious Serious Pistolesi et al Low Low Savarino et al Serious Low Scarabino et al Serious Serious Swobodnik et al Low Low US Buscail et al Low Serious Sensitivity: serious Specificity: very serious Sensitivity: very serious Specificity: serious Not assessed Dramaix et al Low Low Gebel et al Low Low Gmelin et al Low Serious Kremer et al Very serious Serious Lawson et al Low Low Lees et al Serious Low Lin et al Serious Serious Scarabino et al Serious Serious Swobodnik et al Low Low Table 9 GRADE characteristics for each study Sensitivity: serious Specificity: serious Sensitivity: very serious Specificity: not serious Not assessed Sensitivity: serious Specificity: serious Eur Radiol (2017) 27:3820–3844 3837 Table 10 Diagnostic characteristics for each study Study Sensitivity Specificity Accuracy PPV NPV TP TN FP FN Adamek et al MRCP: 88%, ERCP: 90% MRCP: 94%. EUS [All Fields] ERCP: 91% MRCP: 91% ERCP: 90% MRCP: 93%, ERCP:90% MRCP: 90%, ERCP: 91% MRCP:50 ERCP: 51 MRCP: 63 ERCP: 61 MRCP: 4 ERCP: 6 MRCP: 7 ERCP: 6 Albashir et al 84% 100% 87% 100% 57% 16 4 0 3 Alcaraz et al 50% 99% 94% 80% 95% 4 72 1 4 Balci et al 82% 63% 70% 56% 86% 9 12 7 2 Bolog et al 90% 98% 95% 90% 98% 14 86 2 1 Brand et al 42% 96% 84% 71% 86% 10 87 4 14 Buscail et al US: 58%,CT: 75%, ERCP: 74%, EUS: 88% US: 75%, CT: 95%, ERCP: 100%, EUS: 100% US: 65%, CT: 81%, ERCP: 82%, EUS: 92% US: 87%, CT: 97%, ERCP: 100%, EUS: 100% US: 44%, CT: 61%, ERCP: 62%, EUS: 78% US: 26, CT: 33, ERC: 33, EUS: 39 US: 14, CT: 17, ERCP: 18, EUS: 18 US: 4, CT: 1, ERCP: 0, EUS: 0 US: 18, CT: 11, ERCP: 11, EUS: 5 Catalano et al 84% 98% 91% 97% 87% 32 41 1 6 Chong et al 83% 80% 83% 98% 69% 53 5 1 11 Conwell et al 26% 100% 50% 100% 39% 10 18 0 28 Dramaix et al CT: 60%, US: 60% CT: 100% US: 95% CT: 86% US: 82% CT: 100%, US: 90% CT: 76%, US: 76% CT: 11, US: 11 CT: 32, US: 30 CT: 0, US: 2 CT: 7, US: 7 Fusari et al CT: 88%, MRI: 88% CT: 100%, MRI:100%, CT: 98%, MRI: 98% CT: 100%, MRI: 100% CT: 97%, MRI: 97% MRI: 7, CT: 7 MRI: 32, CT: 32 MRI: 0, CT: 0 MRI: 1, CT: 1 Gebel et al US: 82%, ERP: 56% US: 97%, ERP: 97% US: 91%, ERP: 82% US:95%, ERP: 90% US: 89%, ERP: 80% US: 18, ERP: 9 US: 33, ERP: 28 US: 1, ERP: 1 US: 4, ERP: 7 Giovannini et al 94% 56% 81% 80% 83% 16 5 4 1 Glasbrenner et al EUS: 93%, ERCP: 88% EUS: 78%, ERCP: 82% EUS: 85%, ERCP: 85% EUS: 79%, ERCP: 82% EUS: 92%, ERCP: 88% EUS: 38, ERCP: 36 EUS: 34, ERCP: 36 EUS: 10, ERCP: 8 EUS: 3, ERCP: 5 Gmelin et al US: 68%, CT: 84%, ERCP: 89% US: 100%, CT: 91%, ERCP: 91% US: 85%, CT: 89%, ERCP: 90% US: 100%, CT: 89%, ERCP: 89% US: 79%, CT: 87%, ERCP: 91% US: 13, CT: 16, ERCP: 17 US: 22, CT: 20, ERCP: 20 US: 0, CT: 2, ERCP: 2 US: 6, CT: 3. EUS [All Fields] ERCP: 2 Hellerhoff et al MRI: 77%, sMRI: 89% MRI: 100%, sMRI:100% MRI 94%, sMRI: 97% MRI: 100%, sMRI: 100% MRI: 92%, sMRI: 96% MRI: 20, sMRI: 23 MRI: 69, sMRI: 69 MRI: 0, sMRI: 0 MRI: 6, sMRI: 3 Imdahl et al 58% 91% 83% 70% 85% 7 33 3 5 Kremer et al 67% 99% 94% 89% 95% 42 378 5 21 Lammer et al ERCP: 85%, CT: 64% ERCP: 97%, CT: 85% ERCP: 93%, CT: 78% ERCP: 94%, CT: 71% ERCP: 92%, CT: 81% ERCP: 33, CT: 25 ERCP: 66, CT: 58 ERCP: 2, CT: 10 ERCP: 6, CT: 14 Lawson et al US: 38%, ERCP: 73% US: 100%, ERCP: 98% US: 79%, ERCP: 98% US: 100%, ERCP: 95% US: 75%, ERCP: 87% US: 10, ERCP: 19 US: 49, ERCP: 48 US: 0, ERCP: 1 US: 16, ERCP: 7 Lees et al 100% 97% 98% 91% 100% 20 76 2 0 Lin et al US: 86%, EUS: 100% US: 100%, EUS: 100% US: 97%, EUS: 100% US: 100%, EUS: 100% US: 96%, EUS: 100% US: 6, EUS: 7 US: 26, EUS: 26 US: 0, EUS: 0 US: 1, EUS: 0 Nattermann et al 98% 57% 75% 65% 97% 50 36 27 1 Pamos et al 80% 100% 98% 100% 97% 4 36 0 1 Parsi et al 71% 91% 77% 94% 59% 17 10 1 7 Pistolesi et al 58% 81% 74% 58% 81% 18 56 13 13 Pungpapong et al 71% 88% 80% 84% 77% 27 36 5 11 Pungpapong et al EUS: 93%, MRCP: 65% EUS: 93%, MRCP: 90% EUS: 93%, MRCP: 80% EUS: 90%, MRCP: 81% EUS: 95%, MRCP: 79% EUS: 37, MRCP: 26 EUS: 55, MRCP: 53 EUS: 4, MRCP: 6 EUS: 3, MRCP: 14 Rudowicz-Pietruszewska et al 100% 100% 100% 100% 100% 9 79 0 0 Sai et al 60% 79% 68% 77% 60% 10 9 3 6 Savarino et al 90% 59% 76% 73% 83% 53 29 20 6 Scarabino et al ERCP: 83%, US: 42%, CT: 100% ERCP: 67%, US: 34%, CT: 70% ERCP: 70%, US: 35%, CT: 76% ERCP: 37%, US: 13%, CT: 44% ERCP: 94%, US: 71%, CT: 100% ERCP: 10, US: 5, CT: 12 ERCP: 34, US: 17, CT: 36 ERCP: 17, US: 34, CT: 15 ERCP: 2, US: 7, CT: 0 Eur Radiol (2017) 27:3820–3844 383 Eur Radiol (2017) 27:3820–3844 3838 ( ) Study Sensitivity Specificity Accuracy PPV NPV TP TN FP FN Schlaudraff et al MRCP: 67%, sMRCP: 73% MRCP: 93%, sMRCP: 96% MRCP: 89%, sMRCP: 93% MRCP: 63%, sMRCP: 78% MRCP: 95%, sMRCP: 95% MRCP: 6, sMRCP: 7 MRCP: 49, sMRCP: 51 MRCP: 4, sMRCP: 2 MRCP: 3, sMRCP: 2 Stevens et al Radial: 68%, Linear: 44% Radial: 95% Linear: 95% Radial: 84% Linear: 74% Radial: 90%, Linear: 86% Radial: 81%, Linear: 71% 28 56 3 13 Sverko et al 79% 93% 86% 92% 82% 11 14 1 3 Swobodnik et al US: 52%, CT: 74%, ERCP: 93% US: 100%, CT: 98%, ERCP: 100% US: 84%, CT: 90%, ERCP: 98% US: 100%, CT: 95%, ERCP: 100% US: 81%, CT: 88%, ERCP: 96% US: 14, CT: 20, ERCP: 25 US: 54, CT: 53, ERCP: 54 US: 0, CT: 1, ERCP: 0 US: 13, CT: 7, ERCP: 2 Tox et al 77% 75% 76% 66% 84% 50 80 26 15 Trikudanathan et al 61% 75% 63% 92% 29% 34 9 3 22 Triller et al 82% 85% 83% 82% 85% 9 11 2 2 Wiersema et al 80% 86% 84% 83% 84% 24 32 5 6 Zhang et al 92% 75% 84% 81% 88% 22 15 5 2 Zuccaro et al MRCP: 46%, sMRCP: 46% MRCP:85%, sMRCP: 68% MRCP: 70%, sMRCP: 59% MRCP: 68%, sMRCP: 50% MRCP: 70%, sMRCP: 65% MRCP: 13, sMRCP: 13 MRCP: 35, sMRCP: 28 MRCP: 6, sMRCP: 13 MRCP: 15, sMRCP: 15 PPV positive predictive value, NPV negative predictive value, TP true positive, TN true negative, FP false positive, FN false negative Table 11 Imaging characteristics for each study Magnetic resonance imaging (MRI) Study Year Magnetic field Coil type Contrast Secretin enhancement Sequence Scoring criteria Adamek et al 2000 1.0 T Body coil No No T2 Size of common bile and pancreatic duct, the nature and degree of pancreatic duct obstruction, and accuracy in diagnosing pathological findings Alcaraz et al 2000 1.5 T NA No No T2 (HASTE & RARE) NA Balci et al 2006 1.5 T Four-element quadrature phased-array surface coil IV No T1, T2 Increased arterial enhancement pattern, normal gland size and normal ductal morphology (Cambridge classification) Bolog et al 2004 1.0 T Synergy body coil NA No T1, T2 NA Fusari et al 2010 1.5 T Phased-array synergy body coil Oral No T1, T2 1–5 score to identify pancreatic masses (definite benign = 1, probably benign = 2 etc.) Hellerhoff et al 2002 1.5 T Phased-array synergy surface coil Oral No T2 Cambridge classification Pamos et al 1998 1.5 T Body coil NA No T2 NA Pungpapong et al 2007 1.5 T Phased-array surface coil IV/Oral No T1, T2, T2 (HASTE) Presence of 1 or more of the following features: main pancreatic duct dilatation in absence of structural obstruction, dilated side branches, intraductal stones, ( ) sMRCP: 46% sMRCP: 68% sMRCP: 59% sMRCP: 50% sMRCP: 65% sMRCP: 13 sMRCP: 28 sMRCP: 13 sMRCP: 15 PPV positive predictive value, NPV negative predictive value, TP true positive, TN true negative, FP false positive, FN false negative Table 11 Imaging characteristics for each study Magnetic resonance imaging (MRI) Study Year Magnetic field Coil type Contrast Secretin enhancement Sequence Scoring criteria Adamek et al 2000 1.0 T Body coil No No T2 Size of common bile and pancreatic duct, the nature and degree of pancreatic duct obstruction, and accuracy in diagnosing pathological findings Alcaraz et al 2000 1.5 T NA No No T2 (HASTE & RARE) NA Balci et al 2006 1.5 T Four-element quadrature phased-array surface coil IV No T1, T2 Increased arterial enhancement pattern, normal gland size and normal ductal morphology (Cambridge classification) Bolog et al 2004 1.0 T Synergy body coil NA No T1, T2 NA Fusari et al 2010 1.5 T Phased-array synergy body coil Oral No T1, T2 1–5 score to identify pancreatic masses (definite benign = 1, probably benign = 2 etc.) Hellerhoff et al 2002 1.5 T Phased-array synergy surface coil Oral No T2 Cambridge classification Pamos et al 1998 1.5 T Body coil NA No T2 NA Pungpapong et al 2007 1.5 T Phased-array surface coil IV/Oral No T1, T2, T2 (HASTE) Presence of 1 or more of the following features: main pancreatic duct dilatation in absence of structural obstruction, dilated side branches, intraductal stones, Study Sensitivity Specificity Accuracy PPV NPV TP TN FP FN Schlaudraff et al MRCP: 67%, sMRCP: 73% MRCP: 93%, sMRCP: 96% MRCP: 89%, sMRCP: 93% MRCP: 63%, sMRCP: 78% MRCP: 95%, sMRCP: 95% MRCP: 6, sMRCP: 7 MRCP: 49, sMRCP: 51 MRCP: 4, sMRCP: 2 MRCP: 3, sMRCP: 2 Stevens et al Radial: 68%, Linear: 44% Radial: 95% Linear: 95% Radial: 84% Linear: 74% Radial: 90%, Linear: 86% Radial: 81%, Linear: 71% 28 56 3 13 Sverko et al 79% 93% 86% 92% 82% 11 14 1 3 Swobodnik et al US: 52%, CT: 74%, ERCP: 93% US: 100%, CT: 98%, ERCP: 100% US: 84%, CT: 90%, ERCP: 98% US: 100%, CT: 95%, ERCP: 100% US: 81%, CT: 88%, ERCP: 96% US: 14, CT: 20, ERCP: 25 US: 54, CT: 53, ERCP: 54 US: 0, CT: 1, ERCP: 0 US: 13, CT: 7, ERCP: 2 Tox et al 77% 75% 76% 66% 84% 50 80 26 15 Trikudanathan et al 61% 75% 63% 92% 29% 34 9 3 22 Triller et al 82% 85% 83% 82% 85% 9 11 2 2 Wiersema et al 80% 86% 84% 83% 84% 24 32 5 6 Zhang et al 92% 75% 84% 81% 88% 22 15 5 2 Zuccaro et al MRCP: 46%, sMRCP: 46% MRCP:85%, sMRCP: 68% MRCP: 70%, sMRCP: 59% MRCP: 68%, sMRCP: 50% MRCP: 70%, sMRCP: 65% MRCP: 13, sMRCP: 13 MRCP: 35, sMRCP: 28 MRCP: 6, sMRCP: 13 MRCP: 15, sMRCP: 15 PPV positive predictive value, NPV negative predictive value, TP true positive, TN true negative, FP false positive, FN false negative Table 10 (continued) Severe CP: atrophy or diffuse/focal enlargement of the gland, calcification, chronic pseudocysts Ultrasonography (US) Study Year Transducer Scoring criteria Buscail et al 1995 NA NA Dramaix et al 1980 Unirad/Kretz combison 200 NA Gebel et al 1985 ADR 2130 Imager 2380 Sonoline 8000 Duct abnormalities Gmelin et al 1981 Sono fluoroskop 1, unirad model 849 Criteria for PC, CP and normal pancreas were extracted from literature Kremer et al 1977 NA Rettenmaier specified examination technique Lawson et al 1978 13-mm diameter 3.5 Mhz/ 13 or 19-mm diameter 2.25 Mhz Identification of a mass, pseudocyst or generalized glandular enlargement with abnormal parenchymal echogenicity ur Radiol (2017) 27:3820–3844 3839 Eur Radiol (2017) 27:3820–3844 3839 Magnetic resonance imaging (MRI) Study Year Magnetic field Coil type Contrast Secretin enhancement Sequence Scoring criteria ductal irregularity, reduced T1 signal intensity, atrophy of pancreatic parenchyma and reduced secretory response to secretin administration Rudowicz-Pietruszewska et al 2002 0.5 T Body coil NA No T2 NA Schlaudraff et al 2008 1.0 T Dedicated quadrature torso phased-array coil NA No T2, T2 (HASTE) Pancreatic duct stenosis/dilatation, side branch stenosis/dilatation, pseudocysts, extrapancreatic abscess. Based on observers’ judgement Sverko et al 2011 1.0 T NA IV No T1, T2 (HASTE) NA Zhang et al 2003 1.5 T NA IV No T1 Signal intensity by gadolinium (presence of SIR less than 1.73 in the arterial phase) Zuccaro et al 2009 NA Phased array-torso coil IV No T1, T2, T2 (HASTE) Mild CP: secretin-induced T2 intensity significantly reduced; side branch ectasia, mild ductal dilatation. Moderate CP: abnormal enhancement pattern on T1 after gadolinium administration. Severe CP: atrophy or diffuse/focal enlargement of the gland, calcification, chronic pseudocysts Secretin-enhanced magnetic resonance imaging (sMRI) Hellerhoff et al 2002 1.5 T Synerge phased-array surface coil IV Yes T2 Cambridge classification Sai et al 2008 1.5 T Phased-array multi coil IV Yes NA Cambridge classification Schlaudraff et al 2008 1.0 T Dedicated quadrature phased-array torso coil NA Yes T2, T2 (HASTE) Pancreatic duct stenosis/dilatation, side branch stenosis/dilatation, d i b r Radiol (2017) 27:3820–3844 Coil type Contrast Secretin enhancement Sequence Scoring criteria ductal irregularity, reduced T1 signal intensity, atrophy of pancreatic parenchyma and reduced secretory response to secretin administration Body coil NA No T2 NA Dedicated quadrature torso phased-array coil NA No T2, T2 (HASTE) Pancreatic duct stenosis/dilatation, side branch stenosis/dilatation, pseudocysts, extrapancreatic abscess. Table 10 (continued) Table 11 (continued) Magnetic resonance imaging (MRI) Study Year Magnetic field Coil type Contrast Secretin enhancement Sequence Scoring criteria ductal irregularity, reduced T1 signal intensity, atrophy of pancreatic parenchyma and reduced secretory response to secretin administration Rudowicz-Pietruszewska et al 2002 0.5 T Body coil NA No T2 NA Schlaudraff et al 2008 1.0 T Dedicated quadrature torso phased-array coil NA No T2, T2 (HASTE) Pancreatic duct stenosis/dilatation, side branch stenosis/dilatation, pseudocysts, extrapancreatic abscess. Based on observers’ judgement Sverko et al 2011 1.0 T NA IV No T1, T2 (HASTE) NA Zhang et al 2003 1.5 T NA IV No T1 Signal intensity by gadolinium (presence of SIR less than 1.73 in the arterial phase) Zuccaro et al 2009 NA Phased array-torso coil IV No T1, T2, T2 (HASTE) Mild CP: secretin-induced T2 intensity significantly reduced; side branch ectasia, mild ductal dilatation. Moderate CP: abnormal enhancement pattern on T1 after gadolinium administration. Severe CP: atrophy or diffuse/focal enlargement of the gland, calcification, chronic pseudocysts Secretin-enhanced magnetic resonance imaging (sMRI) Hellerhoff et al 2002 1.5 T Synerge phased-array surface coil IV Yes T2 Cambridge classification Sai et al 2008 1.5 T Phased-array multi coil IV Yes NA Cambridge classification Schlaudraff et al 2008 1.0 T Dedicated quadrature phased-array torso coil NA Yes T2, T2 (HASTE) Pancreatic duct stenosis/dilatation, side branch stenosis/dilatation, pseudocysts, extrapancreatic abscess. Based on observers’ judgement Zuccaro et al 2009 NA Phased-array torso coil IV Yes T1, T2, T2 (HASTE) Mild CP: secretin-induced T2 intensity significantly reduced; side branch ectasia, mild ductal dilatation. Moderate CP: abnormal enhancement pattern on T1 after gadolinium administration. Table 10 (continued) Based on observers’ judgement NA IV No T1, T2 (HASTE) NA NA IV No T1 Signal intensity by gadolinium (presence of SIR less than 1.73 in the arterial phase) Phased array-torso coil IV No T1, T2, T2 (HASTE) Mild CP: secretin-induced T2 intensity significantly reduced; side branch ectasia, mild ductal dilatation. Moderate CP: abnormal enhancement pattern on T1 after gadolinium administration. Severe CP: atrophy or diffuse/focal enlargement of the gland, calcification, chronic pseudocysts Synerge phased-array surface coil IV Yes T2 Cambridge classification Phased-array multi coil IV Yes NA Cambridge classification Dedicated quadrature phased-array torso coil NA Yes T2, T2 (HASTE) Pancreatic duct stenosis/dilatation, side branch stenosis/dilatation, pseudocysts, extrapancreatic abscess. Based on observers’ judgement Phased-array torso coil IV Yes T1, T2, T2 (HASTE) Mild CP: secretin-induced T2 intensity significantly reduced; side branch ectasia, mild ductal dilatation. Moderate CP: abnormal enhancement pattern on T1 after gadolinium administration. References Whiting PF, Rutjes AW, Westwood ME et al (2011) QUADAS-2: a revised tool for the quality assessment of diagnostic accuracy stud- ies. Ann Intern Med 155:529–536 26. Gebel M, Stiehl M, Freise J (1985) Value of ultrasonographic im- aging of the pancreas for diagnosis of chronic pancreatitis and pan- creas carcinoma in comparison with ERP. Ultraschall in der Medizin 6:127–130 8. Brozek JL, Akl EA, Jaeschke R et al (2009) Grading quality of evidence and strength of recommendations in clinical practice guide- lines: Part 2 of 3 The GRADE approach to grading quality of evi- dence about diagnostic tests and strategies. Allergy 64:1109–1116 27. Giovannini M, Seitz JF (1994) Endoscopic ultrasonography with a linear-type echoendoscope in the evaluation of 94 patients with pancreatobiliary disease. Endoscopy 26:579–585 9. Guyatt GH, Oxman AD, Vist GE et al (2008) GRADE: an emerg- ing consensus on rating quality of evidence and strength of recom- mendations. BMJ 336:924–926 28. Glasbrenner B, Schwarz M, Pauls S, Preclik G, Beger HG, Adler G (2000) Prospective comparison of endoscopic ultrasound and en- doscopic retrograde cholangiopancreatography in the preoperative assessment of masses in the pancreatic head. Dig Surg 17:468–474 10. Schünemann H BJ, Guyatt G, Oxman A (2013) Handbook for grading the quality of evidence and the strength of recommenda- tions using the GRADE approach. The GRADE Working Group; 2013. Available at: www.guidelinedevelopment.org/handbook. Accessed 28 Jan 2015 29. Gmelin E, Weiss H-D, Fuchs H-D, Reiser M (1981) A comparison of the diagnostic accuracy of ultrasound, computer tomography and ERPC in chronic pancreatitis and carcinoma of the pancreas. Röfo 134:136–141 11. Higgins JP, Thompson SG, Deeks JJ, Altman DG (2003) Measuring inconsistency in meta-analyses. BMJ 327:557–560 12. Reitsma JB, Glas AS, Rutjes AW, Scholten RJ, Bossuyt PM, Zwinderman AH (2005) Bivariate analysis of sensitivity and spec- ificity produces informative summary measures in diagnostic re- views. J Clin Epidemiol 58:982–990 30. Hellerhoff KJ, Helmberger IH, Rosch T, Settles MR, Link TM, Rummeny EJ (2002) Dynamic MR pancreatography after secretin administration: image quality and diagnostic accuracy. Am J Roentgenol 179:121–129 13. Moher D, Liberati A, Tetzlaff J, Altman DG, Group P (2009) Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Ann Intern Med 151:W264 31. Imdahl A, Nitzsche E, Krautmann F et al (1999) Evaluation of positron emission tomography with 2- [18F]fluoro-2-deoxy-D-glu- cose for the differentiation of chronic pancreatitis and pancreatic cancer. Br J Surg 86:194–199 14. References 18. Bolog N, Constantinescu G, Oancea I et al (2004) Magnetic reso- nance imaging of bile and pancreatic ducts: a retrospective study. Rom J Gastroenterol 13:91–97 1. Gardner TB, Kennedy AT, Gelrud A et al (2010) Chronic pancrea- titis and its effect on employment and health care experience: results of a prospective American multicenter study. Pancreas 39:498–501 19. Brand B, Pfaff T, Binmoeller KF et al (2000) Endoscopic ultra- sound for differential diagnosis of focal pancreatic lesions, con- firmed by surgery. Scand J Gastroenterol 35:1221–1228 2. Hall TC, Garcea G, Webb MA, Al-Leswas D, Metcalfe MS, Dennison AR (2014) The socio-economic impact of chronic pan- creatitis: a systematic review. J Eval Clin Pract 20:203–207 20. Buscail L, Escourrou J, Moreau J et al (1995) Endoscopic ultraso- nography in chronic pancreatitis: a comparative prospective study with conventional ultrasonography, computed tomography, and ERCP. Pancreas 10:251–257 3. Mullady DK, Yadav D, Amann ST et al (2011) Type of pain, pain- associated complications, quality of life, disability and resource utilisation in chronic pancreatitis: a prospective cohort study. Gut 60:77–84 21. Catalano MF, Geenen JE (1998) Diagnosis of chronic pancreatitis by endoscopic ultrasonography. Endoscopy 30:A111–A115 22. Chong AK, Hawes RH, Hoffman BJ, Adams DB, Lewin DN, Romagnuolo J (2007) Diagnostic performance of EUS for chronic pancreatitis: a comparison with histopathology. Gastrointest Endosc 65:808–814 4. Olesen SS, Juel J, Nielsen AK, Frokjaer JB, Wilder-Smith OH, Drewes AM (2014) Pain severity reduces life quality in chronic pancreatitis: implications for design of future outcome trials. Pancreatology 14:497–502 23. Conwell DL, Zuccaro G, Purich E et al (2007) Comparison of endoscopic ultrasound chronic pancreatitis criteria to the endoscop- ic secretin-stimulated pancreatic function test. Dig Dis Sci 52: 1206–1210 5. Buchler MW, Martignoni ME, Friess H, Malfertheiner P (2009) A proposal for a new clinical classification of chronic pancreatitis. BMC Gastroenterol 9:93 24. Dramaix JP, Sahel J, Corbeau A (1980) A prospective comparative study of different methods of morphological examination of the pancreas. Place of ultrasonography and computerized tomography. Acta Endoscopica 10:1 6. Schneider A, Lohr JM, Singer MV (2007) The M-ANNHEIM clas- sification of chronic pancreatitis: introduction of a unifying classi- fication system based on a review of previous classifications of the disease. J Gastroenterol 42:101–119 25. Fusari M, Maurea S, Imbriaco M et al (2010) Comparison between multislice CT and MR imaging in the diagnostic evaluation of pa- tients with pancreatic masses. Radiol Med 115:453–466 7. Table 10 (continued) Severe CP: atrophy or diffuse/focal enlargement of the gland, calcification, chronic pseudocysts Scoring criteria NA NA Duct abnormalities Criteria for PC, CP and normal pancreas were extracted from literature Rettenmaier specified examination technique Identification of a mass, pseudocyst or generalized glandular enlargement with abnormal parenchymal echogenicity Scoring criteria NA NA Duct abnormalities Criteria for PC, CP and normal pancreas were extracted from literature Rettenmaier specified examination technique Identification of a mass, pseudocyst or generalized glandular enlargement with abnormal parenchymal echogenicity Eur Radiol (2017) 27:3820–3844 3840 Magnetic resonance imaging (MRI) Study Year Magnetic field Coil type Contrast Secretin enhancement Sequence Scoring criteria Lees et al 1979 2.5 Mhz Appearance of pancreatic parenchyma and duct system/size and shape of the pancreas and from previous reports Lin et al 1989 SAL-90A 3.75 Mhz NA Scarabino et al 1989 NA NA Swobodnik et al 1983 Siemens imager 2300 linear array Organ enlarged or atrophic dense structure, areas of scars or calcification (more echogenic), sonolucent areas only during acute inflammation, dilatation of the pancreatic duct system, symmetric contours, no smooth outlines Computed tomography (CT) Study Year Scanner Contrast Scoring criteria Buscail et al 1995 NA NA NA Dramaix et al 1980 OHIO nuclear - Delta Slan 50FS Oral/IV NA Fusari et al 2010 Marconi MX8000 (four-detector row) IV 1–5 score to identify pancreatic masses (definite benign = 1, probably benign = 2 etc.) Gmelin et al 1981 NA NA Criteria for PC, CP and normal pancreas were extracted from literature Imdahl et al 1999 Somatom Plus 4 helical scanner IV NA Lammer et al 1980 EMI-5005 Oral 3 stadia typical for CP Pistolesi et al 1981 Ohio-Nuclear Delta 50 scanner NA Overall enlargement of the pancreas or calcifications Savarino et al 1980 EMI-5005 Oral Parenchymal atrophy, pancreatic calcifications, pseudocysts or abscesses Scarabino et al 1989 NA NA NA Swobodnik et al 1983 General Electric CT-T8800 Oral/IV Atrophy of the organ (during acute inflammation: segmental enlargement) during acute phase; segments without clear outlines, cysts or calcifications and dense structure Endoscopic ultrasonography (EUS) Study Year Scanner Transducer MHz Scoring criteria Albashir et al 2010 NA NA NA 9 features; >4 diagnostic for CP Brand et al 2000 Olympus GF-UM 3/GF-UM 20/GF-UM 200 Radial NA Own criteria (increased parenchymal lobulations, calcification and/or ductal changes or focal lesion) Buscail et al 1995 Olympus EU-M3 NA 7.5/12 MHz NA Catalano et al 1998 Olympus EU-M3/EU-M20 NA 7.5/12 MHz Wiersema criteria (11 features), own classification system Chong et al 2007 Olympus EU-M20/GF-UM130/GF- UM160/GF-UC30P/GF- UC140P/GF-UCT140 Radial NA 9 features; >3 diagnostic for CP Conwell et al 2007 NA NA NA 9 features; >3 diagnostic for CP Giovannini et al 1994 Pentax FG-32-UA Linear NA NA Glasbrenner et al 2000 Olympus EU-M20 Radial 7.5/12 MHz Wiersema criteria (11 features) Lin et al 1989 Olympus GF-EUM 2/GF-UM2 Radial 7.5 MHz NA Nattermann et al 1993 Olympus GF-UM-3/EU-M3 NA 7.5/12 MHz NA Pungpapong et al 2007 Olympus GF-UE160-AL5/GF-UC140P Radial & linear NA MST criteria; >4 features diagnostic for CP Pungpapong et al 2007 Olympus GF-UC140P/ UCT140-AL5 Linear 7.5 MHz MST criteria; >4 features diagnostic for CP Eur Radiol (2017) 27:3820–3844 3841 ab e (co t ued) Magnetic resonance imaging (MRI) Study Year Magnetic field Coil type Contrast Secretin enhancement Sequence Scoring criteria Stevens et al 2009 Olympus GF-UM-130/GF-UE-160/GF- UC-160P-OL5 Radial & linear NA 9 features; >4 diagnostic for CP Tox et al 2007 Olympus GF-UM20, Pentax EG-3620-UR/EG-3830-UT NA NA Own criteria Trikudanathan 2016 Olympus Linear 7.5 MHz Wiersema criteria (11 features) >4 is CP Wiersema et al 1993 Olympus EU-M3/EU-M20 NA NA Wiersema criteria (11 features) >3 is CP Endoscopic retrograde cholangiopancreatography (ERCP) Study Year Technical features Scoring criteria Adamek et al 2000 NA NA Buscail et al 1995 NA Own criteria (normal/moderate changes (3 abnormal side branches and normal main duct)/marked changes (side and main duct abnormalities)) Gebel et al 1995 NA Deyhle criteria Glasbrenner et al 2000 Olympus Cambridge classification Gmelin et al 1981 NA Criteria according to references Lammer et al 1980 Olympus JFB Loffler criteria Lawson et al 1978 NA Criteria according to references Parsi et al 2008 NA Cambridge classification Scarabino et al 1989 NA NA Swobodnik et al 1983 Olympus JFB-2/3 Own criteria (variation in diameter of the main duct in the whole organ (exception: segmental pancreatitis), cystic dilatation of side branches, kinking of the duct stones in canalicular structures, distension of the main duct) Triller et al 1975 NA NA 3842 Eur Radiol (2017) 27:3820–3844 test, and histology: correlation in chronic pancreatitis. Table 10 (continued) Am J Gastroenterol 105:2498–2503 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 16. Alcaraz MJ, De la Morena EJ, Polo A, Ramos A, De la Cal MA, Gonzalez MA (2000) A comparative study of magnetic resonance cholangiography and direct cholangiography. Rev Esp Enferm Dig 92:427–438 17. Balci NC, Alkaade S, Akduman IE, Bilgin M, Murdock CP, Burton FR (2006) Serial contrast-enhanced MRI of the pancreas. Correlation with secretin-stimulated endoscopic pancreatic function test. Acad Radiol 13:1367–1372 References Adamek HE, Albert J, Breer H, Weitz M, Schilling D, Riemann JF (2000) Pancreatic cancer detection with magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography: a prospective controlled study. Lancet 356:190–193 32. Kremer H, Kellner E, Schierl W, Schumm C, Weidenhiller S, Zollner N (1977) Echographic diagnosis of the pancreas. Catamnesis of 481 cases (author's transl). MMW MunchMed Wochenschr 119:1449–1452 15. Albashir S, Bronner MP, Parsi MA, Walsh RM, Stevens T (2010) Endoscopic ultrasound, secretin endoscopic pancreatic function 33. Lammer J, Lepuschutz H, Sager WD, Kratochvil P, Brandstatter G, Zalaudek G (1980) Endoscopic retrograde cholangio- Eur Radiol (2017) 27:3820–3844 3843 51. Tox U, Hackenberg R, Stelzer A et al (2007) Endosonographic diagnosis of solid pancreatic tumors: a retrospective analysis from a tertiary referral center. Z Gastroenterol 45:307–312 pancreaticography (ERCP) and computer tomography in the diagno- sis of chronic pancreatitis, pseudocysts and carcinoma of the pancreas–a comparison (author's transl). Rontgenblatter 33:602–611 pancreaticography (ERCP) and computer tomography in the diagno- sis of chronic pancreatitis, pseudocysts and carcinoma of the pancreas–a comparison (author's transl). Rontgenblatter 33:602–611 52. Triller J, Voegeli E, Halter F (1975) Selective pancreatic angiogra- phy and retrograde pancreatic cholangiography as a combined ex- amination. Röfo 122:138–144 34. Lawson TL, Irani SK, Stock M (1978) Detection of pancreatic pathology by ultrasonography and endoscopic retrograde cholangiopancreatography. Gastrointest Radiol 3:335–341 53. Wiersema MJ, Hawes RH, Lehman GA, Kochman ML, Sherman S, Kopecky KK (1993) Prospective evaluation of endoscopic ultraso- nography and endoscopic retrograde cholangiopancreatography in patients with chronic abdominal pain of suspected pancreatic origin. Endoscopy 25:555–564 35. Lees WR, Vallon AG, Denyer ME et al (1979) Prospective study of ultrasonography in chronic pancreatic disease. Br Med J 1:162–164 36. Lin JT, Wang JT, Wang TH (1989) The diagnostic value of endo- scopic ultrasonography in pancreatic disorders. Taiwan Yi Xue Hui Za Zhi 88:483–487 54. Zhang XM, Shi H, Parker L, Dohke M, Holland GA, Mitchell DG (2003) Suspected early or mild chronic pancreatitis: enhancement patterns on gadolinium chelate dynamic MRI. Magnetic resonance imaging. J Magn Reson Imaging 17:86–94 37. Nattermann C, Goldschmidt AJW, Dancygier H (1993) Endosonography in chronic pancreatitis - a comparison between endoscopic retrograde pancreatography and endoscopic ultraso- nography. Endoscopy 25:565–570 55. Zuccaro P, Stevens T, Repas K et al (2009) Magnetic resonance cholangiopancreatography reports in the evaluation of chronic pan- creatitis: a need for quality improvement. Pancreatology 9:764–769 38. References Pamos S, Rivera P, Canelles P et al (1998) Magnetic resonance cholangiopancreatography (MRCP) versus endoscopic retrograde cholangiopancreatography (ERCP): diagnostic usefulness. Gastroenterol Hepatol 21:174–180 56. Trikudanathan G, Vega-Peralta J, Malli A et al (2016) Diagnostic performance of endoscopic ultrasound (EUS) for non-calcific chronic pancreatitis (NCCP) based on histopathology. Am J Gastroenterol 111:568–574 39. Parsi MA, Conwell DL, Zuccaro G et al (2008) Findings on endo- scopic retrograde cholangiopancreatography and pancreatic func- tion test in suspected chronic pancreatitis and negative cross- sectional imaging. Clin Gastroenterol Hepatol 6:1432–1436 57. Delhaye M, Van SW, Cesmeli E et al (2014) Belgian consensus on chronic pancreatitis in adults and children: statements on diagnosis and nutritional, medical, and surgical treatment. Acta Gastroenterol Belg 77:47–65 40. Pistolesi GF, Procacci C, Fugazzola C (1981) Place of computed tomography in pancreatic disease. Comparison with other radiolog- ical methods. Comput Tomogr 5:115–137 58. Frulloni L, Falconi M, Gabbrielli A et al (2010) Italian consensus guidelines for chronic pancreatitis. Dig Liver Dis 42:S381–S406 41. Pungpapong S, Noh KW, Woodward TA, Wallace MB, Al-Haddad M, Raimondo M (2007) Endoscopic ultrasound and IL-8 in pancreatic juice to diagnose chronic pancreatitis. Pancreatology 7:491–496 59. Martinez J, Abad-Gonzalez A, Aparicio JR et al (2013) The Spanish Pancreatic Club recommendations for the diagnosis and treatment of chronic pancreatitis: part 1 (diagnosis). Pancreatology 13:8–17 42. Pungpapong S, Wallace MB, Woodward TA, Noh KW, Raimondo M (2007) Accuracy of endoscopic ultrasonography and magnetic resonance cholangiopancreatography for the diagnosis of chronic pancreatitis: a prospective comparison study. J Clin Gastroenterol 41:88–93 60. Schreyer AG, Jung M, Riemann JF et al (2014) S3 guideline for chronic pancreatitis - diagnosis, classification and therapy for the radiologist. Röfo 186:1002–1008 43. Rudowicz-Pietruszewska B, Sasiadek M, Jamrozik-Kruk Z (2002) MRCP with mid-field unit versus ERCP - comparison of 88 cases. Gastroenterol Pol 9:51–58 61. Remedios D, Hierath M, Ashford N et al (2014) Imaging referral guidelines in Europe: now and in the future-EC Referral Guidelines Workshop Proceedings. Insights Imaging 5:9–13 44. Sai JK, Suyama M, Kubokawa Y, Watanabe S (2008) Diagnosis of mild chronic pancreatitis (Cambridge classification): comparative study using secretin injection-magnetic resonance cholangiopancreatography and endoscopic retrograde pancreatography. World J Gastroenterol 14:1218–1221 62. Remedios D, Hierath M, Ashford N et al (2014) European survey on imaging referral guidelines. Insights Imaging 5:15–23 63. Remedios DCP, Ashford N, Grenier P (2014) Referral guidelines for medical imaging - availability and use in the European Union. European Union. doi:10.2833/18118 45. 75. Ahmed Ali U, Nieuwenhuijs VB, van Eijck CH et al (2012) Clinical outcome in relation to timing of surgery in chronic pancre- atitis: a nomogram to predict pain relief. Arch Surg 147:925–932 76. Ahmed Ali U, Issa Y, Bruno MJ et al (2013) Early surgery versus optimal current step-up practice for chronic pancrea- titis (ESCAPE): design and rationale of a randomized trial. BMC Gastroenterol 13:49 References Savarino V, Mansi C, Macchia G (1980) Computed tomography in the diagnosis of pancreatic diseases. Ital J Gastroenterol 12:265– 269 64. Clave P, Boadas J, Gonzalez-Carro P et al (1999) Accuracy of imaging techniques and tumor markers in the diagnosis of pancre- atic cancer. Gastroenterol Hepatol 22:335–341 46. Scarabino T, Petronelli S, Palladino D et al (1989) ERCP in the diagnosis of bilio-pancreatic pathology. Comparison with echography and CT. Radiol Med 77:650–654 65. Orti E, Canelles P, Tome A et al (1993) Diagnostic value of aspiration cytology with ERCP. Rev Esp Enferm Dig 84: 173–177 47. Schlaudraff E, Wagner H-J, Klose KJ, Heverhagen JT (2008) Prospective evaluation of the diagnostic accuracy of secretin- enhanced magnetic resonance cholangiopancreaticography in suspected chronic pancreatitis. Magn Reson Imaging 26:1367– 1373 66. Wang LW, Li ZS, Li SD, Jin ZD, Zou DW, Chen F (2009) Prevalence and clinical features of chronic pancreatitis in China: a retrospective multicenter analysis over 10 years. Pancreas 38:248–254 67. Andriulli A, Loperfido S, Napolitano G et al (2007) Incidence rates of post-ERCP complications: a systematic survey of prospective studies. Am J Gastroenterol 102:1781–1788 48. Stevens T, Zuccaro G, Dumot JA et al (2009) Prospective compar- ison of radial and linear endoscopic ultrasound for diagnosis of chronic pancreatitis. Endoscopy 41:836–841 68. Barthet M, Lesavre N, Desjeux A et al (2002) Complications of endoscopic sphincterotomy: results from a single tertiary referral center. Endoscopy 34:991–997 49. Sverko A, Tripalo-Batos A, Marotti M, Mustapic M, Beslin MB, Kruslin B (2011) Correlation between magnetic resonance imaging and histopathology in differentiation of pancreatic diseases. Acta Clin Croat 50:137–144 69. Cotton PB, Garrow DA, Gallagher J, Romagnuolo J (2009) Risk factors for complications after ERCP: a multivariate analysis of 11,497 procedures over 12 years. Gastrointest Endosc 70:80–88 50. Swobodnik W, Meyer W, Brecht KD (1983) Ultrasound, computed tomography and endoscopic retrograde cholangiopancreatography in the morphologic diagnosis of pancreatic disease. Klin Wochenschr 61:291–296 70. Kim DH, Pickhardt PJ (2007) Radiologic assessment of acute and chronic pancreatitis. Surg Clin North Am 87:1341–1358 3844 Eur Radiol (2017) 27:3820–3844 71. Matos C, Bali MA, Delhaye M, Deviere J (2006) Magnetic reso- nance imaging in the detection of pancreatitis and pancreatic neo- plasms. Best Pract Res Clin Gastroenterol 20:157–178 72. Remer EM, Baker ME (2002) Imaging of chronic pancreatitis. Radiol Clin North Am 40:1229–1242 73. Ly JN, Miller FH (2002) MR imaging of the pancreas: a practical approach. 71. Matos C, Bali MA, Delhaye M, Deviere J (2006) Magnetic reso- nance imaging in the detection of pancreatitis and pancreatic neo- plasms. Best Pract Res Clin Gastroenterol 20:157–178 71. Matos C, Bali MA, Delhaye M, Deviere J (2006) Magnetic reso- nance imaging in the detection of pancreatitis and pancreatic neo- plasms. Best Pract Res Clin Gastroenterol 20:157–178 72. Remer EM, Baker ME (2002) Imaging of chronic pancreatitis. Radiol Clin North Am 40:1229–1242 73. Ly JN, Miller FH (2002) MR imaging of the pancreas: a practical approach. Radiol Clin North Am 40:1289–1306 74. Robinson PJA, Sheridan MB (2000) Pancreatitis: computed tomography and magnetic resonance imaging. Eur Radiol 10: 401–408 72. Remer EM, Baker ME (2002) Imaging of chronic pancreatitis. Radiol Clin North Am 40:1229–1242 References Radiol Clin North Am 40:1289–1306 74. Robinson PJA, Sheridan MB (2000) Pancreatitis: computed tomography and magnetic resonance imaging. Eur Radiol 10: 401–408
https://openalex.org/W3201498379	https://europepmc.org/articles/pmc8461909?pdf=render	English	null	Arthroscopic findings and treatment of maisonneuve fracture complex	BMC musculoskeletal disorders	2,021	cc-by	4,036	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: The Maisonneuve fracture complex (MFC) is a well-known lower leg injury. However, the optimal treat- ment is still not clear and there is limited data on concomitant injuries of cartilage. Therefore, the aim of our study was to report the incidence of incidental cartilage injuries and their management in arthroscopic treatment of MFC. Patients and methods: Between February 2018 and February 2021 all patients presenting with MFC in our depart- ment were treated with diagnostic ankle arthroscopy and percutaneous syndesmotic screw or suture-endobutton fixation. In case of instable cartilage, it was debrided and according to the International Consensus Meeting on Carti- lage Repair of the Ankle, in grade IV lesions < 10 mm or < 100 mm2 area the subchondral bone was microfractured. Results: Eighteen patients, 16 male and two female, with a mean age of 48.1 years, were included. In all cases, insta- bility of the distal tibiofibular articulation was confirmed arthroscopically. Injuries of the cartilage were found in 56% of the cases and in 31% of the patients surgical intervention was required. In three talar and one tibial lesion addi- tional arthroscopic bone marrow stimulation with microfracture of the subchondral bone was performed. Conclusions: Ankle arthroscopy is a helpful method to guide fibular reduction and to detect and address associated cartilage injuries. Due to the high rate of chondral lesions, addressing these arthroscopically may contribute to better postoperative results. Level of evidence: IV Keywords: Maisonneuve fracture, Tibio-fibular instability, Syndesmotic rupture, Ankle arthroscopy, Osteochondral lesion, Cartilage lesion slight supination and in neutral or slight pronation in later stages [2]. Introduction “La fracture du péroné”, or better known as Maison- neuve fracture complex (MFC) was first described by the French surgeon Jacques Gilles Maisonneuve in 1840 [1]. The injury typically consists of a fracture of the proximal fibula with disruption of the distal tibiofibular syndesmo- sis and a deltoid rupture or medial malleolus fracture.h Regarding therapy, some authors reported of nonop- erative treatment: Pankovich [2] treated the MFC nonop- eratively in cases without rupture of the deltoid ligament, interosseous ligaments, or medial malleolus fracture and Merrill [3] suggested that these are often more stable than generally assumed. According to the Lauge-Hansen classification, this could be possible, but sometimes it is difficult to differentiate between partially and total rup- tured syndesmotic ligaments preoperatively. Therefore, MFC should be assumed as an unstable injury, and most authors recommend operative treatment [4–9]. Stufkens [9] defined in their review of literature recommendations The mechanism of the injury was described by Panko- vich as a strong external rotation force with the foot in *Correspondence: mail@dr-fraissler.at 1 Department of Orthopedic Surgery, Klinik Diakonissen Schladming, Salzburger Strasse 777, 8970 Schladming, Austria Full list of author information is available at the end of the article Fraissler et al. BMC Musculoskelet Disord (2021) 22:821 https://doi.org/10.1186/s12891-021-04713-8 Fraissler et al. BMC Musculoskelet Disord (2021) 22:821 https://doi.org/10.1186/s12891-021-04713-8 © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Fraissler et al. BMC Musculoskelet Disord (2021) 22:821 Fraissler et al. BMC Musculoskelet Disord (2021) 22:821 Page 2 of 5 Fraissler et al. BMC Musculoskelet Disord (2021) 22:821 After addressing cartilage damage, avulsed ligaments and debris of the distal tibiofibular syndesmosis was resected with a 4 mm shaver to allow proper position- ing of the fibula into the incisura. Closed reduction was performed with a sharp reduction clamp and controlled arthroscopically and fluoroscopically. Length to the fib- ula was restored and internal rotation as well as medial translation was assessed. Fixation of the fibula was either achieved with percutaneous placement of two tricorti- cal 3.5 mm stainless steel screws or suture-endobuttons (TightRope®, Arthrex, Naples, FL). for treatment of Maisonneuve fractures: the medial malleolus should be fixated, the torn deltoid ligament need not be directly repaired, syndesmotic instability can be treated with one or two 3- or 4-cortical screws which can be placed percutaneously, and the proximal fibular fracture does not require direct internal fixation. How- ever, the optimal operative management is not clear and various options are under debate. Moreover, Hintermann [10] and Loren [11] reported of an incidence of cartilage lesions, including chondral defects and osteochondral lesions, in ankle fractures of 79.2%, respectively 63%. Yoshimura [8] reported that all patients with MFC, who underwent ankle arthroscopy, had cartilaginous damage to the medial section of the talar dome. Results In all 18 cases, a frank instability of the distal tibiofibular articulation was confirmed in intraoperative stress test- ing. Four patients had an arthroscopically confirmed dis- ruption of the deltoid ligament and six patients a fracture of the medial malleolus. Furthermore, 11 patients had a radiologically approved additional posterior fracture of the distal tibia or tibial avulsion fracture of the posterior inferior tibiofibular ligament. MFC was confirmed with radiographs of the ankle and knee. In 5 patients an additional CT-scan and in 3 patients an MRI of the ankle was performed. Preoperatively, prophylactic antibiotics were given intravenously. All Arthroscopies were performed or attended by a national-board certified foot and ankle surgeon in spinal anaesthesia and tourniquet control. Therefore, patients were set in supine position, no ankle distractor was applied, and saline was injected to inflate the ankle joint. Routinely, a 4 × 152.5 mm/30° arthro- scope with standard anteromedial and anterolateral por- tals was used to access the ankle. i Traumatic lesions of the cartilage were found in 10 of 18 ankles (56%). The medial aspect of the talar dome was involved in eight cases, while tibial articulation was affected in 2 patients. A wide range of different (osteo-) chondral lesions was seen intraoperatively (Figs. 2 and 3) and classified as shown in Table 1. With exception of the patient with the grade I talar lesion, additional operative treatment was needed in nine cartilage lesions. There- fore, all nine patients were treated with arthroscopical debridement of ruptured and loose cartilage. In three talar and one tibial lesion additional bone marrow stimu- lation with microfracture of the subchondral bone was necessary. An anterior ankle examination, as described by Ferkel [12], was used to verify syndesmotic instability and to check for cartilage injuries. External rotation stress test was used to assess syndesmotic injury. Frank syndes- mosis instability was defined by ≥2 mm displacement of the lateral malleolus (Fig. 1) [13]. Lesions of the cartilage were classified according to the International Cartilage Repair Society (ICRS) grading system [14]. Postoperative treatment was performed according to our standard protocol with partial weight-bearing for 6 weeks or partial weight-bearing for 2 weeks, followed by 4 weeks of full weight-bearing and screw removal after 6 weeks. Therefore, we started to treat patients with Maisonneuve fracture arthroscopically to detect and address concomitant injuries. The aim of this study was to retrospectively evaluate the incidence of cartilage inju- ries in these patients. Medial malleolar fractures were addressed with open reduction and screw fixation, whereas deltoid ligament disruptions were treated non-operatively in the con- text of postoperative immobilization with cast or walk- ing boot. In cases of arthroscopy and microfracture of talus or tibia, or suture-endobutton fixation of the fibula, patients were instructed for partial weight-bearing (15 kg) for 6 weeks and with screw fixation only partial weight- bearing (15 kg) for 2 weeks, followed by 4 weeks of full weight-bearing. According to our postoperative standard protocol syndesmosis screws were removed 6–8 weeks postoperative. Materials and methods Between February 2018 and February 2021 all 18 patients presenting with MFC, 16 men and two women, were treated consecutively with ankle arthroscopy in our department. The mean age at time of surgery was 48.1 years (range 23 to 74 years). Eleven MFCs were sports related, six occurred as a result of an ankle sprain while walking, and one patient had a motorcycle accident (Table 1). Results Instable cartilage was debrided and, as recommended by the International Consensus Meeting on Cartilage Repair of the Ankle, in lesions < 10 mm or < 100 mm2 area subchondral bone was microfractured [15]. Twelve patients were available for a short-term follow- up. Mean time to follow-up was 15 months (range 3 to Fraissler et al. Results BMC Musculoskelet Disord (2021) 22:821 arthroscopy might be a favourable method to detect and subsequently treat associated injuries. Fig. 1 Diastasis of the distal fibula was proven with a 4 mm shaver. In all cases an apparent syndesmosis instability of at least 4 mm was seen In general, the incidence of complications in ankle arthroscopy is low. Imade [17] reported of one patient with compartment syndrome following ankle arthros- copy after MFC. In our own patient collective, as well as in the report of Yoshimura [8], were no complications as a result of ankle arthroscopy found. Furthermore, the mechanism of injury was supposed to be a strong external rotation force with the foot in slight supination and in neutral or slight pronation in later stages [2]. Based on the fact that there was no dam- age to the posterior malleolus in their patient collective, Yoshimura [8] concluded that there is a strong possibility that the MFC could be a pronation external rotation type fracture according to the Lauge-Hansen classification. In four of our patients MFC occurred in a ski boot while ski- ing; Fritschy [18] also reported of Maisonneuve injuries in professional skiers with rigid ski boots, in which pro- nation or supination is theoretically impossible. There- fore, the mechanism of injury and potentially associated intra-articular damage have to be thought about. Fig. 1 Diastasis of the distal fibula was proven with a 4 mm shaver. In all cases an apparent syndesmosis instability of at least 4 mm was seen 27 months). No perioperative infections or wound com- plications occurred. After 10 months one patient needed revision surgery due to secondary syndesmosis and del- toid ligament diastasis and in one case of TightRope® fixation implant removal and arthroscopic scar debride- ment was necessary. None of the patients complained about secondary cartilage issues. At the time of last fol- low-up all patients were very satisfied or satisfied with the results. Fig. 3 In one patient a 22x4x4 mm osteochondral fragment of the anteromedial edge of the distal tibia had to be removed Fig. 3 In one patient a 22x4x4 mm osteochondral fragment of the anteromedial edge of the distal tibia had to be removed Results BMC Musculoskelet Disord (2021) 22:821 Page 3 of 5 Table 1 Patients characteristics, Surgical Intervention, Trauma mechanism, Finding N/A – Due to an osteochondral comminution fracture of the anterior border of the distal tibia defect si Age [years] Sex Side Diagnosis (in addition to syndesmotic instability) Localization of chondral lesion Size [mm] IC 57 Male right Chondral lesion, medial malleolus fracture, pos- terior tibia fracture Talus medial 5 × 5 IV 46 Male left Medial malleolus fracture 24 Female right Deltoid rupture 27 Male left Chondral lesion Tibia 10 × 4 IV 52 Male left Chondral lesion, medial malleolus frac- ture, Chaput tubercle fracture Talus medial 4 × 4 IV 74 Male right Medial malleolus fracture, posterior tibia fracture 41 Male left Chondral lesion, poste- rior tibia fracture Talus medial 4 × 4 I 49 Male right Deltoid rupture, poste- rior tibia fracture 55 Male right Chondral lesion, del- toid rupture posterior tibia fracture Talus medial 8 × 6 IV 61 Male left Deltoid rupture 23 Male left Medial malleolus fracture, posterior tibia fracture 40 Male right Osteochondral lesion, posterior tibia fracture Tibia N/A IV 43 Male left Osteochondral lesion, medial malleolus fracture, posterior tibia fracture Tibia, Talus medial 22x4x4, 10 × 5 IV II 33 Male left Deltoid rupture, poste- rior tibia fracture 69 Male right Chondral lesion, del- toid rupture, posterior tibia fracture Talus medial 10 × 5 IV 63 Male right 40 Male right 68 Female right Chondral lesion Talus medial 3 × 8 IV Table 1 Patients characteristics, Surgical Intervention, Trauma mechanism, Findings N/A – Due to an osteochondral comminution fracture of the anterior border of the distal tibia defect sizing was not applicable Age [years] Sex Side Diagnosis (in addition to syndesmotic instability) Localization of chondral lesion Size [mm] ICRS Grade Surgical Intervention (in addition to ankle arthroscopy) Trauma 57 Male right Chondral lesion, medial malleolus fracture, pos- terior tibia fracture Talus medial 5 × 5 IV Microfracture, 2x tri- cortical 3.5 mm stain- less steel screws, ORIF medial malleolus Skiing 46 Male left Medial malleolus fracture Suture-endobutton, ORIF medial malleolus Motorcycle riding 24 Female right Deltoid rupture Suture-endobutton Bicycling 27 Male left Chondral lesion Tibia 10 × 4 IV Microfracture, 2x tricortical 3.5 mm stainless steel screws Ski mountaineering 52 Male left Chondral lesion, medial malleolus frac- ture, Chaput tubercle fracture Talus medial 4 × 4 IV Microfracture, percuta- neous fixation medial malleolus, 2x tricortical 3.5 mm stainless steel screws Walking 74 Male right Medial malleolus fracture, posterior tibia fracture 2x tricortical 3.5 mm stainless steel screws Cross-country skiing 41 Male left Chondral lesion, poste- rior tibia fracture Talus medial 4 × 4 I 2x tricortical 3.5 mm stainless steel screws Ski mountaineering 49 Male right Deltoid rupture, poste- rior tibia fracture 2x tricortical 3.5 mm stainless steel screws Martial Arts 55 Male right Chondral lesion, del- toid rupture posterior tibia fracture Talus medial 8 × 6 IV Microfracture, 2x tricortical 3.5 mm stainless steel screws, Hiking 61 Male left Deltoid rupture 2x tricortical 3.5 mm stainless steel screws Hiking 23 Male left Medial malleolus fracture, posterior tibia fracture 2x tricortical 3.5 mm stainless steel screws Walking 40 Male right Osteochondral lesion, posterior tibia fracture Tibia N/A IV Debridement of unsta- ble cartilage/bone, ORIF medial malleolus, 2x tricortical 3.5 mm stainless steel screws Ski mountaineering 43 Male left Osteochondral lesion, medial malleolus fracture, posterior tibia fracture Tibia, Talus medial 22x4x4, 10 × 5 IV, III Debridement of unsta- ble cartilage/bone, ORIF medial malleolus, 2x tricortical 3.5 mm stainless steel screws Walking 33 Male left Deltoid rupture, poste- rior tibia fracture 2x tricortical 3.5 mm stainless steel screws Walking 69 Male right Chondral lesion, del- toid rupture, posterior tibia fracture Talus medial 10 × 5 IV Microfracture, 2x tricortical 3.5 mm stainless steel screws, Walking 63 Male right 2x tricortical 3.5 mm stainless steel screws Hiking 40 Male right 2x tricortical 3.5 mm stainless steel screws Hiking 68 Female right Chondral lesion Talus medial 3 × 8 IV Debridement of unsta- ble cartilage/bone, 2x tricortical 3.5 mm stainless steel screws Walking Table 1 Patients characteristics, Surgical Intervention, Trauma mechanism, Findings N/A – Due to an osteochondral comminution fracture of the anterior border of the distal tibia defect sizing was not applicable Page 4 of 5 Fraissler et al. Ethics approval and consent to participate This publication was performed in accordance with the ethical standards laid down in the 1964 Declarations of Helsinki. Informed consent was obtained orally from all participants. The study design and oral informed consent proce- dure have been approved by the ethics committee of the Medical University Graz, Austria (reference number EK33–305 ex 20/21). g 18. Fritschy D. Une lésion rare de la cheville chez les skieurs de compétition [A rare injury of the ankle in competition skiiers]. Schweiz Z Med Trauma- tol. 1994;(1):13–6. 18. Fritschy D. Une lésion rare de la cheville chez les skieurs de compétition [A rare injury of the ankle in competition skiiers]. Schweiz Z Med Trauma- tol. 1994;(1):13–6. Abbreviations MFC: Maisonneuve fracture complex; ICRS: International Cartilage Repair Society. i 8. Yoshimura I, Naito M, Kanazawa K, Takeyama A, Ida T. Arthroscopic find- ings in Maisonneuve fractures. J Orthop Sci. 2008;13:3–6. 8. Yoshimura I, Naito M, Kanazawa K, Takeyama A, Ida T. Arthroscopic find- ings in Maisonneuve fractures. J Orthop Sci. 2008;13:3–6. Declarations 17. Imade S, Takao M, Miyamoto W, Nishi H, Uchio Y. Leg anterior compart- ment syndrome following ankle arthroscopy after Maisonneuve fracture. Arthrosc J Arthrosc Relat Surg. 2009;25:215–8. Acknowledgements 9. Stufkens SA, van den Bekerom MPJ, Doornberg JN, van Dijk CN, Kloen P. Evidence-based treatment of Maisonneuve fractures. J Foot Ankle Surg. 2011;50:62–7. We are thankful for the research grant from the Paracelsus Medical University Salzburg, Austria, covering the publication fee. 10. Hintermann B, Regazzoni P, Lampert C, Stutz G, Gächter A. Arthroscopic findings in acute fractures of the ankle. J Bone Jt Surg. 2000;82:345–51. 10. Hintermann B, Regazzoni P, Lampert C, Stutz G, Gächter A. Arthroscopic findings in acute fractures of the ankle. J Bone Jt Surg. 2000;82:345–51. Conclusions Ankle arthroscopy is helpful to detect and treat cartilage lesions of talus and tibia in MFC, as well as to guide fibu- lar reduction. To avoid secondary complications, we do not recommend using endobutton fixation for MFC and early screw removal. Furthermore, the long-term results of this technique compared to conventional open surgery need to be evaluated in a larger patient cohort. Nonethe- less, due to the high rate of chondral lesions, addressing these arthroscopically may contribute to better postop- erative results. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 16. Kalyani BS, Roberts CS, Giannoudis PV. The Maisonneuve injury: a com- prehensive review. Orthopedics. 2010;33:190–5. Funding Th h The authors received no financial support for the research, authorship. The publication fee of this article will be paid by the Paracelsus Medical University Salzburg, Austria. g 14. Brittberg M, Peterson L. Introduction of an articular cartilage classification ICRS Newsletter 1998;1:5–8. 15. Hannon CP, Bayer S, Murawski CD, Canata GL, Clanton TO, Haverkamp D, et al. Debridement, curettage, and bone marrow stimulation: proceed- ings of the international consensus meeting on cartilage repair of the ankle. Foot Ankle Int. 2018;39(1_suppl):16S–22S. References 1. Maisonneuve M. Recherches sur la fracture du péroné. Arch Gen Med. 1840;7:165–87. 2. Pankovich AM. Maisonneuve fracture of the fibula. J Bone Joint Surg Am. 1976;58:337–42. 3. Merrill KD. The Maisonneuve fracture of the fibula. Clin Orthop Relat Res. 1993;(287):218–23.i 4. del Castillo J, Geiderman JM. The Frenchman’s fibular fracture (Maison- neuve fracture). J Am Coll Emerg Physicians. 1979;8:404–6. 5. Obeid EM, Amr M, Hirst P, Paul A. Percutaneous fixation of Maisonneuve and Maisonneuve-type fractures: a minimally invasive approach. Injury. 1998;29:619–22. 6. Babis GC, Papagelopoulos PJ, Tsarouchas J, Zoubos AB, Korres DS, Niki- foridis P. Operative treatment for Maisonneuve fracture of the proximal fibula. Orthopedics. 2000;23:687–90. 7. Sproule JA, Khalid M, O’Sullivan M, McCabe JP. Outcome after surgery for Maisonneuve fracture of the fibula. Injury. 2004;35:791–8. 7. Sproule JA, Khalid M, O’Sullivan M, McCabe JP. Outcome after surgery for Maisonneuve fracture of the fibula. Injury. 2004;35:791–8. Authors’ contributions 11. Loren GJ, Ferkel RD. Arthroscopic assessment of occult intra-artic- ular injury in acute ankle fractures. Arthrosc J Arthrosc Relat Surg. 2002;18:412–21. Lukas Fraissler was responsible for conception and design, acquisition and interpretation of data and drafting the manuscript. Lukas A. Holzer and Georg Mattiassich have made substantial contributions to conception and design and helped revising it critically for important intellectual content. Lars Brun- nader was responsible for ethics approval. All authors read and approved the final manuscript. 12. Ferkel RD, Brown DS. Diagnostic arthroscopic examination. In: Ferkel RD, editor. Foot and ankle arthroscopy. 2nd ed. Philadelphia: Wolters Kluwer; 2017. p. 138–54. 13. Lui TH, Ip K, Chow HT. Comparison of radiologic and arthroscopic diag- noses of distal tibiofibular syndesmosis disruption in acute ankle fracture. Arthrosc J Arthrosc Relat Surg. 2005;21:1370.e1–7. 13. Lui TH, Ip K, Chow HT. Comparison of radiologic and arthroscopic diag- noses of distal tibiofibular syndesmosis disruption in acute ankle fracture. Arthrosc J Arthrosc Relat Surg. 2005;21:1370.e1–7. Discussion Treatment of MFC varies widely; traditional therapy con- sists of open or percutaneous techniques with restora- tion of the ankle mortise and syndesmosis screw fixation. Kalyani [16] reported in a review, including 61 patients in 4 studies, of excellent results in 47.54%, good in 40.98%, fair in 4.92% and poor in 6.55% as well as an ankle arthro- sis rate of 16% after an follow-up period from 25.3 months to 6.4 years. Furthermore, a high rate of associated car- tilage lesions in common ankle fractures of up to 79.2% [10, 11] and 100% in MFC [8] have been described. According to this data, a high rate of concomitant carti- lage injuries is confirmed in our study. Therefore, ankle Fig. 3 In one patient a 22x4x4 mm osteochondral fragment of the anteromedial edge of the distal tibia had to be removed Fig. 3 In one patient a 22x4x4 mm osteochondral fragment of the anteromedial edge of the distal tibia had to be removed Fig. 2 A variety of (osteo-)chondral lesions were found arthroscopically: cartilage hematoma (a) as well as instable talar (b) and tibial (c) cartilage injuries, which were debrided and subchondral bone microfractured Fig. 2 A variety of (osteo-)chondral lesions were found arthroscopically: cartilage hematoma (a) as well as instable talar (b) and tibial (c) cartilage Fig. 2 A variety of (osteo-)chondral lesions were found arthroscopically: cartilage hematoma (a) as well as instable talar (b) and tibial (c) cartilage injuries, which were debrided and subchondral bone microfractured Page 5 of 5 Fraissler et al. BMC Musculoskelet Disord (2021) 22:821 Page 5 of 5 Page 5 of 5 Fraissler et al. BMC Musculoskelet Disord (2021) 22:821 Regarding limitations of this study, the small and het- erogenous patient collective has to be disclosed. Further- more, there was no control group, a short follow-up time and no long-time results available. On the other hand, the strength of this paper represents the largest num- ber of study participants with MFC who were treated arthroscopically in current literature and all patients were treated by a national-board certified foot and ankle surgeon. Competing interests Competing interests Competing interests The authors have no competing interests to declare. Competing interests The authors have no competing interests to declare. Author details 1 Department of Orthopedic Surgery, Klinik Diakonissen Schladming, Salz- burger Strasse 777, 8970 Schladming, Austria. 2 Paracelsus Medical University Salzburg, Strubergasse 21, 5020 Salzburg, Austria. 3 AUVA Trauma Center Klagenfurt am Wörthersee, Waidmannsdorfer Strasse 35, 9020 Klagenfurt am Wörthersee, Austria. Received: 17 March 2021 Accepted: 11 September 2021 Received: 17 March 2021 Accepted: 11 September 2021 Consent for publication Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. All methods were carried out in accordance with relevant guidelines and regulations. Images are entirely unidentifiable and there are no details on individuals reported within the manuscript.
https://openalex.org/W309312610	http://www.ecoagri.ac.cn/cn/article/pdf/preview/10.3724/SP.J.1011.2009.00517.pdf	Chinese	null	Temporal variation and spatial distribution of the root systemof corn in a soil profile	Zhongguo shengtai nongye xuebao	2,009	cc-by	1,740	刘晶淼安顺清廖荣伟任三学梁宏 (中国气象科学研究院北京 100081) 摘要玉米根系在土壤剖面中的分布是准确量化植被与气候相互作用不可缺少的参数, 也是玉米生产科学管理和节水农业发展的重要科学依据。在中国气象科学研究院固城生态环境与农业气象实验站内的大型根系观测系统中, 采用地下室玻璃窗观测法和方形整段标本法, 观测了“屯玉46 号”玉米的根深、根宽、根长和根重, 分析了玉米根长、根长密度、根重密度和根系粗度等在土壤剖面中的分布状况。结果表明, 玉米根长、根干重均随土壤深度的增加基本呈递减类型。吐丝期0～40 cm 土层根长占整层根长51.5%, 0～80 cm 土层占 76.2%, 0～120 cm 土层占90.5%。乳熟后期其分布趋势与吐丝期相似。玉米根系粗度随着土壤深度增加, 在上层呈减少分布型, 在下层呈增加分布型。乳熟后期, 玉米最大根深可达230 cm, 根长总量达8.288 km·m−2, 显示出该玉米品种有较发达的根系。通过玻璃窗观测的根深大于远离玻璃窗处的根深。关键词玉米根系分布土壤剖面根长根密度中图分类号: S513 文献标识码: A 文章编号: 1671-3990(2009)03-0517-05 * 科学仪器设备升级改造项目(2005JG100520)、公益性行业(气象)科研专项[GYHY(QX)200706030]和国家自然科学基金项目(40575057) 资助刘晶淼(1957~), 男, 博士, 博士生导师, 主要从事气候变化诊断及模拟和陆面过程参数化的研究。E-mail: jingmiaol@cams.cma.gov.cn 收稿日期: 2008-05-08 接受日期: 2008-08-21 中国生态农业学报 2009 年5 月第17 卷第3 期 Chinese Journal of Eco-Agriculture, May 2009, 17(3): 517−521 中国生态农业学报 2009 年5 月第17 卷第3 期 Chinese Journal of Eco-Agriculture, May 2009, 17(3): 517−521 DOI: 10. 3724/SP.J.1011.2009.00517 DOI: 10. 3724/SP.J.1011.2009.00517 DOI: 10. 3724/SP.J.1011.2009.00517 Temporal variation and spatial distribution of the root system of corn in a soil profile LIU Jing-Miao, AN Shun-Qing, LIAO Rong-Wei, REN San-Xue, LIANG Hong (Chinese Academy of Meteorological Sciences, Beijing 100081, China) Abstract The development of the root system of corn in soil profile is an indispensable parameter for the estimation of corn growth. The distribution of the root system can be used to evaluate the influence of climate on vegetative growth. This constitutes a creative scientific management and development system of water-saving agriculture. In the Gucheng Agro-meteorological Field Experimental Station of Chinese Academy of Meteorological Sciences, root length, root areal reach, root depth and root dry-weight of “Tunyu 46” corn were observed using clod sampling method and installed underground surface glazing. Observation data on the spatial and tem- poral distribution characters of the root system in the soil profile were then analyzed. The results show that root dry-weight and root length decrease with increasing soil depth. In spin silk period, root length in the 40 cm, 80 cm, and 120 cm soil layer is respectively 51.5%, 76.2% and 90.5% of total root length. Root length to total root length ratio in various soil layers is similar for both spin silk and late milk maturity periods. Root thickness decreases in upper soil layer and increases in lower soil layer with increasing soil depth. In the late milk maturity period, root depth may reach 230 cm, and total combined root length can reach 8.288 km· m−2. Clearly thus, root depth and the total root length of “Tunyu 46” are larger than those of other corn varieties. Root distribution charac- teristics show that the root system of “Tunyu 46” is a lot more developed and robust for defending drought. Based on data obtained from the installed glazing, root depth is much deeper than that observed far from the glazing. Key words Corn, Root distribution, Soil profile, Root length, Root density (Received May 8, 2008; accepted Aug. Temporal variation and spatial distribution of the root system of corn in a soil profile 21, 2008) 玉米根系在土壤剖面中的分布决定了其吸收养分和水分的能力, 也是准确量化植被与气候间相互作用不可缺少的参数。因此, 研究玉米根系在土壤剖面中的分布对玉米生产的科学管理和节水农业发 * 科学仪器设备升级改造项目(2005JG100520)、公益性行业(气象)科研专项[GYHY(QX)200706030]和国家自然科学基金项目(40575057) 资助刘晶淼(1957~), 男, 博士, 博士生导师, 主要从事气候变化诊断及模拟和陆面过程参数化的研究。E-mail: jingmiaol@cams.cma.gov.cn 收稿日期: 2008-05-08 接受日期: 2008-08-21 中国生态农业学报 2009 518 第17 卷第17 卷统中进行。该系统包括地下根系观测室、地上作物种植小区和大型电动防雨棚3 个主要部分。地下根系观测室南北走向, 深3.2 m, 宽2.5 m。在东西两侧各有12 个钢化玻璃观测窗(高3 m, 宽1.5 m, 厚度 20 mm), 每个玻璃观测窗与相应的种植小区土壤紧密相连。观测室顶部是用钢筋作骨架并覆盖泡沫塑料、沥青板和土, 起保温遮光作用。地下室南进入口设有两道防盗门, 地下室地面有两条排水沟。在24 个玻璃观测窗中选取8 个(东西侧各4 个) 安装根系微型观测管(长2.2 m, 外径6.0 cm), 在每个玻璃观测窗的垂直中线部位自上而下在40 cm、80 cm、120 cm、160 cm、200 cm 处安装5 根管子, 每个管向上倾斜10° 29′ 插入作物种植小区土壤内, 用电子窥镜在管内观测小区不同土壤深度的根系生长状况。展具有重要意义, 也可为全球变化研究中的区域土壤-植被-大气系统碳、水分、养分传输过程提供基础性资料。展具有重要意义, 也可为全球变化研究中的区域土壤-植被-大气系统碳、水分、养分传输过程提供基础性资料。性资料。虽然因作物根系生长在地下而导致研究其在土壤剖面中的分布存在许多困难, 但随着科学技术的发展以及农业生产和科学研究的迫切需要, 国内外对玉米根系的研究, 特别是在土壤剖面中分布规律的研究仍取得了显著进展。在国外, Jackson 等[1]研究了全球范围陆地生态系统中包括玉米在内的主要植物群落的根系分布、密度和生物量, 并比较了不同植物群落根系差异。Gale 等[2]给出描述根系在土壤剖面中分布的关系式。Teare 等[3]对玉米根深及其分布进行了定量描述, 玉米根系扩展范围一般为1.25 m, 大部分根系可达1.6 m, 有的接近2 m, 并指出玉米根系具有一种潜在逃避受旱机能。Liedgens 等[4] 总结了玉米根系在土壤剖面中分布的4 种类型, 即 ①随土壤深度稳定减少型; ②从土壤表层到较深土层根系急剧减少型; ③从土壤上层向下到最大根系生长深度, 随着深度增加根量增加, 再向下根量减少型; ④根系随深度分布呈无规律型。并指出, 这些分布型是受玉米植株发育和土壤环境条件影响而形成的。国外在玉米根系研究方法上也有明显进展, 近年来应用微型根系观测管技术观测根生长的空间和时间类型受到重视。与传统的土壤采样技术相比, 微型根系观测管是透明界面, 且观测时不破坏土壤和根系。Schröder 等[5]应用微型根系观测管技术系统地描述了玉米根系的空间分布, 还有一些科学家应用微型根系观测管技术研究了干旱、灌溉、施肥和耕作对玉米根系在土壤中分布的影响。种植小区共24 个, 东西两侧各12 个, 每个小区长4 m, 宽2 m, 各小区之间用水泥墙隔离, 深度为 3 m, 小区内土壤为原状土。在安装有微型根系观测管的8 个小区内, 安装有观测土壤湿度的TDR 和土壤温度自动观测仪。小区土壤为壤土, 0～200 cm 土层平均田间持水量为22.7%(占干土重), 平均凋萎湿度为5.0%(占干土重), 容重为1.37 g· cm−3。大型电动防雨棚长30 m, 宽15 m, 高5 m, 降水时覆盖小区, 无雨时退出。 1.2 玉米品种、特性及生育期 2007 年5 月12 日在24 个小区播种“ 屯玉46 号” 杂交中晚熟玉米, 行距50 cm, 株距30 cm, 人工灌溉供水, 及时除草。玉米生育期为5 月20 日出苗, 5 月27 日3 叶, 6 月10 日7 叶, 6 月21 日拔节, 7 月17 日抽雄, 7 月20 日开花, 7 月23 日吐丝, 8 月8 日乳熟, 8 月27 日成熟, 整个生育期108 d。 1.2 玉米品种、特性及生育期 2007 年5 月12 日在24 个小区播种“ 屯玉46 号” 杂交中晚熟玉米, 行距50 cm, 株距30 cm, 人工灌溉供水, 及时除草。玉米生育期为5 月20 日出苗, 5 月27 日3 叶, 6 月10 日7 叶, 6 月21 日拔节, 7 月17 日抽雄, 7 月20 日开花, 7 月23 日吐丝, 8 月8 日乳熟, 8 月27 日成熟, 整个生育期108 d。在国内, 李少昆等[6]研究了玉米根系干重在不同生育期的侧向和垂直分布模型, 并指出玉米成熟期根深可达160 cm。张喜英[7]研究了太行山山前平原冬小麦、夏玉米、谷子和高粱等作物的根系生长发育状况, 指出夏玉米最大根深为1.2 m, 80%以上的根系集中在0～40 cm 土层中。国内在根系研究方法上长期以来主要应用土壤采样技术, 近年来少数单位开始应用地下玻璃窗观测技术和微型根系观测管技术[8]。本文根据在中国气象科学研究院固城生态环境和农业气象实验站大型根系观测系统所观测的部分资料, 分析研究了“ 屯玉46 号” 玉米根系在土壤剖面中的分布状况。在国内, 李少昆等[6]研究了玉米根系干重在不同生育期的侧向和垂直分布模型, 并指出玉米成熟期根深可达160 cm。张喜英[7]研究了太行山山前平原冬小麦、夏玉米、谷子和高粱等作物的根系生长发育状况, 指出夏玉米最大根深为1.2 m, 80%以上的根系集中在0～40 cm 土层中。国内在根系研究方法上长期以来主要应用土壤采样技术, 近年来少数单位开始应用地下玻璃窗观测技术和微型根系观测管技术[8]。本文根据在中国气象科学研究院固城生态环境和农业气象实验站大型根系观测系统所观测的部分资料, 分析研究了“ 屯玉46 号” 玉米根系在土壤剖面中的分布状况。 1.3 观测项目及方法观测项目包括玉米生育期、根深、根宽、根长、根重等。在地下室玻璃窗上观测玉米根深和根宽, 6 月17 日开始观测直到成熟期, 共观测7 次。同时, 在地下观测室应用微型根系观测管技术观测不同深度根数、根长及根长密度。分别于8 月4 日(吐丝期) 和8 月25 日(成熟期)应用方形整段标本法, 在种植小区选取有代表性植株, 以其为中心, 取长50 cm, 宽30 cm 的长方形, 沿四周垂直挖土, 形成有根样的土柱, 一直挖到无根为止, 然后沿土柱自上而下每10 cm 取一土样。将土样用水浸泡、漂选和清洗, 得到干净根系样品。之后吸干根样表面水分, 用万分之一精度电子天平称量根重。再对粗根和细根进行分类, 粗根直接测量; 细根先测量其总重量, 然后选出一部分进行称重测量长度, 根据得出的长度 1 材料和方法 1.1 观测地点及设施观测工作在河北省定兴县中国气象科学研究院固城生态环境与农业气象试验站的大型根系观测系 1.1 观测地点及设施观测工作在河北省定兴县中国气象科学研究院固城生态环境与农业气象试验站的大型根系观测系刘晶淼等: 玉米根系在土壤剖面中的分布研究第3 期第3 期 519 2.2 玉米根长密度在土壤中的分布根长密度是单位土壤体积中的根长, 单位为 cm· cm−3, 它是根系长度的一种常用表示方法。本文每20 cm 厚土壤中的根长密度用式(1)求得: 3 20 cm cm cm 50 30 20 L ρ − ⋅ = × × 每层总根长（） (1) 图2 8 月4 日(a)和8 月25 日(b)玉米根长密度在土壤中的分布 Fig. 2 Distribution of corn root length density in various soil layer depths on August 4(a) and August 25(b), 2007 按照重量比例换算出细根总长度。最后在电烘箱中烘干, 称其干重。这样测出每层土壤中的根长、根重, 计算根长密度和根重密度。 2 结果与分析玉米根系一般按其发生时期、部位、形态和功能, 分为初生根(胚根)、次生根和支持根。根量的大小主要表现在根重和根长上。根重是抗倒性、抗旱性的主要指标之一。根长是衡量根系生长和吸收能力的重要指标。单位体积中的根长即根长密度是评估根系吸收水分和养分能力的重要依据。单株根长是衡量根系生长能力强弱的重要指标。 2.1 玉米根长在土壤中的分布 2007 年8 月4 日和25 日两次应用方形整段标本法所取得的土壤逐层根长资料表明, 玉米根长基本随土壤深度增加而递减, 如8 月4 日(吐丝期, 图 1a)0～20 cm 土层根长占整层根长的34.5%, 20～40 cm 土层占17.0%, 40～60 cm 土层占11.0%, 60～80 cm 土层占13.7%, 80～100 cm 土层占7.2%, 100～ 120 cm 土层占7.1%, 120～140 cm 土层占5.7%, 140～160 cm 土层占2.9%, 160～170 cm 土层仅占 0.3%。该期0～40 cm 土层玉米根长占整层根长的 51.5%, 0～80 cm 土层占整层的76.2%, 0～120 cm 土层占整层的90.5%。8 月25 日(乳熟后期, 图1b)玉米根长在土壤中的分布趋势与8 月4 日相似, 只是最大深度达到230 cm。玉米成熟期玉米根长总量达 8.288 km· m−2。 · 。 · 。由图2 可知, 随着深度增加, 玉米根长密度基本上呈递减型。8 月4 日0～20 cm 土层的根长密度最大, 达1.08 cm· cm−3, 140～160 cm 土层最小, 仅为0.09 cm· cm−3。8 月25 日与8 月4 日相比, 相应层次的根长密度均较大, 0～20 cm 土层达1.48 cm· cm−3, 比8 月4 日大0.40 cm· cm−3。160 cm 以下土层根长密度均小于0.042 cm· cm−3。 2.3 玉米根重(干重)密度在土壤中的分布根重密度是指单位体积内的根重, 以g· cm−3 为单位。文中每20 cm 厚土层的根重密度计算与根长密度相同。其计算式为: 3 20 cm (g cm ) 50 30 20 g ρ − ⋅ = × × 土层内根重 (2) 由图3 可知, 玉米根重密度基本上随土壤深度增加而减少。8 月4 日0～20 cm 土层根重密度最大, 为0.000 66 g· cm−3, 60～80 cm 土层减少到0.000 14 g· cm−3, 140～160 cm 土层最小, 仅为0.000 02 g· cm−3。8 月25 日玉米根重密度在土壤剖面的分布趋势与8 月4 日基本相似, 只是深度达到230 cm。图1 8 月4 日(a)和8 月25 日(b)不同厚度土层玉米根长占整层根长的比例 Fig. 1 The proportions of corn root length to total length in various soil layer depths on August 4(a) and August 25(b), 2007 (2) 图1 8 月4 日(a)和8 月25 日(b)不同厚度土层玉米根长占整层根长的比例 Fig. 1 The proportions of corn root length to total length in various soil layer depths on August 4(a) and August 25(b), 2007 520 中国生态农业学报 2009 第17 卷第17 卷图4 8 月4 日(a)和8 月25 日(b)玉米根系粗度在土壤中的分布 Fig. 4 Distribution of corn root thickness in various soil layer depths on August 4(a) and August 25(b), 2007 图3 8 月4 日(a)和8 月25 日(b)玉米根重密度在土壤中的分布 Fig. 3 Distribution of corn root dry weight density in various soil layer depths on August 4(a) and August 25(b), 2007 Fig. 3 Distribution of corn root dry weight density in various soil layer depths on August 4(a) and August 25(b), 2007 图4 8 月4 日(a)和8 月25 日(b)玉米根系粗度在土壤中的分布 Fig. 4 Distribution of corn root thickness in various soil layer depths on August 4(a) and August 25(b), 2007 图4 8 月4 日(a)和8 月25 日(b)玉米根系粗度在土壤中的分布 Fig. 4 Distribution of corn root thickness in various soil layer depths on August 4(a) and August 25(b), 2007 下生长速率最大, 为5.39 cm· d−1。其次为6 月17 日到6 月29 日期间, 即7 叶期到拔节期为3.17 cm· d−1, 生长速率最小的为7 月7 日到7 月17 日, 即拔节期到抽雄期, 仅为0.7 cm· d−1。其他23 个小区的观测结果大多与EN01 小区类似。参考文献 [1] Jackson R. B., Canadell J., Ehleringer J. R., et al. A global analysis of root distributions for terrestrial biomes[J]. Oecologia, 1996, 108: 389−411 [2] Gale M. R., Grigal D. F. Vertical root distributions of northern tree species in relation to successional status[J]. Canadian Journal of Forest Research, 1987, 17: 829−834 [3] Teare I. D., Peet M. M. Crop-Water Relations[M]. New York: A. Wiley Interscience Publication, 1982: 186−208 [4] Liedgens M., Richner W. Minirhizotron observations of the spatial distribution of the maize root system[J]. Agronomy Journal, 2001, 93: 1097−1104 [5] Schröder U., Söndgerath D. The concept of biological time for computing the switching points of a growth model for winter wheat[J]. Ecological Modelling, 1996, 88(1/3): 1−8 减少再增加分布。通过玻璃窗观测的根深大于远离玻璃窗处根深, 玉米成熟期最大根深可达230 cm, 上层土壤(0～40 cm土层)集中了50%的根量, 0～100 cm 土层集中了80%以上的根量。比一般夏玉米根量随深度减少的程度缓和, 显示出“ 屯玉46 号” 玉米根系发达, 有利于吸收深层水分。减少再增加分布。通过玻璃窗观测的根深大于远离玻璃窗处根深, 玉米成熟期最大根深可达230 cm, 上层土壤(0～40 cm土层)集中了50%的根量, 0～100 cm 土层集中了80%以上的根量。比一般夏玉米根量随深度减少的程度缓和, 显示出“ 屯玉46 号” 玉米根系发达, 有利于吸收深层水分。土层根长占整层根长仅51.5%, 乳熟后期根长总量达8.288 km· m−2。这些明显差异可能与玉米品种特性有关, 因为根系的大小和深度是由遗传决定的, 且随地上部分的改变而扩大。“ 屯玉46 号”玉米生育期比一般夏玉米长, 属于大株型, 根系发达。另外, 通过地下室玻璃窗观测的玉米根深和根宽量值能否代表实际的状况呢？如果玻璃窗与小区土壤剖面紧密相连, 则所观测的结果应该能代表。但本文中地下室玻璃窗在安装时因与小区土壤剖面有间隙, 只得用细土填在空隙中, 因此在玻璃窗与土壤剖面连接处, 环境条件与远离玻璃窗的小区土壤的环境条件有差异, 因为又是第1 年进行观测, 故观测结果可能与实际状况有差异。这从8 月25 日在EN01 小区用两种方法同时观测的根深资料可看出, 该日通过地下室玻璃窗观测的最大根深为290 cm, 而在该小区应用方形整段标本法可获得的最大根深为230 cm, 前者比后者深60 cm。这种差异随着时间的推移, 当界面处土壤环境与远离玻璃小区土壤环境接近时, 观测结果就会接近真实状况。参考文献 (3) 由图4 可知, 玉米根系粗度随土壤深度增加呈先减少后增加趋势。8 月4 日0～20 cm 土层根系粗度为0.001 163 g· cm−1, 60～80 cm 土层减少到 0.000 258 g· cm−1, 到140～160 cm 土层增加到 0.000 542 g· cm−1。8 月25 日玉米根系粗度在土壤剖面的分布趋势与8 月4 日基本相似, 只是在200～ 220 cm 土层仍达到0.000 447 g· cm−1。根宽是玉米根系生长状况的另一个重要方面, 它反映根系在水平方向上的分布状况, 影响水分和养分对植株的有效性。由地下室24 个小区玻璃窗上的观测结果显示: 根宽随着玉米根的加深而变小。对于EN07 小区的单株玉米根系, 6 月17 日根深80 cm, 根宽为125 cm; 6 月29 日根深85 cm, 根宽为 100 cm; 7 月7 日根深100 cm, 根宽为71 cm; 7 月17 日根深122 cm, 根宽为75 cm; 8 月4 日根深215 cm, 根宽65 cm; 8 月14 日根深243 cm, 根宽65 cm; 8 月 25 日根深290 cm, 根宽65 cm。 2.5 玉米根系深度和根宽状况根系深度与作物水分供给关系密切。地下观测室24 个小区的玻璃窗7 次观测资料显示, 玉米根系深度随生育期的延长而加深, 大多小区在成熟期根深达300 cm。如东侧自北数第1 小区(EN01), 6 月 17 日(7 叶期)根深为90 cm, 6 月29 日(拔节期)为128 cm, 7 月7 日(拔节后期)为138 cm, 7 月17 日(抽雄期) 为145 cm, 8 月4 日(吐丝期)为242 cm, 8 月14 日(乳熟期)为270 cm, 8 月25 日(乳熟后期)为290 cm。玉米在不同生育期间其根系向下生长的速率存在差异, 其中7 月17 日到8 月4 日即抽雄期到吐丝期向 2.5 玉米根系深度和根宽状况根系深度与作物水分供给关系密切。地下观测室24 个小区的玻璃窗7 次观测资料显示, 玉米根系深度随生育期的延长而加深, 大多小区在成熟期根深达300 cm。如东侧自北数第1 小区(EN01), 6 月 17 日(7 叶期)根深为90 cm, 6 月29 日(拔节期)为128 cm, 7 月7 日(拔节后期)为138 cm, 7 月17 日(抽雄期) 为145 cm, 8 月4 日(吐丝期)为242 cm, 8 月14 日(乳熟期)为270 cm, 8 月25 日(乳熟后期)为290 cm。玉米在不同生育期间其根系向下生长的速率存在差异, 其中7 月17 日到8 月4 日即抽雄期到吐丝期向从以上分析结果可以看出,“ 屯玉46 号”玉米根量(根长和根干重)在土壤剖面中的分布基本上是随着深度加深呈减少型, 这与大多数玉米根量分布的研究结论相类似。但在数量和根深方面存在不同, 如张喜英在太行山山前平原测得的夏玉米最大根深为1.2 m 左右, 80%以上根系集中在0～40 cm 土层, 成熟期根长总量为4 km· m−2。而本文“ 屯玉46 号” 玉米8 月25 日实测的最大根深达230 cm, 0～40 cm 刘晶淼等: 玉米根系在土壤剖面中的分布研究第3 期第3 期 521 3 小结 [6] 李少昆, 涂华玉, 张旺峰. 玉米健壮素对玉米生长和产量的影响[J]. 新疆农业科学, 1991 (6): 243−246 本文应用地下室玻璃窗观测法和方形整段标本法获得“ 屯玉4 号” 玉米的根系资料, 该玉米品种根量(根长和根干重)在土壤剖面中随深度增加呈减少分布型, 根系粗度在土壤剖面中随深度增加呈先 [7] 张喜英. 作物根系与土壤水分利用[M]. 北京: 气象出版社, 1999: 171−186, 44−45 [8] 佟屏亚, 程延年. 玉米生育和产量模型[M]. 北京: 中国农业科技出版社, 1997: 90−97
https://openalex.org/W4376603127	https://www.researchsquare.com/article/rs-2915108/latest.pdf	English	null	Lipid metabolism disturbances in papillary thyroid cancer patients and the relationship with iodine nutrition status	Research Square (Research Square)	2,023	cc-by	8,121	Lipid metabolism disturbances in papillary thyroid cancer patients and the relationship with iodine nutrition status Yuqian Zhai Chinese Center for Disease Control and Prevention, Harbin Medical University Xuebin Wang Chinese Center for Disease Control and Prevention, Harbin Medical University Jie Luo Chinese Center for Disease Control and Prevention, Harbin Medical University Xueqian Wang The Harbin Medical University Cancer Hospital, Harbin Medical University Zhonghao Liu The Harbin Medical University Cancer Hospital, Harbin Medical University Junrong Wang The Harbin Medical University Cancer Hospital, Harbin Medical University Zhiyong Liu Chinese Center for Disease Control and Prevention, Harbin Medical University Xionghui Mao The Harbin Medical University Cancer Hospital, Harbin Medical University Lijun Fan (  fanlijun@hrbmu.edu.cn ) Chinese Center for Disease Control and Prevention, Harbin Medical University Research Article Keywords: Iodine nutrition, Thyroid cancer, Thyroid nodules, Serum lipids Posted Date: May 15th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2915108/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Chinese Center for Disease Control and Prevention, Harbin Medical University Jie Luo Lipid metabolism disturbances in papillary thyroid cancer patients and the relationship with iodine nutrition status 1. Introduction Thyroid cancer is the most common malignancy of the endocrine system and its incidence has shown a marked increase in recent years [1]. Thyroid tumors are mainly classified into papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), medullary thyroid cancer (MTC) and undifferentiated or anaplastic thyroid cancer (ATC) according to histology subtypes [2]. Among them, PTC is the most common subtype, accounting for more than 90% of all thyroid cancers. According to the 1975–2016 SEER Cancer Statistics Review, the incidence of PTC alone increased from 4.8 per 100,000 people to 14.9 per 100,000 people [3]. Although PTC is usually a well-differentiated type of thyroid cancer with a favorable prognosis, it has wide biological and clinical repercussions. Studies have shown that patients with thyroid cancer are more likely to coexist with other types of cancer [4, 5], and other studies have demonstrated that thyroid cancer patients frequently coexist with pathoglycemia and dyslipidemia [6, 7]. However, the pathophysiological effects of PTC, particularly on lipid metabolism, are not yet completely understood. Iodine is an indispensable component of thyroid hormone biosynthesis [8], and it plays a crucial role in physiological metabolism, especially in lipid synthesis and decomposition [9]. The effect of iodine on health is bi-directional. Insufficient iodine intake can result in iodine deficiency disease, including goiter, hypothyroidism, hypothyroxinemia and other physical impairments, whereas excessive iodine intake can result in hyperthyroidism, hypothyroidism, goiter, autoimmune diseases, etc [10]. Thyroid cancer is related to both iodine nutrition and its metabolism, and these relationships are mutual. Iodine may have an influence on the occurrence of thyroid cancer. Blomberg M et al. found a substantial rise in the incidence of thyroid cancer after salt iodization was implemented [11]. This finding was also supported by a domestic study conducted by Liu L et al, in a population-based epidemiological survey, it was found that the prevalence of PTC was much higher in high iodine areas than in moderate iodine areas, and that excessive iodine intake increased the risk of PTC [12]. In addition, the development of thyroid cancer may resulted in alterations in iodine metabolism related to the cancer-damaged thyroid. Xing et al. believed that the iodide-handling machinery is often compromised in thyroid cancer, especially in advanced thyroid cancers, resulted in aberrant iodine metabolism [13]. [13]. Although the physiological functions of iodine are mainly realized through thyroid gland, iodine can also affect the body through oxidative stress [14]. Research Article Page 1/11 Abstract Objective: Cancer patients are frequently accompanied by problems in lipid metabolism. Uncertainty exists as to whether changes in serum lipids occur in patients with papillary thyroid cancer (PTC) and their relationship with iodine nutrition remains obscure. The aim of this study was to explore lipid metabolism disturbances in PTC patients and their relationship with iodine nutrition status. Methods: A total of 909 patients who were initially diagnosed with PTC and 183 patients who were initially diagnosed with benign thyroid nodules were enrolled in this study. The serum iodine concentration (SIC), the urine iodine concentration (UIC) and nine serum lipids indicators were measured. The generalized linear model (GLM) together with other statistical methods were used to determine whether there were differences in serum lipids between patients with PTC and those with benign thyroid nodules. Results: After adjusting for baseline information, triglycerides (TG) levels in the control group (4.29±1.21) were significantly higher than in the cancer group (1.59±1.25). The rate of abnormal thyroid function was significantly lower in the patients with PTC than in the patients with benign nodules. In the PTC patients, different clinicopathological features had an impact on thyroid function, as reflected by a significant increase in FT3 in PTC with lymph node metastases, a significant increase in TSH, TGAb, and TPOAb, and a significant decrease in FT4 in PTC with AITD. Correlation analysis revealed weak to moderate correlations between iodine nutritional status, thyroid function, and serum lipids. In benign thyroid nodule patients, LDL-C and ApoB values in patients with benign thyroid nodules were significantly higher in the high SIC group than in the adequate and deficient groups. In PTC patients. ApoE levels in the low UIC group were significantly higher than in the middle and high UIC groups. Mediating effects were used to analyze the effect of iodine nutrition on the serum lipids, it showed that the total and direct effects of iodine nutritional status on serum lipids were significant, and the mediating effect of thyroid function was not significant. Conclusion: TG levels in the control group were significantly higher than in the PTC group. Iodine nutritional status influences lipids, and an excess or deficient iodine nutrition increases the risk of dyslipidemia in patients with thyroid nodule. Iodine nutritional status had a direct effect on serum lipids. 2.1 Study population Patients of various ages with initially diagnosed as thyroid nodules were enrolled between November 2015 and April 2019 in the Third Affiliated Hospital of Harbin Medical University. The inclusion criteria were as follows: (1) patients newly diagnosed as thyroid nodules, and (2) those who will undergo surgical treatment at the Third Affiliated Hospital of Harbin Medical University. Some subjects were excluded based on the following criteria: (1) inability to undergo an operation, (2) previous diagnosis of other thyroid disorders, (3) abnormal liver or kidney functions, and (4) consumption of iodine-rich food within 48 hours before blood collection. In total, 1092 patients were included in the study. Among them, 909 were diagnosed with PTC, and 183 patients were diagnosed with benign thyroid nodule. The Ethics Committee of Harbin Medical University approved this study (HRBMUECDC 20210410). It was conducted in accordance with the provisions of the Declaration of Helsinki. Written informed consent was obtained from all participants. The Ethics Committee of Harbin Medical University approved this study (HRBMUECDC 20210410). It was condu Declaration of Helsinki. Written informed consent was obtained from all participants. 1. Introduction Consequently, an abnormal intake of iodine could disturb lipid metabolism. As for iodine deficiency, an epidemiological study by Rönnefarth et al. showed that adolescents with iodine-deficient goiter had higher total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels compared with non-goiter controls [15]. Wang et al. indicated that low iodine intake decreased thyroid hormone levels and increased levels of apolipoprotein B (ApoB), TC, non-high- density lipoprotein cholesterol (HDL-C), and LDL-C [16]. As for iodine excess, Liu et al. showed adults living in areas with high iodine levels (urinary iodine greater than 300 µg/L) may have an increase in blood glucose and blood pressure, which may increase their risk of hypertension and diabetes [14]. Another study found that the rate of HDL-C abnormalities was considerably higher in residents with household drinking water iodine levels above 300 µg/L than in the control group. High iodine was a risk factor for abnormally low HDL-C, and drinking highly iodized water increased the risk of dyslipidemia [17]. In addition, abnormal serum lipid levels can arise in thyroid cancer patients. Li D et al. reported higher levels of triglycerides (TG) and LDL-C/HDL-C, and significantly lower levels of apolipoprotein A1 (ApoA1) and HDL-C in women with thyroid cancer compared to controls [2]. Cari M et al. found that the components of the metabolic syndrome can influence the development, progression, and prognosis of thyroid cancer through several mechanisms, and a study from Iceland indicated that men with high triglycerides had a higher risk of developing thyroid cancer [18]. Many studies have demonstrated that iodine metabolism disorders or serum dyslipidemia can occur in thyroid cancer, but their relationship between them has not been looked at in depth very often. In order to examine lipid alternations in thyroid cancer patients and the effect of iodine on serum lipids, this study examined 1092 patients with thyroid disease. We anticipate that our results will provide clues for the regulation of serum lipids in patients with thyroid disease. Page 2/11 Page 2/11 2.3 Laboratory test Urinary iodine concentration was measured using arsenic-cerium catalytic spectrophotometry according to the China national health industry standard WS/T107.1-2016 [19]. Serum iodine concentration was detected using arsenic-cerium catalytic spectrophotometry according to the China national health industry standard WS/T572-2017 [20]. A 3100 automated biochemistry instrument (manufactured by Hitachi, Japan) was used to test lipid parameters ApoA1, ApoB, apolipoprotein E (ApoE), TG, TC, HDL-C, LDL-C, lipoprotein a (Lp(a)), and non-esterified fatty acids (NEFA). According to the World Health Organization (WHO) recommendations for assessing individual iodine status, adults with serum iodine concentration below 45 µg/L were deemed iodine deficient, those between 45 and 90 µg/L were deemed iodine adequate, and those above 300 µg/L were deemed iodine excessive. Urine iodine concentrations were grouped into three categories, < 100 µg/L, 100–299 µg/L, and > 300 µg/L. Reference ranges for thyroid function were 2.8–7.1 pmol/L for free triiodothyronine (FT3), 12–22 pmol/L for free thyroxine (FT4), 0.27–4.2 mIU/L for thyroid stimulating hormone (TSH), 0 ~ 115IU/mL for Thyroglobulin antibody (TgAb), and 0 ~ 34IU/mL for Thyroid peroxidase antibody (TPOAb). Reference levels for serum lipid concentrations were: NEFA: 0.1– 0.77 mmol/L, HDL-C: > 1.04 mmol/L, LDL-C: 0–3.12 mmol/L, ApoA1: 1–1.6 g/L, ApoB: 0.6–1.2 g/L, ApoE: 2.7–4.9 mg/dL; Lp(a): 0–300 mg/L, TC: 0–5.2 mmol/L, TG: 0–1.7 mmol/L, LDL-C/HDL-C: 0–2.5mmol/L. 2.2 Sample collection From each participant, 5 mL of whole blood and 10 mL of morning urine were collected, after fasting and avoiding alcohol and medicine for 12 h before any medical treatment. For the blood samples, 1 h after centrifugation, the serum was extracted, and 400 µL aliquots of each sample were stored at − 80°C until use. The urine samples were centrifuged at 3,000 rpm for 15 min and then separated into 10 tubes, 1 of which was analyzed for urine iodine concentration, while and the others were used for other experiment. All of the samples were immediately frozen and stored at − 80°C until analysis. Basic information, comprising demographic characteristics (including gender, age, smoking history, drinking history, etc.), clinical information (including thyroid function indicators), and pathological information (including pathologic characteristics, etc.), was extracted from the electronic medical record. 2.3 Laboratory test 2.4 Statistical analyses The differences in continuous (BMI, age, urine iodine concentration, serum iodine concentration) and categorical variables (gender, smoking and alcohol consumption) across groups were analyzed using the t-test / rank sum test and the chi-squared test, respectively. The general linear model (GLM) was used to analyze whether there were differences in serum lipids status between PTC patients and benign controls, as well as between the various PTC patients statuses. Pearson correlation analyses were used to describe correlations between iodine nutritional status, thyroid function, and serum lipids. All the statistical analyses except correlation were performed using SAS software version 9.1.3 (SAS Institute, Incorporation, Cary, North Carolina, USA), and correlation analyses were performed by ggcorrplot package in R platform (version 4.0.5). All statistical tests were two-sided using an alpha level of 0.05. 3.1 Baseline characteristics A total of 1092 subjects were included in the study, including 909 PTC patients (PTC group), and 183 patients with benign thyroid nodules (benign group). Table 1 presents the baseline characteristics of the study populations. The PTC group had a lower average age than the benign group (P < 0.05). The rate of smoking was decreased in the PTC group compared to the benign group (p < 0.05). Other indicators did not differ significantly between the two groups. Page 3/11 Page 3/11 Table 1 Baseline characteristics of the study populations based on group variable cancer control statistic value P n = 907 n = 183 Age (y) 45.92±10.48 52.52±9.87 59.39 < 0.0001 Gender male 117(79.05%) 31(20.95%) female 615(83.56%) 121(16.44%) 1.76 0.1854 BMI (cm/kg2) 24.63±3.69 24.08±3.34 3.25 0.0718 smoke yes 68(9.52%) 27(18.36%) no 646(90.48%) 120(81.63%) 9.80 0.0018 Alcohol consumption yes 28(75.68%) 9(24.32%) no 685(83.43%) 136(16.57%) 1.52 0.2184 The iodine nutrition status, thyroid function indicators and lipid indicators were presented in Table 2. In the PTC group, the median of urine iodine concentration (UIC) was 143.36 µg/L (95% confidence intervals: 94.08–227.94 µg/L), slightly lower than in the control group (median: 146.28 µg/L, 95% confidence intervals: 112.89–236.07 µg/L). There was no significant difference between the median of serum iodine concentration (SIC) of the PTC group and the benign group, which were 77.61 µg/L (65.22–89.80 µg/L) and 77.94 µg/L (66.50–93.33 µg/L), respectively. Even after adjusting for age, gender, smoking, and alcohol consumption, the median UIC between the PTC group and the benign group remained significantly different. The iodine nutrition status, thyroid function indicators and lipid indicators were presented in Table 2. In the PTC group, the median of urine iodine concentration (UIC) was 143.36 µg/L (95% confidence intervals: 94.08–227.94 µg/L), slightly lower than in the control group (median: 146.28 µg/L, 95% confidence intervals: 112.89–236.07 µg/L). There was no significant difference between the median of serum iodine concentration (SIC) of the PTC group and the benign group, which were 77.61 µg/L (65.22–89.80 µg/L) and 77.94 µg/L (66.50–93.33 µg/L), respectively. Even after adjusting for age, gender, smoking, and alcohol consumption, the median UIC between the PTC group and the benign group remained significantly different. 3.1 Baseline characteristics Table 2 Serum lipid and thyroid function of the study populations based on group variable cancer control F P n = 907 n = 183 Iodine nutrition UIC (µg/L) 143.36 (94.08, 227.94) 146.28 (112.89, 236.07) 4.16 0.0417 SIC (µg/L) 77.61 (65.22, 89.80) 77.94 (66.50, 93.33) 0.06 0.8088 Lipid serum NEFA (mmol/L) 0.57±0.26 0.57±0.29 0.17 0.6779 Lp (a) (mg/L) 109(55,220) 106(60,222) 0.67 0.4149 TC (mmol/L) 4.64±0.90 4.78±1.12 0.19 0.6613 TG (mmol/L) 1.59±1.25 4.29±1.21 6.31 0.0126 HDL-C (mmol/L) 1.56±0.38 1.58±0.37 0.25 0.6138 LDL-C (mmol/L) 3.38±1.01 3.47±0.96 0.87 0.3516 LDL-C/HDL-C (mmol/L) 2.25±0.72 2.27±0.70 0.96 0.3285 ApoA1 (g/L) 1.21±0.25 1.22±0.23 0.07 0.8254 ApoB (g/L) 0.85±0.24 0.87±0.22 2.41 0.1211 ApoE (mg/dL) 5.10±1.45 5.13±1.57 0.10 0.7465 Thyroid function TSH (mIU/L) 2.90±5.50 2.68±6.75 0.16 0.6882 FT3 (pmol/L) 5.46±2.30 5.57±2.29 0.09 0.7701 FT4 (pmol/L) 16.70±6.35 16.64±3.55 0.14 0.7127 TgAb 23.26(14.61,52.22) 23.31(15.30,38.13) 0.76 0.3824 TPOAb 15.51(11.27,22.26) 15.03(10.07,22.44) 0.11 0.7380 Levels of thyroid function did not differ significantly between the two groups. However, the abnormal rate of thyroid function was significantly lower (χ2 = 5.23, P = 0.0222) in the PTC group (14.70%) than in the benign nodule group (20.80%). The antibody abnormality rate was 17.97% in the PTC group and 12.56% in the benign nodule group, with no statistical difference between the two groups (P > 0.05). Comparing the changes in thyroid function under different clinicopathological features of PTC, the results were shown in Table 3–5. After adjusting for age, gender, et al., FT3 was significantly higher in the PTC patients with lymph node metastasis than in the patients without lymphoid metastasis group (F = 5.42, P = 0.0206). TSH, TGAb, and TPOAb were significantly higher in the PTC patients combined with AITD than in those without AITD (F = 23.24, P < 0.0001; F = 81.55, P < 0.0001; F = 475.96, P < 0.0001), and FT4 was significantly lower in the PTC patients combined with AITD than those without AITD (F = 4.94, P = 0.0266). PTC patients combined with AITD than those without AITD (F = 4.94, P = 0.0266). 3.1 Baseline characteristics Table 3 Effect of the presence of metastatic lymph nodes on thyroid function Variables Metastatic lymph nodes F P Fa Pa yes no TSH 3.01±4.97 2.56±2.04 1.81 0.1795 1.55 0.2142 FT3 5.11±0.63 4.99±0.70 3.58 0.0591 5.42 0.0206 FT4 16.83±2.45 16.71±2.76 0.21 0.6503 1.19 0.2763 TGAb 23.59(14.86,35.05) 26.08(16.54,70.78) 0.09 0.7614 0.11 0.7413 TPOAb 15.37(11.66,23.61) 16.87(12.71,23.76) 0.71 0.4001 0.18 0.6749 Table 4 Effect of the presence of AITD on thyroid function Variables AITD F P Fa Pa yes no TSH 5.00±11.50 2.40±1.44 27.59 < 0.0001 23.24 < 0.0001 FT3 5.06±1.51 5.44±2.13 2.99 0.0843 1.72 0.1903 FT4 15.38±3.48 16.81±6.47 4.94 0.0266 2.93 0.0873 TGAb 127.80(47.38,353.80) 20.84(13.81,31.28) 124.22 < 0.0001 81.55 < 0.0001 TPOAb 139.20(74.92,244.53) 14.18(10.35,18.16) 671.00 < 0.0001 475.96 < 0.0001 Table 5 Effect of cancer unilateral and bilateral on thyroid function in the PTC group Variables cancer unilateral and bilateral F P Fa Pa yes no TSH 2.71±1.84 2.64±3.41 0.10 0.7563 0.03 0.8623 FT3 5.24±1.58 5.45±2.24 1.50 0.2215 1.30 0.2545 FT4 16.69±7.18 16.60±5.67 0.03 0.8553 0.37 0.5452 TGAb 23.12(14.78,40.11) 23.26(14.61,51.75) 0.01 0.9079 0.28 0.5977 TPOAb 15.26(10.97,22.57) 15.66(11.95,22.20) 0.01 0.9207 0.04 0.8416 dicators, the rates of dyslipidemia in the PTC group and in the benign nodule group were 60.46% and 61.54%, respectively, with no tween the two groups. TG was significant lower in the PTC group compared to the benign group. The other lipid indicators did not een the two groups. After adjusting for age, gender, smoking, and alcohol consumption, TG levels remained significantly different and the benign group. PTC patients were grouped according to the different pathological characteristics, and there was no statistical indicators. n between iodine, thyroid function and serum lipids in patients with thyroid dings of a Pearson correlation analysis performed to explore the relationships between iodine nutrition, thyroid function, and serum e observed between several lipid indicators. 3.1 Baseline characteristics ApoA1 and HDL-C exhibited a correlation coefficient of 0.87, while ApoB and LDL-C ffi i t f 0 94 With l ti ffi i t f 0 11 th l ti b t SIC d UIC k Th l ti b t th Table 3 Effect of the presence of metastatic lymph nodes on thyroid function Variables Metastatic lymph nodes F P Fa Pa yes no TSH 3.01±4.97 2.56±2.04 1.81 0.1795 1.55 0.2142 FT3 5.11±0.63 4.99±0.70 3.58 0.0591 5.42 0.0206 FT4 16.83±2.45 16.71±2.76 0.21 0.6503 1.19 0.2763 TGAb 23.59(14.86,35.05) 26.08(16.54,70.78) 0.09 0.7614 0.11 0.7413 TPOAb 15.37(11.66,23.61) 16.87(12.71,23.76) 0.71 0.4001 0.18 0.6749 Table 4 Effect of the presence of AITD on thyroid function Variables AITD F P Fa Pa yes no TSH 5.00±11.50 2.40±1.44 27.59 < 0.0001 23.24 < 0.0001 FT3 5.06±1.51 5.44±2.13 2.99 0.0843 1.72 0.1903 FT4 15.38±3.48 16.81±6.47 4.94 0.0266 2.93 0.0873 TGAb 127.80(47.38,353.80) 20.84(13.81,31.28) 124.22 < 0.0001 81.55 < 0.0001 TPOAb 139.20(74.92,244.53) 14.18(10.35,18.16) 671.00 < 0.0001 475.96 < 0.0001 Table 4 Effect of the presence of AITD on thyroid function Variables AITD F P Fa Pa yes no TSH 5.00±11.50 2.40±1.44 27.59 < 0.0001 23.24 < 0.0001 FT3 5.06±1.51 5.44±2.13 2.99 0.0843 1.72 0.1903 FT4 15.38±3.48 16.81±6.47 4.94 0.0266 2.93 0.0873 TGAb 127.80(47.38,353.80) 20.84(13.81,31.28) 124.22 < 0.0001 81.55 < 0.0001 TPOAb 139.20(74.92,244.53) 14.18(10.35,18.16) 671.00 < 0.0001 475.96 < 0.0001 Table 5 Effect of cancer unilateral and bilateral on thyroid function in the PTC group Variables cancer unilateral and bilateral F P Fa Pa yes no TSH 2.71±1.84 2.64±3.41 0.10 0.7563 0.03 0.8623 FT3 5.24±1.58 5.45±2.24 1.50 0.2215 1.30 0.2545 FT4 16.69±7.18 16.60±5.67 0.03 0.8553 0.37 0.5452 TGAb 23.12(14.78,40.11) 23.26(14.61,51.75) 0.01 0.9079 0.28 0.5977 TPOAb 15.26(10.97,22.57) 15.66(11.95,22.20) 0.01 0.9207 0.04 0.8416 Regarding serum lipid indicators, the rates of dyslipidemia in the PTC group and in the benign nodule group were 60.46% and 61.54%, respectively, with no significant difference between the two groups. TG was significant lower in the PTC group compared to the benign group. The other lipid indicators did not differ significantly between the two groups. After adjusting for age, gender, smoking, and alcohol consumption, TG levels remained significantly different between the PTC group and the benign group. PTC patients were grouped according to the different pathological characteristics, and there was no statistical difference in serum lipid indicators. 3.1 Baseline characteristics p 3.2 Correlation between iodine, thyroid function and serum lipids in patients with thyroid nodule Figure 1 displays the findings of a Pearson correlation analysis performed to explore the relationships between lipids. Figure 1 displays the findings of a Pearson correlation analysis performed to explore the relationships between iodine nutrition, thyroid function, and serum lipids. Several correlations were observed between several lipid indicators. ApoA1 and HDL-C exhibited a correlation coefficient of 0.87, while ApoB and LDL-C exhibited a correlation coefficient of 0.94. With a correlation coefficient of 0.11, the correlation between SIC and UIC was weak. The correlations between the thyroid function indicators were also weak, with correlation coefficients of less than 0.3. Page 5/11 Page 5/11 Concerning iodine nutrition status, SIC was positively correlated with ApoB, Lp (a), NEFA, TG, LDL-C, LDL-C/HDL-C, FT3 and FT4, however, the correlation was weak (correlation coefficients were less than 0.3). No significant association existed between UIC and these indicators. Positive correlations were seen between FT3 and NEFA, LDL-C/HDL-C, and negative correlation were seen between FT3 and ApoA1, TC, and HDL-C. FT4 had a positive correlation with NEFA, and negative correlation with TG. However, the correlations were weak (correlation coefficients were less than 0.3). There was only a negative correlation between TSH and FT4, and no significant association between TSH and other indicators. 3.3 Effect of iodine nutrition on serum lipids and thyroid function in patients with thyroid d l 3.3 Effect of iodine nutrition on serum lipids and thyroid function in patients with thyroid nodule Table 6 presents the effects of SIC on thyroid function and serum lipids in PTC group and control group. There was no significant difference in the effect of SIC on thyroid function. Analysis of the effect of SIC on serum lipids after correction of thyroid function, in PTC group, ApoE was significantly different among groups with different SIC. ApoE was significantly higher in the group with SIC < 45 µg/L compared to the group with SIC in the range of 45–90 µg/L, indicating that low SIC was more likely to result in high ApoE. In the benign thyroid patients, LDL-C and ApoB were significantly different among groups with different SIC. The effect of UIC on thyroid function and serum lipids is presented in Table 7. Analysis of the effect of UIC on serum lipids after correction of thyroid function. There was no significant difference in the effect of UIC on serum lipids and thyroid function in the cancer group. In the control group, ApoE was significantly different at different UIC. Compared to UIC (100–300 µg/L), ApoE was significantly higher in the UIC < 100 µg/L group, and ApoE was significantly lower in the UIC > 300 µg/L group. The results indicated that low UIC was more likely to lead to high ApoE, while high UIC was more likely to lead to low ApoE. 3.1 Baseline characteristics Compared to the group with SIC in the range of 45–90 µg/L, LDL-C was significantly higher in the group with SIC > 90 µg/L, high SIC is more likely to lead to high LDL-C; ApoB was significantly higher in the SIC > 90 µg/L group, high SIC is more likely to lead to high ApoB. Table 6 Effect of serum iodine concentration on serum lipids and thyroid function variable cancer F P control SIC < 45µg/L SIC(45–90µg/L) SIC > 90µg/L SIC < 45µg/L SIC(45–90µg/L) SIC > 90µg/L Thyroid function TSH 2.56±1.76 2.70±3.77 2.77±2.23 0.04 0.9647 1.91±1.38 3.11±8.61 2.12±1.86 FT3 5.01±0.64 5.38±2.32 5.91±3.05 2.72 0.0670 6.34±3.96 5.43±1.80 5.71±2.72 FT4 16.74±2.69 16.57±6.07 17.04±9.77 0.12 0.8912 14.82±5.05 16.67±3.10 16.85±4.02 TgAb 24.93(20.39,36.31) 23.79(14.64,52.33) 21.06(13.39,53.79) 0.04 0.9623 20.39(11.50,32.74) 23.30(16.03,36.14) 24.50(13.26,42 TPOAb 14.58(11.06,17.51) 15.89(11.19,23.09) 15.46(11.95,22.36) 0.32 0.7270 11.50(7.98,19.79) 14.70(9.83,21.54) 17.22(12.32,27 Lipid serum NEFA 0.58±0.35 0.57±0.25 0.58±0.26 0.16 0.8485 0.58±0.32 0.60±0.31 0.54±0.27 Lp (a) 152(66,266) 99(53,206) 115(62,208) 0.95 0.3863 76(40,224) 120.5(62.5,227) 119.5(64,289) TC 4.60±0.98 4.69±0.92 4.51±1.03 0.44 0.6453 3.74±0.14 4.56±1.25 5.00±0.99 TG 1.69±1.06 1.60±1.35 1.63±1.07 0.11 0.8951 3.58±3.40 1.65±2.33 3.05±4.72 HDL-C 1.57±0.31 1.57±0.38 1.55±0.36 0.14 0.8685 1.49±0.37 1.55±0.37 1.64±0.33 LDL-C 3.04±0.95 3.45±1.02 3.33±1.04 2.00 0.1360 3.35±0.76 3.33±0.87 3.83±1.13 LDL- C/HDL- C 2.04±0.79 2.29±0.73 2.22±0.68 2.37 0.0948 2.34±0.63 2.25±0.71 2.39±0.75 ApoA1 1.24±0.20 1.21±0.25 1.23±0.24 0.76 0.4689 1.18±0.27 1.19±0.22 1.27±0.21 ApoB 0.79±0.22 0.87±0.24 0.85±0.24 1.38 0.2530 0.83±0.18 0.84±0.20 0.95±0.24 ApoE 5.72±2.16 5.14±1.45 4.87±1.22 4.07 0.0178 4.38±0.97 5.15±1.73 5.51±1.54 The effect of UIC on thyroid function and serum lipids is presented in Table 7. Analysis of the effect of UIC on serum lipids after correction of thyroid function. There was no significant difference in the effect of UIC on serum lipids and thyroid function in the cancer group. In the control group, ApoE was significantly different at different UIC. 3.1 Baseline characteristics Discussion Table 8 Bootstrap analysis of the intermediary effect Effect Value Boot SE Boot LLCI Boot ULCI Total effect 20.6090 7.7608 0.0082 5.3209 Direct effect 20.6340 7.7757 0.0082 5.3567 Intermediary Effect -0.0251 0.3141 -0.8343 0.5371 3.1 Baseline characteristics Compared to UIC (100–300 µg/L), ApoE was significantly higher in the UIC < 100 µg/L group, and ApoE was significantly lower in the Page 6/11 Table 7 Table 7 Effect of urinary iodine concentration on serum lipids and thyroid function variable cancer F P control UIC < 100µg/L UIC(100–300µg/L) UIC > 300µg/L UIC < 100µg/L UIC(100–300µg/L) UIC > 300µg/ Thyroid function TSH 3.73±9.54 2.43±1.80 3.03±2.78 2.19 0.1127 2.22±1.75 2.28±2.02 4.90±16.01 FT3 5.28±1.87 5.65±2.59 5.44±2.57 0.56 0.5700 5.31±2.02 5.56±2.16 6.09±3.31 FT4 16.30±3.45 17.01±8.31 16.31±3.29 0.34 0.7104 16.12±3.14 16.89±3.45 15.88±4.46 TgAb 28.53(15.85,115.30) 22.63(14.55,35.81) 21.09(12.57,37.10) 2.18 0.1201 23.40(16.03,40.26) 23.41(14.71,35.09) 23.24(13.26,8 TPOAb 16.53(12.11,24.40) 15.17(11.25,20.75) 14.84(11.46,22.62) 0.65 0.5200 14.70(10.69,21.77) 14.84(9.68,21.81) 15.17(8.13,24 Lipid serum NEFA 0.59±0.25 0.57±0.27 0.53±0.24 0.50 0.6041 0.54±0.27 0.60±0.33 0.57±0.26 Lp (a) 97(52,189) 109(54,231) 108(55,202) 1.00 0.3694 139(76,243) 106.5(56,218) 77.5(47,222) TC 4.61±0.87 4.57±0.98 4.82±0.74 0.72 0.4866 4.56±1.63 4.77±0.86 5.14±1.22 TG 1.61±1.13 1.58±1.31 1.53±0.69 0.06 0.9420 3.21±4.18 2.30±3.92 1.92±1.46 HDL-C 1.59±0.39 1.53±0.34 1.53±0.39 0.46 0.6312 1.56±0.35 1.60±0.37 1.56±0.32 LDL-C 3.36±0.97 3.36±1.05 3.51±1.15 1.55 0.2126 3.72±1.07 3.44±0.93 3.57±0.92 LDL- C/HDL- C 2.17±0.63 2.27±0.77 2.37±0.72 2.22 0.1104 2.49±0.82 2.23±0.68 2.39±0.75 ApoA1 1.27±0.26 1.19±0.22 1.18±0.27 2.58 0.0771 1.25±0.25 1.23±0.22 1.19±0.18 ApoB 0.87±0.22 0.85±0.25 0.88±0.26 0.99 0.3739 0.88±0.26 0.87±0.21 0.90±0.22 ApoE 5.19±1.46 5.11±1.51 4.85±1.20 0.44 0.6427 6.29±2.68 5.08±1.32 4.74±1.03 3 4 Th ff t f i di t iti l t t li id 3.4 The effects of iodine nutritional status on serum lipids Mediation analysis was used to further analyze the effect of iodine nutrition level on blood lipid levels in the study subjects (Table 8). The results showed that iodine nutrition level did have an effect on serum lipid levels, and the effect was mainly direct. Mediation analysis was used to further analyze the effect of iodine nutrition level on blood lipid levels in the study subjects (Table 8). The results showed that iodine nutrition level did have an effect on serum lipid levels, and the effect was mainly direct. Mediation analysis was used to further analyze the effect of iodine nutrition level on blood lipid iodine nutrition level did have an effect on serum lipid levels, and the effect was mainly direct. Table 8 Bootstrap analysis of the intermediary effect Effect Value Boot SE Boot LLCI Boot ULCI Total effect 20.6090 7.7608 0.0082 5.3209 Direct effect 20.6340 7.7757 0.0082 5.3567 Intermediary Effect -0.0251 0.3141 -0.8343 0.5371 4. 4. Discussion In our study, urinary iodine and serum iodine levels were classified as low, normal, and high according to the recommendations of WHO guidelines, respectively. Spot urinary iodine concentration usually reflects a population’s iodine nutritional status. As this indicator is influenced by several factors, it is not ideal for assessingin individuals. In this study, morning UIC was detected to reflect short-term iodine nutritional status, and the indicator is better than spot urinary iodine. Compared to urinary iodine, serum iodine is more stable and reflects the long-term iodine nutrition status [24]. In this study, both serum iodine and morning urine iodine levels were measured, in order to comprehensively evaluate iodine nutritional status. The results of studies examining relationship between iodine nutrition and blood lipids are inconsistent. Zhao's study found that in healthy rats, iodine overdose leaded to an increase in serum cholesterol levels, particularly LDL cholesterol levels, and a significant dose-time effect relationship [25]. Han et al. also found high iodine intake could lead to an increase in the expression of the LDL receptor gene in the liver, resulting in dyslipidemia [26]. Nevertheless, Gao’s study found a statistically significant downward trend in TC and LDL-C with the increase of water iodine concentration (WIC), suggesting that high WIC is a protective factor against dyslipidemia [27]. Wang et al. found an association between serum lipid levels and WIC. In circumstances of iodine excess, the association between WIC and dyslipidemia is beneficial [28]. All of these studies were all conducted on healthy people or animals, and to the best of our knowledge, there has seldom been a study on the effects of iodine nutrition on lipids mechanisms in patients with thyroid diseases. Our analysis of several blood lipid indicators revealed that high iodine nutrition had a negative effect on serum lipid indicators. LDL-C and ApoB were significantly higher in the group with high iodine level compared to the group with suitable iodine level, indicating that iodine excess is more likely to resulted in abnormal lipid metabolism. High iodine levels appear to increase the risk of dyslipidemia in patients with PTC and patients with benign thyroid nodules. Low iodine nutrition has significant effects on serum lipid indicators in iodine insufficiency. A randomized controlled trial (RCT) showed that moderate to severe iodine deficiency in overweight women was associated with a higher atherogenic lipid profile and that iodine supplementation in this group would reduce the prevalence of hypercholesterolemia. 4. Discussion By analyzing data from the US Nutrition and Health Survey from 2007–2012, Lee et al. found that low iodine intake led to a reduction in thyroid hormone synthesis and an increase in abnormal lipid metabolism [29]. A Finnish study showed that iodine deficiency was the sole cause of hypercholesterolemia in the population of eastern Finland, after removing the influence of various dietary factors [30]. Herter-Aeberli’s study showed iodine prophylaxis may reduce the risk of cardiovascular disease in overweight adults [31]. The randomized controlled trial conducted by Zimmermann's on randomized controlled trial study in Moroccan children showed that iodine supplementation improved lipid levels, particularly by significantly reducing LDL-C [32]. Consistent with the findings of several previous studies, our study showed that ApoE was significantly higher in the iodine deficiency group (with low SIC) among PTC patients, but ApoB was significantly higher in the iodine deficiency group among thyroid benign group (with low UIC). Many researchers believe that iodine impacts serum lipids through its effect on thyroid hormones, as these hormones regulate lipid synthesis, absorption, and metabolism in numerous ways [33]. Studies have reported an association between thyroid function and serum lipid concentrations [34–36], and poor thyroid function influences lipid metabolism [37–39]. Leonidas H et al. also showed that elevated TSH levels were associated with elevated LDL-C, LDL/HDL-C, and TC levels, and that TSH abnormalities were a risk factor for abnormal lipid metabolism [40]. Moreover, supplementation with thyroid hormone reversed the increase in lipid levels [41–43]. In this study, it was found that there was no significant difference in thyroid function in different iodine nutrition levels, and after adjusting for thyroid function, iodine nutrition was still observed to influence blood lipids. It indicated that abnormal iodine nutrition may not affect blood lipids through thyroid function in people with thyroid nodules. Beisides, in this study, the results showed that iodine could directly affect the serum lipid levels instead of affecting through thyroid function. The possible reason is that the iodine nutrition of the study population in this study is generally adequate. When the iodine nutrition of the population is suitable, the iodine will directly work on lipids metabolism. Although our study had a large sample size and included multiple iodine nutrition indicators and comprehensive lipid indicators, some limitations still existed. First, our research was carried out in an area of China with iodine supplementation. 4. Discussion The thyroid gland is an important organ that is closely related to the body’s metabolism. Abnormal morphology and function of the thyroid, as well as thyroid cancer, must have a negative impact on an organism's metabolism, particularly lipid metabolism. Thyroid cancer is the most common endocrine malignancy, and it can lead to alterations in thyroid function and abnormal iodine metabolism. Uncertainty exists as to whether abnormal lipid metabolism occurs in thyroid cancer and whether it is affected by iodine nutritional status. In this study, a large sample case-control study was conducted to explore the relationship between thyroid cancer, iodine nutritional status, and lipid metabolism in order to provide useful clues for a comprehensive understanding of the effects of thyroid cancer on body metabolism. Many studies focus on the relationship between abnormal blood lipid metabolism and cancer. Recent studies have revealed that lipid metabolism disorders occur in carcinogenesis and the development of cancer, as cancer induces abnormalities in the expression of various genes and proteins, as well as in the regulation of cytokines and signaling pathways [21, 22]. A study by Li et al. found that the TC levels were significantly different between patients with thyroid cancer and control groups, and a disordered lipid profile occurred in patients with thyroid cancer [2]. Other studies have concluded that lipid metabolism is not significantly associated with cancer, including thyroid cancer. For example, Martin Almquist et al. reported that thyroid cancer was not linked to the development of the metabolic syndrome and that there were no statistically significant changes in lipid indicators in patients with thyroid cancer [23]. Page 7/11 Page 7/11 In our study, urinary iodine and serum iodine levels were classified as low, normal, and high according to the recommendations of WHO guidelines, respectively. Spot urinary iodine concentration usually reflects a population’s iodine nutritional status. As this indicator is influenced by several factors, it is not ideal for assessingin individuals. In this study, morning UIC was detected to reflect short-term iodine nutritional status, and the indicator is better than spot urinary iodine. Compared to urinary iodine, serum iodine is more stable and reflects the long-term iodine nutrition status [24]. In this study, both serum iodine and morning urine iodine levels were measured, in order to comprehensively evaluate iodine nutritional status. 4. Discussion Although the participants in this study did not alter their eating habits prior to blood and urine collection, multi-center studies are still needed to validate the findings. Second, the samples were not evenly disturbed between the PTC group and the benign thyroid nodule group; for our future research, we will include more samples for in-depth research. 5. Conclusion In conclusion, compared to benign controls, PTC may influence the metabolism of blood lipids. In people with thyroid nodules (both benign and malignant tumor), abnormal iodine nutritional status has a negative impact on lipids, and both excess and deficient iodine nutrition increase the risk of dyslipidemia. Declarations Acknowledgements Acknowledgements L.F. and X.M.conceived and designed the study. Y.Z. wrote the first draft of the article. X.W., J.L., Z.L. and J.W. performed the experiments. Z.L. and X.W. performed the statistical analysis. All authors contributed to acquisition, analysis, or interpretation of data. All authors revised the report and approved the final version before submission. Competing interests The authors declare no competing interests. Data Availability The datasets generated during and analyzed during the current study are not publicly available due to protect participant’s privacy but are available from the corresponding author on reasonable request. Ethics Approval The present study was approved by the Ethics Committee of the Center for Endemic Disease Control of the Harbin Medical University (HRBMUECDC20211002). All procedures performed in the study involving human participants were in accordance with the 1964 Helsinki Declaration and its later amendments. Page 8/11 Consent to Participate Informed consent was obtained from all individual participants included in the study. Written informed consent was signed by participants. Consent for Publication The authors affirm that human research participants provided informed consent for the publication of their data results. Consent for Publication The authors affirm that human research participants provided informed consent for the publication of their data results. Consent for Publication The authors affirm that human research participants provided informed consent for the publication of their data results. References 1. Sipos JA, Mazzaferri EL (2010) Thyroid cancer epidemiology and prognostic variables. Clin Oncol (R Coll Radiol) 22(6):395–404. https://doi.org/10.1016/j.clon.2010.05.004 2. Li D, Zhou L, Ma C, Chen W, Zhang Y, Yu S et al (2019) Comparative analysis of the serum proteome profiles of thyroid cancer: An initial focus on the lipid profile. Oncol Lett 18(3):3349–3357. https://doi.org/10.3892/ol.2019.10655 3. Weng SS, Cao Y, Tang XJ, Zhu LZ, Tan YN, Dong CX et al (2017) Epidemiological features of lung giant cell carcinoma and therapy for patients with EGFR mutations based on case reports and the surveillance, epidemiology, and end results (SEER) database. Oncotarget 8(15):25323–25333. https://doi.org/10.18632/oncotarget.15831 4. Bolf EL, Sprague BL, Carr FE (2019) A Linkage Between Thyroid and Breast Cancer: A Common Etiology. Cancer Epidemiol Biomarkers Prev 28(4):643– 649. https://doi.org/10.1158/1055-9965.EPI-18-0877 5. Nielsen SM, White MG, Hong S, Aschebrook-Kilfoy B, Kaplan EL, Angelos P et al (2016) The Breast-Thyroid Cancer Link: A Systematic Review and Meta- analysis. Cancer Epidemiol Biomarkers Prev 25(2):231–238. https://doi.org/10.1158/1055-9965.EPI-15-0833 6. Hu S, Zhang X (2014) Correlation between abnormal thyroid function and blood level. J Hainan Med Univ 20(08 7. Ulmer H, Borena W, Rapp K, Klenk J, Strasak A, Diem G et al (2009) Serum triglyceride concentrations and cancer risk in a large cohort study in Austria. Br J Cancer 101(7):1202–1206. https://doi.org/10.1038/sj.bjc.6605264 8. Zimmermann MB, Jooste PL, Pandav CS (2008) Iodine-deficiency disorders. Lancet 372(9645):1251–1262. https://doi.org/10.1016/S0140- 6736(08)61005-3 91) The regulation and role of epidermal lipid synthesis. Adv Lipid Res 24:57–82. https://doi.org/10.1016/b978-0 9. Feingold KR (1991) The regulation and role of epidermal lipid synthesis. Adv Lipid Res 24:57–82. https://doi.org/10.1016/b978-0-12-024924-4.50007-9 9. Feingold KR (1991) The regulation and role of epidermal lipid synthesis. Adv Lipid Res 24:57–82. https://doi.o 0. Zhao Y, Lin X (2006) Progress of studies on effects of iodine deficiency and iodine excess on cells and organis 11. Blomberg M, Feldt-Rasmussen U, Andersen KK, Kjaer SK (2012) Thyroid cancer in Denmark 1943–2008, before and after iodine supplementation. Int J Cancer 131(10):2360–2366. https://doi.org/10.1002/ijc.27497 12. Liu L, Li Y, Zheng X, Wei F, Liu J, Gao M (2011) Detection and clinical analysis of urinary iodine in patients with thyroid tumor in Tianjin. Chin J Clin Oncol 38(01):24–27. https://doi.org/ 12. Liu L, Li Y, Zheng X, Wei F, Liu J, Gao M (2011) Detection and clinical analysis of urinary iodine in patients with thyroid tumor in Tianjin. Chin J Clin Oncol 38(01):24–27. https://doi.org/ 13. Xing M (2013) Molecular pathogenesis and mechanisms of thyroid cancer. References Nat Rev Cancer 13(3):184–199. https://doi.org/10.1038/nrc3431 13. Xing M (2013) Molecular pathogenesis and mechanisms of thyroid cancer. Nat Rev Cancer 13(3):184–199 13. Xing M (2013) Molecular pathogenesis and mechanisms of thyroid cancer. Nat Rev Cancer 13(3):184–199. https://doi.org/10.1038/nrc3431 14. Liu J, Liu L, Jia Q, Zhang X, Jin X, Shen H (2019) Effects of Excessive Iodine Intake on Blood Glucose, Blood Pressure, and Blood Lipids in Adults. Biol Trace Elem Res 192(2):136–144. https://doi.org/10.1007/s12011-019-01668-9 14. Liu J, Liu L, Jia Q, Zhang X, Jin X, Shen H (2019) Effects of Excessive Iodine Intake on Blood Glucose, Blood Pressure, and Blood Lipids in Adults. Biol Trace Elem Res 192(2):136–144. https://doi.org/10.1007/s12011-019-01668-9 15. Rönnefarth G, Kauf E, Deschner F, Forberger M (1996) [Therapy of iodine deficiency goiter in adolescents with iodine or a combination of iodine and levothyroxine with special reference to lipid parameters]. Klin Padiatr 208(3):123–128. https://doi.org/10.1055/s-2008-1046460 15. Rönnefarth G, Kauf E, Deschner F, Forberger M (1996) [Therapy of iodine deficiency goiter in adolescents with iodine or a combination of iodine and levothyroxine with special reference to lipid parameters]. Klin Padiatr 208(3):123–128. https://doi.org/10.1055/s-2008-1046460 16. Wang X, Xian T, Zhang L, Jia X, Man F, Liu L et al (2019) Associations between urinary iodine concentration, lipid profile and other cardiometabolic risk factors in adolescents: a cross-sectional, population-based analysis. Br J Nutr 121(9):1039–1048. https://doi.org/10.1017/S0007114518003860 16. Wang X, Xian T, Zhang L, Jia X, Man F, Liu L et al (2019) Associations between urinary iodine concentration, lipid profile and other cardiometabolic risk factors in adolescents: a cross-sectional, population-based analysis. Br J Nutr 121(9):1039–1048. https://doi.org/10.1017/S0007114518003860 7. Liu M, Li S, Li X, Wang P, Liang P, Li S (2009) Exploratory study on the association between high iodine intake a 18. Kitahara CM, McCullough ML, Franceschi S, Rinaldi S, Wolk A, Neta G et al (2016) Anthropometric Factors and Thyroid Cancer Risk by Histological Subtype: Pooled Analysis of 22 Prospective Studies. Thyroid 26(2):306–318. https://doi.org/10.1089/thy.2015.0319 19. Arsenic-cerium-catalyzed (2016) spectrophotometric method for the determination of urinary iodine WS/T107.1-2016[S]. Health Industry Standard of the People's Republic of China 20. Determination of iodine in (2017) serum by arsenic-cerium-catalyzed spectrophotometric method WS/T 572–2017[S]. Health Industry Standard of the People's Republic of China 21. Long J, Zhang CJ, Zhu N, Du K, Yin YF, Tan X et al (2018) Lipid metabolism and carcinogenesis, cancer development. Am J Cancer Res 8(5):778–791 22. References Wang D, Wan S, Liu P, Meng F, Ren B, Qu M et al (2021) Associations between water iodine concentration and the prevalence of dyslipidemia in Chinese adults: A cross-sectional study. Ecotoxicol Environ Saf 208:111682. https://doi.org/10.1016/j.ecoenv.2020.111682 29. Lee KW, Shin D, Song WO (2016) Low Urinary Iodine Concentrations Associated with Dyslipidemia in US Adults. Nutrients 8(3):171. https://doi.org/10.3390/nu8030171 29. Lee KW, Shin D, Song WO (2016) Low Urinary Iodine Concentrations Associated with Dyslipidemia in US Adults. Nutrients 8(3):171. https://doi.org/10.3390/nu8030171 30. ROINE P, PEKKARINEN M, KARVONEN MJ, KIHLBERG J (1958) Diet and cardiovascular disease in Finland. Lancet 2(7039):173–175. https://doi.org/10.1016/s0140-6736(58)91523-x 30. ROINE P, PEKKARINEN M, KARVONEN MJ, KIHLBERG J (1958) Diet and cardiovascular disease in Finland. Lancet 2(7039):173–175. https://doi.org/10.1016/s0140-6736(58)91523-x 31. Herter-Aeberli I, Cherkaoui M, El Ansari N, Rohner R, Stinca S, Chabaa L et al (2015) Iodine Supplementation Decreases Hypercholesterolemia in Iodine- Deficient, Overweight Women: A Randomized Controlled Trial. J Nutr 145(9):2067–2075. https://doi.org/10.3945/jn.115.213439 31. Herter-Aeberli I, Cherkaoui M, El Ansari N, Rohner R, Stinca S, Chabaa L et al (2015) Iodine Supplementation Decreases Hypercholesterolemia in Iodine- Deficient, Overweight Women: A Randomized Controlled Trial. J Nutr 145(9):2067–2075. https://doi.org/10.3945/jn.115.213439 32. Zimmermann MB, Aeberli I, Melse-Boonstra A, Grimci L, Bridson J, Chaouki N et al (2009) Iodine treatment in children with subclinical hypothyroidism due to chronic iodine deficiency decreases thyrotropin and C-peptide concentrations and improves the lipid profile. Thyroid 19(10):1099–1104. https://doi.org/10.1089/thy.2009.0001 32. Zimmermann MB, Aeberli I, Melse-Boonstra A, Grimci L, Bridson J, Chaouki N et al (2009) Iodine treatment in children with subclinical hypothyroidism due to chronic iodine deficiency decreases thyrotropin and C-peptide concentrations and improves the lipid profile. Thyroid 19(10):1099–1104. https://doi.org/10.1089/thy.2009.0001 33. Kang MJ, Hwang IT, Chung HR (2018) Excessive Iodine Intake and Subclinical Hypothyroidism in Children and Adolescents Aged 6–19 Years: Results of the Sixth Korean National Health and Nutrition Examination Survey, 2013–2015. Thyroid 28(6):773–779. https://doi.org/10.1089/thy.2017.0507 34. Zhu X, Cheng SY (2010) New insights into regulation of lipid metabolism by thyroid hormone. Curr Opin Endocrinol Diabetes Obes 17(5):408–413. https://doi.org/10.1097/MED.0b013e32833d6d46 35. Peppa M, Betsi G, Dimitriadis G (2011) Lipid abnormalities and cardiometabolic risk in patients with overt and subclinical thyroid disease. J Lipids 575840. https://doi.org/10.1155/2011/575840 36. Raziel A, Rosenzweig B, Botvinic V, Beigel I, Landau B, Blum I (1982) The influence of thyroid function on serum lipid profile. Atherosclerosis 41(2–3):321– 326. https://doi.org/10.1016/0021-9150(82)90196-4 36. Raziel A, Rosenzweig B, Botvinic V, Beigel I, Landau B, Blum I (1982) The influence of thyroid function on serum lipid profile. References Nikitin PV, Potapov AA, Ryzhova MV, Shurkhay VA, Kulikov EE, Zhvanskiy ES et al (2018) [The role of lipid metabolism disorders, atypical isoforms of 21. Long J, Zhang CJ, Zhu N, Du K, Yin YF, Tan X et al (2018) Lipid metabolism and carcinogenesis, cancer dev 21. Long J, Zhang CJ, Zhu N, Du K, Yin YF, Tan X et al (2018) Lipid metabolism and carcinogenesis, cancer development. Am J Cancer Res 8(5):778–791 22. Nikitin PV, Potapov AA, Ryzhova MV, Shurkhay VA, Kulikov EE, Zhvanskiy ES et al (2018) [The role of lipid metabolism disorders, atypical isoforms of protein kinase C, and mutational status of cytosolic and mitochondrial forms of isocitrate dehydrogenase in carcinogenesis of glial tumors]. Zh Vopr Neirokhir Im N N Burdenko 82(3):112–120. https://doi.org/10.17116/neiro2018823112 23. Almquist M, Johansen D, Björge T, Ulmer H, Lindkvist B, Stocks T et al (2011) Metabolic factors and risk of thyroid cancer in the Metabolic syndrome and Cancer project (Me-Can). Cancer Causes Control 22(5):743–751. https://doi.org/10.1007/s10552-011-9747-2 24. Gao F, Diao X, Liu D (2004) Survey of urinary and serum iodine levels in children. Journal of Jining medical college (01): 58 4. Gao F, Diao X, Liu D (2004) Survey of urinary and serum iodine levels in children. Journal of Jining medical col 5. Zhao L (2010) Effect of iodine excess on lipid metabolism and its underlying mechanism. In: Sun Xiufa, ed (11 26. Han H, Xin P, Zhao L, Xu J, Xia Y, Yang X et al (2012) Excess iodine and high-fat diet combination modulates lipid profile, thyroid hormone, and hepatic LDLr expression values in mice. Biol Trace Elem Res 147(1–3):233–239. https://doi.org/10.1007/s12011-011-9300-x 26. Han H, Xin P, Zhao L, Xu J, Xia Y, Yang X et al (2012) Excess iodine and high-fat diet combination modulates lipid profile, thyroid hormone, and hepatic LDLr expression values in mice. Biol Trace Elem Res 147(1–3):233–239. https://doi.org/10.1007/s12011-011-9300-x 27. Gao J, Zhang M, Wang X, Wang M, Zhang B, Jiang W et al (2021) Effects of long-term excessive iodine intake on blood lipids in Chinese adults: a cross- sectional study. Eur J Clin Nutr 75(4):708–714. https://doi.org/10.1038/s41430-020-00773-6 27. Gao J, Zhang M, Wang X, Wang M, Zhang B, Jiang W et al (2021) Effects of long-term excessive iodine intake on blood lipids in Chinese adults: a cross- sectional study. Eur J Clin Nutr 75(4):708–714. https://doi.org/10.1038/s41430-020-00773-6 Page 9/11 Page 9/11 Page 9/11 28. References Pearce EN, Wilson PW, Yang Q, Vasan RS, Braverman LE (2008) Thyroid function and lipid subparticle sizes in patients with short-term hypothyroidism and a population-based cohort. J Clin Endocrinol Metab 93(3):888–894. https://doi.org/10.1210/jc.2007-1987 43. Pearce EN, Wilson PW, Yang Q, Vasan RS, Braverman LE (2008) Thyroid function and lipid subparticle sizes in patients with short-term hypothyroidism and a population-based cohort. J Clin Endocrinol Metab 93(3):888–894. https://doi.org/10.1210/jc.2007-1987 References Atherosclerosis 41(2–3):321– 326. https://doi.org/10.1016/0021-9150(82)90196-4 37. Sasaki S, Kawai K, Honjo Y, Nakamura H (2006) [Thyroid hormones and lipid metabolism]. Nihon Rinsho 64 38. Berti JA, Amaral ME, Boschero AC, Nunes VS, Harada LM, Castilho LN et al (2001) Thyroid hormone increases plasma cholesteryl ester transfer protein activity and plasma high-density lipoprotein removal rate in transgenic mice. Metabolism 50(5):530–536. https://doi.org/10.1053/meta.2001.22514 38. Berti JA, Amaral ME, Boschero AC, Nunes VS, Harada LM, Castilho LN et al (2001) Thyroid hormone increases plasma cholesteryl ester transfer protein activity and plasma high-density lipoprotein removal rate in transgenic mice. Metabolism 50(5):530–536. https://doi.org/10.1053/meta.2001.22514 39. Huesca-Gómez C, Franco M, Luc G, Montaño LF, Massó F, Posadas-Romero C et al (2002) Chronic hypothyroidism induces abnormal structure of high- density lipoproteins and impaired kinetics of apolipoprotein A-I in the rat. Metabolism. 2002;51(4):443 – 50. https://doi.org/10.1053/meta.2002.31323 39. Huesca-Gómez C, Franco M, Luc G, Montaño LF, Massó F, Posadas-Romero C et al (2002) Chronic hypothyroidism induces abnormal structure of high- density lipoproteins and impaired kinetics of apolipoprotein A-I in the rat. Metabolism. 2002;51(4):443 – 50. https://doi.org/10.1053/meta.2002.31323 40. Duntas LH, Brenta G (2018) A Renewed Focus on the Association Between Thyroid Hormones and Lipid Metabolism. Front Endocrinol (Lausanne) 9:511. https://doi.org/10.3389/fendo.2018.00511 40. Duntas LH, Brenta G (2018) A Renewed Focus on the Association Between Thyroid Hormones and Lipid Metabolism. Front Endocrinol (Lausanne) 9:511. https://doi.org/10.3389/fendo.2018.00511 41. Santi A, Duarte MM, Moresco RN, Menezes C, Bagatini MD, Schetinger MR et al (2010) Association between thyroid hormones, lipids and oxidative stress biomarkers in overt hypothyroidism. Clin Chem Lab Med 48(11):1635–1639. https://doi.org/10.1515/CCLM.2010.309 41. Santi A, Duarte MM, Moresco RN, Menezes C, Bagatini MD, Schetinger MR et al (2010) Association between thyroid hormones, lipids and oxidative stress biomarkers in overt hypothyroidism. Clin Chem Lab Med 48(11):1635–1639. https://doi.org/10.1515/CCLM.2010.309 42. Khatri P, Neupane A, Banjade A, Sapkota S, Kharel S, Chhetri A et al (2021) Lipid Profile Abnormalities in Newly Diagnosed Primary Hypothyroidism in a Tertiary Care Centre of Western Nepal: A Descriptive Cross-sectional Study. JNMA J Nepal Med Assoc 59(240):783–786. https://doi org/10 31729/jnma 6809 42. Khatri P, Neupane A, Banjade A, Sapkota S, Kharel S, Chhetri A et al (2021) Lipid Profile Abnormalities in Newly Diagnosed Primary Hypothyroidism in a Tertiary Care Centre of Western Nepal: A Descriptive Cross-sectional Study. JNMA J Nepal Med Assoc 59(240):783–786. https://doi.org/10.31729/jnma.6809 43. Figures Figures Page 10/11 Figure 1 The interrelationship between iodine nutrition status, thyroid function, and serum lipids. Figure 1 The interrelationship between iodine nutrition status, thyroid function, and serum lipids. Figure 1 The interrelationship between iodine nutrition status, thyroid function, and serum lipids. Page 11/11
https://openalex.org/W2165863321	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/6BC8205E18A4C06B66E1FEA4D9DBD298/S1758320900004455a.pdf/div-class-title-psychiatric-medications-are-no-more-effective-div.pdf	English	null	Psychiatric medications are no more effective	The psychiatrist	2,013	cc-by	1,147	1 Martin F, Elworthy T. Scottish psychiatrists’ attitudes to electroconvulsive therapy: survey analysis. Psychiatrist 2013; 37: 261-6. Psychiatrists as neurologists . . . or biologists? 4 Bauer MS, Mitchner L. What is a ‘mood stabilizer’? An evidence-based response. Am J Psychiatry 2004; 161: 3-18. 4 Bauer MS, Mitchner L. What is a ‘mood stabilizer’? An evidence-based response. Am J Psychiatry 2004; 161: 3-18. Michael Fitzgerald thinks that ‘All future psychiatrists should be neuropsychiatrists’1 - and, what’s more, should only concern themselves with diagnosis and prescribing, leaving psychological treatments to non-psychiatrists. I disagree. Don’t get me wrong, I enjoy ‘hunt the lesion’ as much as anyone I know. And I have valued my medical school-level neurology on the few occasions when it has come in really handy. But to hive off all psychological interventions to other professions is where I take issue. Let us look at the two ‘core’ tasks Fitzgerald suggests and try to take the psychology out of them. Richard Braithwaite, Consultant Psychiatrist, Isle of Wight NHS Trust, email: richard.braithwaite@iow.nhs.uk doi:10.1192/pb.37.12.403 Psychiatric medications are no more effective can easily be challenged and Dr Braithwaite’s example of reboxetine is a good one. can easily be challenged and Dr Braithwaite’s example of reboxetine is a good one. Martin & Elworthy report that the biggest reason for prescribing electroconvulsive therapy (ECT) less frequently than before is the perception among psychiatrists that ‘more effective medication’ now exists.1 Unfortunately, the authors collude with this exaggerated view, claiming that ‘psychiatric medications have undoubtedly become more effective over recent years’. Their bold statement references a 2002 story in The New York Times. 1 Cipriani A, La Ferla T, Furukawa TA, Signoretti A, Nakagawa A, Churchill R, et al. Sertraline versus other antidepressive agents for depression. Cochrane Database Syst Rev 2009; 2: CD006117 1 Cipriani A, La Ferla T, Furukawa TA, Signoretti A, Nakagawa A, Churchill R, et al. Sertraline versus other antidepressive agents for depression. Cochrane Database Syst Rev 2009; 2: CD006117 1 Cipriani A, La Ferla T, Furukawa TA, Signoretti A, Nakagawa A, Churchill R, et al. Sertraline versus other antidepressive agents for depression. Cochrane Database Syst Rev 2009; 2: CD006117 Fiona Martin and Tim Elworthy, University of Dundee Medical School, Ninewells Hospital and Medical School, Dundee, UK, email: ﬁona.martin5@nhs.net doi:10.1192/pb.37.12.403a doi:10.1192/pb.37.12.403a Meta-analysis shows that the current ﬁrst-line treatments for depressive disorder, selective serotonin reuptake inhibitors, are marginally less effective than older tricyclic antidepressants (TCAs), while serotonin-noradrenaline reuptake inhibitors show no statistically signiﬁcant advantage over TCAs.2 One newer drug, reboxetine, does not work at all,3 yet is inexplicably still licensed as an antidepressant. Psychiatry needs more psychotherapy 3 Eyding D, Lelgemann M, Grouven U, Hrter M, Kromp M, Kaiser T, et al. Reboxetine for acute treatment of major depression: systematic review and meta-analysis of published and unpublished placebo and selective serotonin reuptake inhibitor controlled trials. BMJ 2010; 341: c4737. 3 Eyding D, Lelgemann M, Grouven U, Hrter M, Kromp M, Kaiser T, et al. Reboxetine for acute treatment of major depression: systematic review and meta-analysis of published and unpublished placebo and selective serotonin reuptake inhibitor controlled trials. BMJ 2010; 341: c4737. doi:10.1192/pb.37.12.403b doi:10.1192/pb.37.12.403 Author response: Dr Braithwaite is correct in challenging the view that antidepressants have become more effective. Our study did show that this is a dominant view within the profession that may contribute to reduced ECT prescribing rates, and articles such as those referenced may help to perpetuate this view. We concede that our use of the general media to support this assertion reﬂects clumsy referencing on our part. There are, however, peer-reviewed studies that support the view of increased effectiveness of some newer antidepressants over some older antidepressants.1 This may in part be related to efﬁcacy but also better tolerability, and pharmaceutical company inﬂuence could also be a factor. However, the perceived belief that new equates to better In spite of the golden dawn promised over the course of my career, there are still no physical investigations that usefully inform the most common issues of psychiatric diagnosis. The main instrument of investigation continues to be conversation. William Osler stated one of the fundamental principles in this area - ‘Listen to your patient, he is telling you the diagnosis’. This sounds simple, but it clearly is not. The patient will only tell the doctor the necessary information if the patient feels that they are being taken seriously and listened to.2 Some of us 403 Psychiatry needs more psychotherapy I could not disagree more strongly with Michael Fitzgerald’s letter1 asserting that all future psychiatrists should be neuropsychiatrists. Having worked as a medical psychotherapist for over 20 years, my job changed and I had a choice between resigning and becoming a community psychiatrist. I found instead that what many of my colleagues and particularly the junior doctors seemingly had difﬁculty with was precisely that lack of certainty, the need to listen with minute attention to what the patient was saying, which of course is the bedrock of psychotherapy. We need more, not less, psychotherapy to be embedded into psychiatry. We desperately need medical psychotherapists to act as role models for trainees or else we will lose the essence of our art - and yes, I use the word advisedly - and we will become gloriﬁed technicians. Is this really what we want? Lithium remains the only true mood stabiliser: it is the only drug with efﬁcacy in treating acute manic and depressive symptoms and in prophylaxis of manic and depressive symptoms in bipolar disorder.4 One has to conclude that the prevailing delusion that treatments across psychiatry have become more effective has been mediated by the pharmaceutical industry. Psychiatrists should take their evidence from meta-analyses in peer- reviewed journals, not from advertising representatives and certainly not from the newsstand. 1 Fitzgerald M. All future psychiatrists should be neuropsychiatrists. Psychiatrist 2013; 37: 309. 1 Fitzgerald M. All future psychiatrists should be neuropsychiatrists. Psychiatrist 2013; 37: 309. 2 Machado M, Iskedjian M, Ruiza I, Einarson TR. Remission, dropouts, and adverse drug reaction rates in major depressive disorder: a meta-analysis of head-to-head trials. Curr Med Res Opin 2006; 22: 1825-37. 2 Machado M, Iskedjian M, Ruiza I, Einarson TR. Remission, dropouts, and adverse drug reaction rates in major depressive disorder: a meta-analysis of head-to-head trials. Curr Med Res Opin 2006; 22: 1825-37. Teresa J. Black, Consultant Psychiatrist, Complex Care Team, Steps to Health, Black Country Partnership NHS Foundation Trust, Wolverhampton, email: Teresa.Black@bcpft.nhs.uk doi:10.1192/pb.37.12.403b Teresa J. Black, Consultant Psychiatrist, Complex Care Team, Steps to Health, Black Country Partnership NHS Foundation Trust, Wolverhampton, email: Teresa.Black@bcpft.nhs.uk 3 Eyding D, Lelgemann M, Grouven U, Hrter M, Kromp M, Kaiser T, et al. Reboxetine for acute treatment of major depression: systematic review and meta-analysis of published and unpublished placebo and selective serotonin reuptake inhibitor controlled trials. BMJ 2010; 341: c4737. https://doi.org/10.1192/pb.37.12.403 Published online by Cambridge University Press https://doi.org/10.1192/pb.37.12.403 Published online by Cambridge University Press https://doi.org/10.1192/pb.37.12.403 Published online by Cambridge University Press
https://openalex.org/W3037108128	https://www.nature.com/articles/s41598-020-67417-4.pdf	English	null	Removal of an abluminal lining improves decellularization of human umbilical arteries	Scientific reports	2,020	cc-by	8,945	OPEN Ho‑Yi Tuan‑Mu1,2, Yi‑Hao Chang3 & Jin‑Jia Hu4* The decellularization of long segments of tubular tissues such as blood vessels may be improved by perfusing decellularization solution into their lumen. Particularly, transmural flow that may be introduced by the perfusion, if any, is beneficial to removing immunogenic cellular components in the vessel wall. When human umbilical arteries (HUAs) were perfused at a transmural pressure, however, very little transmural flow was observed. We hypothesized that a watertight lining at the abluminal surface of HUAs hampered the transmural flow and tested the hypothesis by subjecting the abluminal surface to enzyme digestion. Specifically, a highly viscous collagenase solution was applied onto the surface, thereby restricting the digestion to the surface. The localized digestion resulted in a water- permeable vessel without damaging the vessel wall. The presence of the abluminal lining and its successful removal were also supported by evidence from SEM, TEM, and mechanical testing. The collagenase-treated HUAs were decellularized with 1% sodium dodecyl sulfate (SDS) solution under either rotary agitation, simple perfusion, or pressurized perfusion. Regardless of decellularization conditions, the decellularization of HUAs was significantly enhanced after the abluminal lining removal. Particularly, complete removal of DNA was accomplished in 24 h by pressurized perfusion of the SDS solution. We conclude that the removal of the abluminal lining can improve the perfusion- assisted decellularization. Atherosclerosis is the primary cause of vascular disease worldwide, resulting in heart attacks, stroke, and periph- eral arterial disease1,2. Heart disease, most commonly due to atherosclerotic coronary disease, is the first cause of death in the US3. Balloon angioplasty with or without stenting has been regularly performed to open up affected arteries. In the case of peripheral arterial occlusion or severe narrowing, however, by-pass surgery may be preferred or required to restore blood flow and preserve functions of downstream tissues. Although synthetic vascular grafts made of ePTFE or Dacron have been successfully used for the reconstruction of large-diameter blood vessels, small-diameter synthetic grafts usually suffer from early-stage complications. Autologous arteries or veins remain the gold standard for small-diameter bypass grafts, which, however, are not always available. Tissue engineering potentially offers a solution for the lack of grafts in small-diameter bypass surgery.ff ft Decellularized tissue scaffolds, among a variety of scaffolding materials used in tissue engineering, have shown promise in creating tissue substitutes including tendons4, bones5, nerves6, heart valves7, and blood vessels8,9. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports OPEN Decellularization is a process to remove immunogenic cellular components from native tissues or organs, while minimizing the associated adverse effects on the chemical composition, biological activity, and mechanical properties of their extracellular matrix (ECM). Except for thin tissues, the process is, in general, time consum- ing, and usually requires a combination of physical, chemical and enzymatic treatments to achieve satisfactory results10. Decellularized tissue scaffolds are essentially composed of ECM components and exhibit excellent biocompatibility11–13. As the ECM therein is largely preserved after decellularization, less efforts are required for recreating the composition and microstructure of native tissues, which are the results of a series of biological processes during the development and maturation of the tissue14. Recently, it has been found that non-chemically cross-linked decellularized tissues, when implanted, are able to modulate host response toward a constructive tissue remodeling15. 1Department of Physical Therapy, Tzu Chi University, Hualien, Taiwan. 2Department of Sports Medicine Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan. 3Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan. 4Department of Mechanical Engineering, National Chiao Tung University, #1001 University Rd., Hsinchu 300, Taiwan. email: jjhu@nctu.edu.tw \| https://doi.org/10.1038/s41598-020-67417-4 Scientific Reports \| (2020) 10:10556 www.nature.com/scientificreports/ Table 1. Compositions of collagenase solutions of different viscosity. a Collagenase type I (CLS-1, Worthington, Lakewood, NJ) was used. Collagenase conc. (%) Collagenasea (mg) Sucrose (g) HBSS (ml, final volume) Viscosity (centipoise, cP) 0.1 10 0 10 Low viscosity ~ 1.0 0.2 20 0.1 10 3 10 Medium viscosity ~ 2.8 0.2 20 0.1 10 5 10 High viscosity ~ 7.5 0.2 20 Table 1. Compositions of collagenase solutions of different viscosity. a Collagenase type I (CLS-1, Worthington, Lakewood, NJ) was used. Particularly because of their advantageous mechanical properties, decellularized native blood vessels have been used as a scaffold to create tissue-engineered vascular grafts (TEVGs)11,16,17. Many xenogeneic9,18 and allogeneic19,20 blood vessels have been decellularized for this purpose. Specifically, decellularized human umbili- cal arteries (HUAs), which have an inner diameter less than 6 mm, represent an attractive scaffold for preparing small-diameter TEVGs because they are widely available and sufficiently long grafts without branches can be easily prepared. y p p For a general decellularization procedure, tissues to be processed are immersed in a decellularization solution and subjected to some form of agitation. OPEN The agitation increases the concentration of the decellularization agent and reduces the concentration of released cellular components at the tissue surface, facilitating the diffusion of the decellularization agent and cellular components into and out of the tissue, respectively. The agitation, however, has little influence on the interstitial transport within the tissue. That is, the effectiveness of agitation- based decellularization is determined mainly by passive diffusion of molecules21. Our previous study on decel- lularizing HUAs used short segments of HUAs that can be well decellularized by rotary agitation22. The rotary agitation may not be effective in delivering the decellularization solution into the lumen of the HUAs that are long enough for bypass surgery. Also, the diffusion of cellular components out of a long vessel may be limited to the abluminal side of the vessel.f In an effort to more consistently improve decellularization of human umbilical veins (HUVs), McFetridge’s group, instead of relying on agitation, perfused the vein at an elevated transmural pressure21. The improved decel- lularization efficiency was attributed to the transmural convective flow induced by the pressurized perfusion21. Supposedly, transmural flow in the vessel wall, if any, is beneficial to removing immunogenic cellular components therein. When we attempted to decellularize long segments of HUAs using a similar setup, however, very little transmural flow was observed. We hypothesized that a watertight lining at the abluminal side of HUAs hampered the transmural flow and tested the hypothesis by subjecting the abluminal surface of HUAs to enzyme digestion. Specifically, a highly viscous collagenase solution was applied onto the abluminal surface, thereby restricting the digestion to the surface. The vessel permeability, mechanical properties, and microstructure of collagenase- treated HUAs were examined in comparison with those of untreated HUAs in order to demonstrate the presence of the abluminal lining. Furthermore, both collagenase-treated and untreated HUAs were decellularized with 1% sodium dodecyl sulfate (SDS) solution under either rotary agitation, simple perfusion, or pressurized perfusion. Decellularization efficiency was examined by histology and residual DNA quantification over time. Materials and methods Preparation of human umbilical arteries. Human umbilical cords were obtained from the AN-AN Women and Children Clinic (Tainan, Taiwan) with patients’ consents and all the experimental protocols and the inform consent form were approved by the institutional review board/ethics committee of National Cheng Kung University Hospital (ER-99-060). Immediately after delivery, the cords were placed in cold Hank’s buffer saline solution and transferred to the laboratory for processing. Intact HUAs were isolated from the cords on ice using blunt dissection. Each HUA was cut in half; one half directly underwent decellularization or mechanical testing and the other half was subjected to the abluminal lining removal prior to further processing or testing. All methods were carried out in accordance with relevant guidelines and regulations. Enzymatic digestion of the abluminal surface. The abluminal surface of HUAs was treated with colla- genase solutions of different viscosity (Table 1) to remove the abluminal lining. Prior to the treatment, a silicone tube was inserted into the lumen of the HUA to maintain its tubular shape. Both ends of the HUA were then cannulated with plugged Luer adaptors to prevent the collagenase solution from entering the luminal space. For those HUAs that were to be treated with the collagenase solution of low viscosity, the HUA was immersed and incubated therein at 37 °C in a humidified CO2 incubator for 30, 60 or 120 min. For those HUAs that were to be treated with the collagenase solution of medium or high viscosity, the collagenase solution was applied evenly onto the abluminal surface of the HUA to ensure that enzyme digestion occurred from the surface (Fig. 1). The HUA was then incubated at 37 °C in a humidified CO2 incubator for 30, 60 or 120 min. After the treatment, the collagenase was removed by washing with phosphate buffer. In this study, the HUA treated with the collagenase solution of high viscosity for 60 min was designated as Col-HUA. The HUA that was not treated with collagenase solutions (a control group) was designated as Unt-HUA. Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4 www.nature.com/scientificreports/ Figure 1. Schematics of the collagenase treatment procedures for removing the abluminal lining of HUAs (a) and of the perfusion system for decellularizing HUAs (b). Figure 1. Schematics of the collagenase treatment procedures for removing the abluminal lining of HUAs (a and of the perfusion system for decellularizing HUAs (b). Permeability of the vessel wall. Materials and methods For permeability testing, HUAs were first cannulated with Luer adaptors at both ends. Upon expelling air in the lumen, one end of the HUA was plugged and the other end was connected to a bottle filled with deionized water. The HUA was then pressurized to 30 cmH2O by raising the water level in the bottle. Transmural flow was measured with transmural pressure fixed at 30 cmH2O. The permeability ( k ) of the HUA vessel wall was calculated using Darcy’s law, which describes the fluid flow through porous media as k = QLµ AsP , where As is the luminal surface area of the HUA (m2), P is the pressure drop across the vessel wall (mmHg), µ is the viscosity of deionized water (Pa s), Q is the transmural flow (m3/s), and L is the wall thickness measured by a custom-made high frequency ultrasound (lateral/axial resolution = 180/42 μm). Scanning electron microscopy. Scanning electron microscopy (SEM) was employed to examine the surface topography of Col-HUAs and Unt-HUAs cross-section. The fractured cross-section of the HUA was prepared by breaking the vessel in liquid nitrogen. The HUA was rinsed with PBS buffer, fixed in 2.5% glutar- aldehyde solution for 1 h, rinsed again with deionized water, and then dehydrated with graded alcohols. Dried specimens were sputter-coated with platinum (Sputter E-1045, Hitachi, Japan) and viewed using a scanning electron microscope (S-4100, Hitachi, Japan). Transmission electron microscopy. Transmission electron microscopy (TEM) was employed to reveal the ultrastructure of Col-HUAs and Uni-HUAs cross-section near the abluminal surface. The HUA was rinsed with PBS buffer, fixed in 2.5% glutaraldehyde solution overnight at 4 °C, rinsed again with deionized water, and then dehydrated with graded alcohols. The dried specimens were prepared into ultra-thin sections and stained with uranyl acetate. Images were acquired by a transmission electron microscope (JEM-1400, JEOL Ltd., Japan). Decellularization. SDS was selected as the decellularization agent to evaluate the effect of the abluminal lining removal on the decellularization of HUAs22. SDS (Mallinckrodt Baker, Phillipsburg, NJ) was dissolved in deionized water to prepare 1% (w/v) SDS solution. Both Col-HUAs and Unt-HUAs were decellularized with 1% SDS solution under three decellularization conditions (i.e., rotary agitation, simple perfusion, and pressurized perfusion) for 12, 24, or 48 h. For the simple agitation group, the HUA (~ 6 cm) was immersed in the SDS solu- tion on an orbital shaker at 100 rpm at room temperature22. www.nature.com/scientificreports/ groups, the HUA (~ 6 cm) was cannulated with luer adapters using 5-O suture and coupled to a custom-built perfusion system, which consists of a peristatic pump, a chamber accommodating the vessels to be decellular- ized, a solution reservoir, a pressure transducer (Model 80A-005G, 0–5 psi, Sensormate, Taiwan) and screw clamps (Fig. 1). During decellularization, 1 L of the SDS solution was continuously circulated at an average flow rate of 30 mL/min and a pulse frequency of 1 Hz at room temperature. The transmural pressure was set at 30 ± 10 mmHg (± 10 was caused by pulse flow) by adjusting the screw clamp in the flow loop for the pressurized perfusion group whereas the screw clamp was not engaged for the simple perfusion group. The pressure was selected based on our pilot study in which significant condensation of the ECM was observed in the Col-HUAs perfused at a transmural pressure greater than 30 mmHg. After the specified period of time, specimens of 2 mm length were cut at least 1 cm away from the sutured ends, washed in deionized water with agitation at least five times until no bubbles were found in the water to remove residual SDS, and then processed for either histology or DNA quantification. Histology and image quantification. The specimens were fixed in 10% neutral-buffered formalin over- night at room temperature. After standard processing, the HUA was embedded in paraffin to enable exami- nation of cross sections. Five micron sections were cut using a microtome (Leitz 1512, Leica, Germany) and collected on positively charged slides. The sections were stained with H&E, Alcian blue, and picro-sirius red (PSR) for illustration of nuclei, glycosaminoglycans (GAGs), and collagen, respectively. Histological images were acquired by an optical microscope (DM2500P, Leica, Germany) equipped with a CCD camera (DFC295 digital camera, Leica, Germany). Specifically, the PSR-stained sections were imaged under polarized light. Ten 24-bit color images of 2048 × 1563 pixel resolution were acquired under polarized light and normal light, respectively, for each specimen with a 40 × objective and saved in TIFF for semi-quantification of collagen and GAG. Relative collagen content was analyzed by a LabVIEW routine previously developed for quantifying collagen in PSR- stained sections23. Relative GAG content was analyzed similarly by another LabVIEW routine. DNA quantification. Quant-iT PicoGreen dsDNA assay kit (Invitrogen, USA) was used to quantify resid- ual DNA in the specimen. www.nature.com/scientificreports/ Similar to the previously published protocol22, the specimen was lyophilized at − 40 °C for 24 h and its dry weight was measured. The dried specimen was then incubated in a papain solution, which contained 20 U/mL papain (Worthinton, Lakewood, NJ), 1.1 mM EDTA (Panreac, Spain), 5.5 mM cysteine-HCl (Panreac, Spain) and 0.067 mM 2-mercaptoethanol (Alfa Aesar, England) overnight at 60 °C until the HUA was completely digested. The solution was diluted with 0.2 M Tris–EDTA buffer and then incubated with the work- ing solution of the kit in a 96-well plate. The fluorescence of the sample was measured using a fluorometer (exci- tation: 485 nm, emission: 538 nm; Fluoroskan Ascent, Thermo Fisher Scientific, Waltham, MA) and compared with that of a λ dsDNA standard (0–10 ng/mL) to determine the weight of residual DNA in the HUA. Finally, the weight of the residual DNA was normalized by the dry weight of the specimen. Mechanical testing of HUAs. The mechanical properties of Unt-HUAs, Col-HUAs, and decellularized Col-HUAs were evaluated by pressure-diameter tests using a custom-built mechanical tester24. Five HUAs were used; one from each of five donors. Every HUA was cut into three ~ 25-mm segments for preparing the three kinds of HUAs. First, the vessel was cannulated with Luer adaptors and then coupled to the loading frame of the tester. Specifically, every vessel was cannulated with a mechanically negligible, size-matched PDMS tube inserted in its lumen as water leakage from Col-HUAs and decellularized Col-HUAs during the test was expected24. The vessel was preconditioned by cyclic pressurization between 0 and 150 mmHg ten times. After preconditioning, the vessel was decoupled from the loading frame and recoupled at its updated unloaded configuration (the luminal pressure was about 10 mmHg, and the axial load was about 0 mN). The outer diameter of the vessel was recorded at the unloaded configuration. The axially constrained vessel was then subjected to cyclic pressuriza- tion at the unloaded length. Data from the loading phase of the cycle were analyzed for the mechanical proper- ties of the vessel as follows.h The compliance of the vessel was calculated by the Eq. (1). The compliance of the vessel was calculated by the Eq. (1). Materials and methods For the simple perfusion and pressurized perfusion Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Time courses of permeability changes in the vessel wall of the HUAs treated with the collagenase solution containing no thickening agent (low viscosity), 30% sucrose (medium viscosity), or 50% sucrose (high viscosity). The HUAs treated with the collagenase solution of low viscosity for 120 min leaked tremendously when pressurized thus no permeability coefficient was calculated. Each error bar should be on both sides of the mean; only one side is shown for simplicity. (3) a = b2 −(B2 −A2 z ) (3) where z = l L is the axial stretch ratio in which l is the deformed length of the vessel. Given a and b , the mean circumferential stress, σθ , was calculated using the Eq. (4), where z = l L is the axial stretch ratio in which l is the deformed length of the vessel. Given a and b , the mean circumferential stress, σθ , was calculated using the Eq. (4), (4) σθ = Pa h σθ = Pa h (4) where P is the transmural pressure, and h = (b −a) the deformed wall thickness. The associated mean circumferential stretch ratio was determined by the Eq (5) where P is the transmural pressure, and h = (b −a) the deformed wall thickness. The associated mean circumferential stretch ratio, θ , was determined by the Eq. (5) p , ( ) The associated mean circumferential stretch ratio, θ , was determined by the Eq. (5), (5) θ = (a + b) 2 (A + B) 2 θ = (a + b) 2 (A + B) 2 (5) The burst pressure and suture retention strength of the three groups of HUAs were also examined using the same mechanical tester with a 100 psi pressure transducer (Model 80A, 0–100 psi, Sensormate, Taiwan) and a 50 N load cell (MOB-10, Transducer Technique, Temecula, CA) as larger maximum pressure and maximum force were expected. The burst pressure of the HUA, the maximum pressure that the vessel could bear before failure, was measured by gradually increasing the transmural pressure of a preconditioned vessel. Suture retention tests were performed on ~ 20-mm HUA segments with a single 6-0 polypropylene suture placed at 2 mm away from one end of the vessel. The suture was connected to the load cell and the other end of the vessel was fixed to the loading frame. www.nature.com/scientificreports/ As the vessel was stretched at an extension rate of 0.25 mm/s, the force was recorded until the suture pulled through the vessel. Statistical analysis. Data were presented as mean ± standard deviation (SD). The mechanical properties of Unt-HUAs, Col-HUAs, and decellularized Col-HUAs were compared by one-way ANOVA with repeated measures in conjunction with Tukey post-hoc procedure. The collagen and GAG contents of the three HUAs groups as well as the residual DNA in either the Unt-HUAs or the Col-HUAs decellularized under the three decellularization conditions were compared by one-way ANOVA with Tukey post-hoc. The significance level was set at p = 0.05. www.nature.com/scientificreports/ (1) Compliance (% per 100 mmHg) = (D1 −D2) D2(P1 −P2) × 104 (1) where Pi and Di ( i = 1 or 2) respectively represent the transmural pressure and the corresponding outer diameter of the vessel. In this study, the compliance was calculated between 70 and 120 mmHg. That is, P1 = 120 mmHg and P2 = 70 mmHg. As the compliance can be influenced by the vessel wall thickness, it is more informative to examine the mechanical properties of the HUAs in terms of stress-stretch relationship. p p p The stress was determined based on the dimensions of the vessel at its deformed state. The unloaded thickness of the vessel, H , was measured from the histological section of the vessel using Image J (NIH). The wall volume ( V ) of the vessel was then determined by the Eq. (2), (2) V = π B2 −A2 L (2) where B is the unloaded outer radius, A = (B −H) the unloaded inner radius, and L the unloaded length of the HUA. where B is the unloaded outer radius, A = (B −H) the unloaded inner radius, and L the unloaded length of the HUA. Although not measurable, the deformed inner radius, a , at any deformed state can be computed by the Eq. (3) given the on-line measurement of deformed outer radius, b , with the assumption of incompressibility. Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4 www.nature.com/scientificreports/ Figure 2. Time courses of permeability changes in the vessel wall of the HUAs treated with the collagenase solution containing no thickening agent (low viscosity), 30% sucrose (medium viscosity), or 50% sucrose (high viscosity). The HUAs treated with the collagenase solution of low viscosity for 120 min leaked tremendously when pressurized thus no permeability coefficient was calculated. Each error bar should be on both sides of the mean; only one side is shown for simplicity. Figure 2. Time courses of permeability changes in the vessel wall of the HUAs treated with the collagenase solution containing no thickening agent (low viscosity), 30% sucrose (medium viscosity), or 50% sucrose (high viscosity). The HUAs treated with the collagenase solution of low viscosity for 120 min leaked tremendously when pressurized thus no permeability coefficient was calculated. Each error bar should be on both sides of the mean; only one side is shown for simplicity. Figure 2. Results Figure 2 shows the temporal changes in the permeability of the HUAs treated with the collagenase solution containing no sucrose, 30% sucrose, or 50% sucrose. The collagenase solution containing no sucrose apparently digested the whole vessel wall. Note that 120 min of incubation therein resulted in severe water leakage from the vessels during permeability tests and thus no permeability coefficient was calculated. HUAs remained intact in appearance after being treated with the collagenase solution containing 30% or 50% sucrose for up to 2 h. The collagenase treatment loosened the connective tissue at the abluminal surface of HUAs. This was well illustrated on the H&E-stained sections of Col-HUAs; the abluminal surface of which was smoother than that of Unt-HUAs (Supplementary Fig. 1). Histology also demonstrated that the treatment of the collagenase solution containing sucrose did not damage the media; the collagenase digestion appeared to be localized, limiting its effect to the https://doi.org/10.1038/s41598-020-67417-4 Scientific Reports \| (2020) 10:10556 \| www.nature.com/scientificreports/ Figure 3. Representative SEM images of Col-HUA (a) and Unt-HUA (b,c) cross-sections. The arrows indicate the microstructure that was absent in Col-HUAs. Panel (c) shows higher magnification view of the box outlined in panel (b). Fi 3 R t ti SEM i f C l HUA ( Figure 3. Representative SEM images of Col-HUA (a) and Unt-HUA (b,c) cross-sections. The arrows indicate the microstructure that was absent in Col-HUAs. Panel (c) shows higher magnification view of the box outlined in panel (b). abluminal surface. Regardless of sucrose concentrations, the permeability coefficients of HUAs increased with the collagenase concentration and duration of digestion. The permeability variations were smaller, in particular, for the HUAs treated with the collagenase solution containing 50% sucrose. Therefore, for all the subsequent experiments involving the abluminal lining removal, HUAs were treated with the collagenase solution containing 50% sucrose for 60 min as the treatment resulted in acceptable vessel permeability with better consistency. When Unt-HUAs were examined for permeability, no water leaked out of the pressurized Unt-HUAs; the permeability coefficient of Unt-HUAs was zero. abluminal surface. Regardless of sucrose concentrations, the permeability coefficients of HUAs increased with the collagenase concentration and duration of digestion. The permeability variations were smaller, in particular, for the HUAs treated with the collagenase solution containing 50% sucrose. Results Therefore, for all the subsequent experiments involving the abluminal lining removal, HUAs were treated with the collagenase solution containing 50% sucrose for 60 min as the treatment resulted in acceptable vessel permeability with better consistency. When Unt-HUAs were examined for permeability, no water leaked out of the pressurized Unt-HUAs; the permeability coefficient of Unt-HUAs was zero. fi Figures 3 and 4 show, respectively, the representative SEM and TEM images of Col-HUAs and Unt-HUAs near the abluminal surface. The SEM image of the Unt-HUA revealed a protective lining at the abluminal surface, which was absent from the SEM image of the Col-HUA. Note also that the vessel wall of the Col-HUA remained intact, indicating that collagenase digestion was restricted to the abluminal surface. The TEM image of the Unt- HUA cross-section revealed an electron dense lining near the abluminal surface, which was also absent from the mostly electron lucent Col-HUA cross section. In another TEM image (Supplementary Fig. 2), the lining tissue was separated from the underlying tissue probably due to tissue sectioning, suggesting that the lining tissue is structurally distinct from the underlying tissue. Based on TEM observation, the lining appeared to be acellular and had a thickness of 2–3 μm. μ Figure 5 shows the mechanical properties of Unt-HUAs and Col-HUAs. Both the pressure-diameter curves (mechanical behavior including both structural and material contributions) and stress-stretch curves (mechanical behavior free of structural contribution) shifted to the right after collagenase treatment. As the collagenase treat- ment did not change the vessel thickness, the shifting of both curves could be due to either changes in materials or the removal of some structure that has a negligible thickness. Note that the toe region of the stress-stretch curve became more significant and the slope of the stress-stretch curve at the linear region remained unchanged after collagenase treatment. That is, the collagenase treatment did not change the main load bearing capability of HUAs. Together with the histological finding that the media was not affected by collagenase treatment (will be shown below), the changes in mechanical behavior of HUAs suggest the removal of a stiff abluminal lining. Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4 www.nature.com/scientificreports/ ntificreports/ Figure 4. Representative TEM images of Col-HUA (a) and Unt-HUA (b,c) cross-sections near the abluminal surface. The arrows indicate the microstructure that was absent in Col-HUAs. Panel c shows higher magnification view of the box outlined in panel (b). Results Figure 4. Representative TEM images of Col-HUA (a) and Unt-HUA (b,c) cross-sections near the abluminal surface. The arrows indicate the microstructure that was absent in Col-HUAs. Panel c shows higher magnification view of the box outlined in panel (b). Figure 6 shows the representative H&E-stained cross-sections of the Col-HUAs and Unt-HUAs that were decellularized with the SDS solution under rotary agitation, simple perfusion, or pressurized perfusion for 24 h. A few nuclei remained visible on the Unt-HUAs decellularized under simple agitation. While no nucleus was observed on the sections of the Unt-HUAs decellularized under simple perfusion or pressurized perfusion, there appeared blue diffuse smears in their media. The finding is consistent with previous reports19,25, in which HUAs were decellularized by surfactants only. The smears were also seen on the sections of the Col-HUAs decellularized under simple agitation whereas they are absent from the sections of the Col-HUAs decellularized under simple perfusion and pressurized perfusion. Simple perfusion, in general, appeared to be as effective as pressurized perfusion based on the histological findings.hfi p gi g The efficiency of decellularization can be more objectively evaluated by quantifying residual DNA in the vessel. Figure 7 shows that the amount of DNA remaining in both Unt-HUAs and Col-HUAs depended on Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4 www.nature.com/scientificreports/ Figure 5. Average pressure-diameter curves (a) and circumferential stress-stretch curves (b) of Unt-HUAs and Col-HUAs. Data are presented as mean ± SD (n = 5). Figure 5. Average pressure-diameter curves (a) and circumferential stress-stretch curves (b) of Unt-HUAs and Col-HUAs. Data are presented as mean ± SD (n = 5). Figure 6. Illustration of residual DNA in Unt-HUAs (a,c,e) and Col-HUAs (b,d,f) decellularized with 1% sucrose solution under rotary agitation, simple perfusion, or pressurized perfusion for 24 h with H&E stained histological sections. Arrows indicate nuclei debris. Figure 6. Illustration of residual DNA in Unt-HUAs (a,c,e) and Col-HUAs (b,d,f) decellularized with 1% sucrose solution under rotary agitation, simple perfusion, or pressurized perfusion for 24 h with H&E stained histological sections. Arrows indicate nuclei debris. Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4 www.nature.com/scientificreports/ Figure 7. Time course of residual DNA in Unt-HUAs (a) and Col-HUAs (b) decellularized with 1% SDS solution under rotary agitation, simple perfusion, or pressurized perfusion. Data are presented as mean ± SD (n = 5). p < 0.025 vs. pressurized perfusion; †p < 0.025 vs. simple perfusion. Figure 7. Results Time course of residual DNA in Unt-HUAs (a) and Col-HUAs (b) decellularized with 1% SDS solution under rotary agitation, simple perfusion, or pressurized perfusion. Data are presented as mean ± SD (n = 5). p < 0.025 vs. pressurized perfusion; †p < 0.025 vs. simple perfusion. the decellularization conditions in the order: rotary agitation > simple perfusion > pressurized perfusion for all the time points. Regardless of decellularization conditions, however, an appreciable amount of residual DNA remained in the Unt-HUAs decellularized for up to 48 h. When compared with Fig. 7a, Fig. 7b shows that decel- lularization efficiency was significantly improved upon the abluminal lining removal. Particularly, complete removal of DNA was accomplished within 24 h by pressurized perfusion of the SDS solution. Results of residual DNA quantification were consistent with the histological findings. qi gi g Figure 8 shows the representative images of PSR-stained and Alcian blue-stained cross sections of Unt-HUAs, Col-HUAs, and decellularized Col-HUAs and their corresponding semi-quantitative analysis. The PSR images revealed that birefringence intensity on the Col-HUA and Unt-HUA sections was comparable whereas the intensity decreased after decellularization. The corresponding image quantification (Fig. 8d) also showed that the staining intensity on the decellularized CT-HUA sections was significantly lower than that on the Unt-HUA and Col-HUA sections, indicating that the collagen in the media was not affected by collagenase treatment and some collagen was removed by decellularization. Similarly, the Alcian blue staining on the Col-HUA section was comparable to that on the Unt-HUA section and the staining decreased after decellularization, which was verified by image quantification (Fig. 8h); that is, the collagenase treatment did not change the GAGs content but decellularization reduced the GAG content significantly.ttt gi y Figure 9 shows both the pressure-diameter and stress–strain curves shifted to the left after decellularization; that is, the ECM components removed by decellularization might contribute to the mechanical behavior of HUAs. Figure 9c show the compliance, burst pressure and suture retention strength of Unt-HUAs, Col-HUAs, and decellularized Col-HUAs. The compliance (i.e., the reciprocal of structural stiffness) of Col-HUAs was sig- nificantly greater than that of Unt-HUAs and decellularized Col-HUAs. Note that decellularized Col-HUAs had a compliance similar to that of Unt-HUAs. No significant differences in the burst pressure were found among the three groups of HUAs. The suture retention strength, however, significantly decreased in Col-HUAs and decellularized Col-HUAs compared to that of Unt-HUAs. Discussion We demonstrated for the first time the presence of a watertight barrier at the abluminal side of HUAs by the abluminal collagenase treatment, which rendered HUAs water permeable without damaging the underlying media. The presence of the abluminal lining was further supported by comparing the SEM and TEM images, and the mechanical properties of Unt-HUAs and Col-HUAs. In our pilot study to test if there is a watertight barrier at the luminal side of HUAs, highly viscous collagenase solution was delivered to and confined within the luminal space, allowing the collagenase to work on luminal surface only. Also, HUAs were turned inside-out and the external luminal surface was then treated with collagenase. In both cases, the vessel wall of the treated HUAs remained impermeable to water, indicating that the watertight barrier is not at the luminal side of HUAs.h The addition of sucrose, serving as a thickening agent, in collagenase solution increased its viscosity (i.e., reduced its fluidity), which resulted in the spatially restricted activity of collagenase and hence made localized digestion possible. The highly viscous collagenase solution was effective in removing the abluminal lining while preserving the underlying media. The dependence of the vessel permeability on the collagenase concentration and digestion duration indicates that the watertight lining is composed of some substrates of collagenase. In our pilot study, highly viscous hyaluronidase and elastase solutions were also prepared and tested, separately. Neither was effective in making HUAs water permeable, though.i f g p g Similar to the adventitia found in other arteries, the abluminal lining appeared to have a specific role in the mechanical behavior of HUAs. The substantial right-shifting of the stress-stretch curve upon collagenase treat- ment indicates that the abluminal lining may prevent HUAs from over-dilation at low pressure. On the other hand, the independence of the burst pressure on the abluminal lining, indicates that the failure of the abluminal Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4 www.nature.com/scientificreports/ Figure 8. Representative PSR stained (a–c) and Alcian blue stained (e–g) of Unt-HUAs (a,e), Col-HU and decellularized Col-HUAs (c,g). Comparisons of relative collagen (d) and GAG (h) contents among groups. Data are presented as mean ± SD (n = 3). p < 0.025. Scale bar 100 μm. Figure 8. Representative PSR stained (a–c) and Alcian blue stained (e–g) of Unt-HUAs (a,e), Col-HUA and decellularized Col-HUAs (c,g). Discussion Comparisons of relative collagen (d) and GAG (h) contents among r p D t r pr nt d m n±SD (n 3) p<0 025 S l b r 100 m Figure 8. Representative PSR stained (a–c) and Alcian blue stained (e–g) of Unt-HUAs (a,e), Col-HUAs (b,f), and decellularized Col-HUAs (c,g). Comparisons of relative collagen (d) and GAG (h) contents among the three groups. Data are presented as mean ± SD (n = 3). p < 0.025. Scale bar 100 μm. lining may occur earlier than the whole HUA in response to increasing pressure. Although the suture retention strength of HUAs reduced after the abluminal lining removal, it remained sufficient for a successful anastomosis. The removal of cellular components is essential to prevent severe immune responses for in vivo use of decel- lularized tissue scaffolds10,26. We showed in our previous study that 48 h of decellularization under rotary agitation reduced residual DNA in short segments of HUAs to the detection limit of the picoGreen assay22. The rotary agitation was not effective for decellularizing long segments of HUAs, however. The decellularization of long segments of HUAs may be improved by perfusing decellularization solution into their lumen. Transmural flow lining may occur earlier than the whole HUA in response to increasing pressure. Although the suture retention strength of HUAs reduced after the abluminal lining removal, it remained sufficient for a successful anastomosis. The removal of cellular components is essential to prevent severe immune responses for in vivo use of decel- lularized tissue scaffolds10,26. We showed in our previous study that 48 h of decellularization under rotary agitation reduced residual DNA in short segments of HUAs to the detection limit of the picoGreen assay22. The rotary agitation was not effective for decellularizing long segments of HUAs, however. The decellularization of long segments of HUAs may be improved by perfusing decellularization solution into their lumen. Transmural flow lining may occur earlier than the whole HUA in response to increasing pressure. Although the suture retention strength of HUAs reduced after the abluminal lining removal, it remained sufficient for a successful anastomosis. The removal of cellular components is essential to prevent severe immune responses for in vivo use of decel- lularized tissue scaffolds10,26. We showed in our previous study that 48 h of decellularization under rotary agitation reduced residual DNA in short segments of HUAs to the detection limit of the picoGreen assay22. Discussion The rotary agitation was not effective for decellularizing long segments of HUAs, however. The decellularization of long segments of HUAs may be improved by perfusing decellularization solution into their lumen. Transmural flow Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4 www.nature.com/scientificreports/ Figure 9. Average pressure-diameter curves (a) and circumferential stress-stretch curves (b) of Unt-HUA, Col- HUAs and decellularized Col-HUAs. (c) Comparisons of the compliance, burst pressure, and suture retention strength among Unt-HUAs, Col-HUAs, and decellularized Col-HUAs. Data are presented as mean ± SD (n = 5). p < 0.05. Figure 9. Average pressure-diameter curves (a) and circumferential stress-stretch curves (b) of Unt-HUA, Col- HUAs and decellularized Col-HUAs. (c) Comparisons of the compliance, burst pressure, and suture retention strength among Unt-HUAs, Col-HUAs, and decellularized Col-HUAs. Data are presented as mean ± SD (n = 5). p < 0.05. that may be introduced by the perfusion, if any, is beneficial to removing immunogenic cellular components in the vessel wall. It was not until the removal of the abluminal lining and hence the associated increase in vessel permeability that the perfusion-assisted decellularization of long segments of HUAs was significantly improved. Residual DNA was almost undetectable by the picoGreen assay in the Col-HUAs decellularized under pres- surized perfusion for 24 h. This is a considerable improvement in terms of efficiency for the decellularization of HUAs19,22,25 or other blood vessels27–31. The improved decellularization efficiency could significantly reduce the cost of production. Additionally, as SDS has been reported to have adverse effects on the ECM, the reduced duration of SDS treatment is favorable. One of the advantages of using decellularized tissue scaffolds for tissue engineering is the availability of bioactive molecules in the scaffold. The reduction in process time may also be beneficial to the preservation of bioactive molecules such as basement membrane components and ECM-bound growth factors (e.g., bFGF32 and VEGF33). The former could facilitate the recellularization of the vessel with anti- thrombotic endothelial cells as well as smooth muscle cells. The latter could also support reendothelialization and help maintain a functional endothelium. that may be introduced by the perfusion, if any, is beneficial to removing immunogenic cellular components in the vessel wall. It was not until the removal of the abluminal lining and hence the associated increase in vessel permeability that the perfusion-assisted decellularization of long segments of HUAs was significantly improved. www.nature.com/scientificreports/ might complicate decellularization and future cell seeding, respectively. Because of the pressure drop between upstream and downstream, the length for the whole vessel to be completely decellularized using the current system at the specified conditions without over compression of the ECM is limited (~ 6 cm). Other factors that may cause uneven transmural interstitial flow in the vessel include irregular microstructure and locally varying wall thickness of the vessel. These factors should be borne in mind when decellularizing long tubular tissues by pressurized perfusion. might complicate decellularization and future cell seeding, respectively. Because of the pressure drop between upstream and downstream, the length for the whole vessel to be completely decellularized using the current system at the specified conditions without over compression of the ECM is limited (~ 6 cm). Other factors that may cause uneven transmural interstitial flow in the vessel include irregular microstructure and locally varying wall thickness of the vessel. These factors should be borne in mind when decellularizing long tubular tissues by pressurized perfusion. In vitro recellularization of decellularized blood vessels remains challenging because of their dense micro- structure, which hinders cell migration. Shimizu et al. used magnetite nanoparticles to facilitate cell-seeding into decellularized porcine carotid arteries; however, cells were only found on the external surface of the vessel (i.e., no infiltration of cells into the media)37. Yazdani et al. showed that the removal of adventitia from decellularized porcine carotid arteries could improve cell seeding efficiency31. In their study, the adventitia was mechanically removed, however, which may damage the arterial structure. We attempted to recellularize in vitro the decel- lularized HUAs in our pilot study. Unfortunately, cells were only found to attach on the external surface of decellularized Col-HUAs without infiltrating into the media (data not shown).h i There are a few limitations in this study. We used 1% SDS solution as the decellularizing agent to see if the enhanced permeability benefits the decellularization of HUAs. Other agents (e.g., Triton X-100, and sodium deoxycholate) and their combinations may work better and should be tested for optimal HUA decellularization. It is unclear if the abluminal lining of HUAs has biological roles or physiological functions. The host response of the decellularized Col-HUAs and Unt-HUAs following implantation warrants a detailed study. Conclusion h d In this study, we demonstrate the presence of an abluminal watertight lining in HUAs that renders the vessel water impermeable. The removal of the lining without affecting the underlying media was successfully achieved by digesting the abluminal surface of the vessel using a highly viscous collagenase solution. The establishment of transmural flow was verified upon the abluminal lining removal. Evidence from SEM, TEM, and mechanical testing also supported the presence of the abluminal lining. More importantly, the removal of the abluminal lining significantly improved the perfusion-assisted decellularization of HUAs. Received: 19 December 2019; Accepted: 2 June 2020 www.nature.com/scientificreports/ Note, however, that the different protocols used for decellularizing Col-HUAs and Unt-HUAs, in addition to the presence or absence of the abluminal lining, could lead to different in vivo remodeling outcomes of the two tissues. Because of the many interesting characteristics of the abluminal lining, a detailed proteomic analysis should be conducted to fully understand its composition. Finally, the effectiveness of decellularized Col-HUAs or recellularized Col- HUAs to be used as a vascular graft should be evaluated using vascular reconstructive animal models. References 1. Herrington, W., Lacey, B., Sherliker, P., Armitage, J. & Lewington, S. Epidemiology of atherosclerosis and the potential to reduce the global burden of atherothrombotic disease. Circ. Res. 118, 535–546. https://doi.org/10.1161/Circresaha.115.307611 (2016). 1. Herrington, W., Lacey, B., Sherliker, P., Armitage, J. & Lewington, S. Epidemiology of atherosclerosis and the potential to reduce the global burden of atherothrombotic disease. Circ. Res. 118, 535–546. https://doi.org/10.1161/Circresaha.115.307611 (2016). g . Libby, P. et al. Atherosclerosis. Nat. Rev. Dis. Primers 5, 56. https y p g 3. Benjamin, E. J. et al. Heart disease and stroke statistics-2017 update a report from the American Heart Association. Circulation 135, E146–E603. https://doi.org/10.1161/Cir.0000000000000485 (2017). y p g 3. Benjamin, E. J. et al. Heart disease and stroke statistics-2017 update a report from the American Heart Association. Circulation 135, E146–E603. https://doi.org/10.1161/Cir.0000000000000485 (2017). 4. Youngstrom, D. W., Barrett, J. G., Jose, R. R. & Kaplan, D. L. Functional characterization of detergent-decellularized equine tendon extracellular matrix for tissue engineering applications. PLoS ONE 8, 1 (2013).t 5. Grayson, W. L. et al. Engineering anatomically shaped human bone grafts. Proc. Natl. Acad. Sci. USA 107, 3299–3304 (2010 6. Hudson, T. W. et al. Optimized acellular nerve graft is immunologically tolerated and supports regeneration. Tissue Eng. 10 1641–1651 (2004).th 7. Elkins, R. C., Dawson, P. E., Goldstein, S., Walsh, S. P. & Black, K. S. Decellularized human valve allografts. Ann. Thorac. Surg. 71 S428–S432 (2001). 8. Cho, S. W. et al. Small-diameter blood vessels engineered with bone marrow-derived cells. Ann. Surg. 241, 506–515. https://doi org/10.1097/01.sla.0000154268.12239.ed (2005).t g 9. Xiong, Y. et al. Decellularized porcine saphenous artery for small-diameter tissue-engineered conduit graft. Artif. Organs 37 E74–E87. https://doi.org/10.1111/Aor.12014 (2013). p g ( ) 10. Crapo, P. M., Gilbert, T. W. & Badylak, S. F. An overview of tissue and whole organ decellularization processes. Biomaterials 32, 3233–3243. https://doi.org/10.1016/j.biomaterials.2011.01.057 (2011).f p g j ( ) 1. Dahl, S. L. M., Koh, J., Prabhakar, V. & Niklason, L. E. Decellularized native and engineered arterial scaffolds for transplantation Cell Transplant. 12, 659–666 (2003).ffi 12. Rieder, E. et al. Decellularization protocols of porcine heart valves differ importantly in efficiency of cell removal and susceptibility of the matrix to recellularization with human vascular cells. J. Thorac. Cardiovasc. Surg. 127, 399–405. https://doi.org/10.1016/j. jtcvs.2003.06.017 (2004).i j 3. Kerdjoudj, H. et al. Decellularized umbilical artery treated with thin polyelectrolyte multilayer films: potential use in vascular engineering. Bio-Med. Mater. Eng. 16, S123–S129 (2006). g g g 14. Discussion Residual DNA was almost undetectable by the picoGreen assay in the Col-HUAs decellularized under pres- surized perfusion for 24 h. This is a considerable improvement in terms of efficiency for the decellularization of HUAs19,22,25 or other blood vessels27–31. The improved decellularization efficiency could significantly reduce the cost of production. Additionally, as SDS has been reported to have adverse effects on the ECM, the reduced duration of SDS treatment is favorable. One of the advantages of using decellularized tissue scaffolds for tissue engineering is the availability of bioactive molecules in the scaffold. The reduction in process time may also be beneficial to the preservation of bioactive molecules such as basement membrane components and ECM-bound growth factors (e.g., bFGF32 and VEGF33). The former could facilitate the recellularization of the vessel with anti- thrombotic endothelial cells as well as smooth muscle cells. The latter could also support reendothelialization and help maintain a functional endothelium.hl p The convective transmural flow was thought to improve the decellularization of HUVs21,34–36. When infused by water, Unt-HUAs were found to be watertight; i.e., no transmural flow. The controversial finding may be due to the difference in nature between HUVs and HUAs. Note, however, that HUVs in their study were trimmed to have a uniform wall thickness by using a cryo lathe prior to decellularization21. The machining process could have removed the abluminal watertight lining of HUVs, if any. Nevertheless, an abluminal watertight lining in HUVs was not identified in their studies. i Transmural pressure is expected to decrease gradually along the length of the vessel because of the perme- able vessel wall. In our perfusion system, pressure was measured and manipulated downstream from the ves- sel (see Fig. 1). The pressure, which was set at 30 mmHg for the group of pressurized perfusion, thus ensured transmural flow throughout the entire vessel despite the decreasing transmural flow along the vessel. Histology at three locations of the vessel (upstream, middle, and downstream) showed that no residual nuclei were found at all locations (data not shown). The ECM at the upstream location of vessels that were longer than 6 cm, however, appeared to be condensed after decellularization (data not shown), which might be attributed to rela- tively higher transmural pressure therein. The reduced vessel permeability and pore size in the condensed ECM Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4 11 www.nature.com/scientificreports/ www.nature.com/scientificreports/ et al. The use of high-hydrostatic pressure treatmen 31. Yazdani, S. K. et al. Smooth muscle cell seeding of decellularized scaffolds: the importance of bioreactor preconditioning to opment of a more native architecture for tissue-engineered blood vessels. Tissue Eng. Part A 15, 827–840 (2009).i 32. Rogelj, S. et al. Basic fibroblast growth-factor is an extracellular-matrix component required for supporting the proliferation of vascular endothelial-cells and the differentiation of Pc12 cells. J Cell Biol 109, 823–831 (1989).h f 33. Olaya, C. M. et al. The umbilical cord, preeclampsia and the VEGF family. Int. J. Womens Health 10, 783–795. https://doi. org/10.2147/IJWH.S174734 (2018).hft g 4. Abousleiman, R. I., Reyes, Y., McFetridge, P. & Sikavitsas, V. The human umbilical vein: a novel scaffold for musculoskeletal sof tissue regeneration. Artif. Organs 32, 735–742. https://doi.org/10.1111/j.1525-1594.2008.00598.x (2008). 35. Crouzier, T., McClendon, T., Tosun, Z. & McFetridge, P. S. Inverted human umbilical arteries with tunable wall thicknesses for nerve regeneration. J. Biomed. Mater. Res., Part A 89A, 818–828. https://doi.org/10.1002/Jbm.A.32103 (2009).f 36. Rodriguez, M., Juran, C., McClendon, M., Eyadiel, C. & McFetridge, P. S. Development of a mechanically tuneable 3D scaffold for vascular reconstruction. J. Biomed. Mater. Res., Part A 100A, 3480–3489. https://doi.org/10.1002/Jbm.A.34267 (2012).f p g 37. Shimizu, K. et al. Effective cell-seeding technique using magnetite nanoparticles and magnetic force onto decellularized b vessels for vascular tissue engineering. J. Biosci. Bioeng. 103, 472–478. https://doi.org/10.1263/Jbb.103.472 (2007). References Chen, F. M. & Liu, X. H. Advancing biomaterials of human origin for tissue engineering. Prog. Polym. Sci. 53, 86–168 (2016).f g g F. M. & Liu, X. H. Advancing biomaterials of human origin for tis g g g 14. Chen, F. M. & Liu, X. H. Advancing biomaterials of human or g g g 4. Chen, F. M. & Liu, X. H. Advancing biomaterials of human origin for tissue engineering. Prog. Polym. Sci. 53, 86–168 (2016). 5. Londono, R. & Badylak, S. F. Biologic scaffolds for regenerative medicine: mechanisms of in vivo remodeling. Ann. Biomed. Eng 43 577 592 h //d i /10 1007/ 10439 014 1103 8 (2015) g g g g g y 15. Londono, R. & Badylak, S. F. Biologic scaffolds for regenerative medicine: mechanisms of in vivo remodeling. Ann. Biomed. 43, 577–592. https://doi.org/10.1007/s10439-014-1103-8 (2015). p g 16. Dahl, S. L. M., Rhim, C., Song, Y. C. & Niklason, L. E. Mechanical properties and compositions of tissue engineered and native arteries. Ann. Biomed. Eng. 35, 348–355 (2007).f 17. Daniel, J., Abe, K. & McFetridge, P. S. Development of the human umbilical vein scaffold for cardiovascular tissue engineering applications. ASAIO J. 51, 252–261. https://doi.org/10.1097/01.Mat.0000160872.41871.7e (2005). pp p g 8. Dahan, N. et al. Porcine small diameter arterial extracellular matrix supports endothelium formation and media remodeling forming a promising vascular engineered biograft. Tissue Eng. Part A 18, 411–422 (2012). g p g g gt g ( ) 19. Gui, L. Q., Muto, A., Chan, S. A., Breuer, C. K. & Niklason, L. E. Development of decellularized human umbilical arteries as small- diameter vascular grafts. Tissue Eng. Part A 15, 2665–2676. https://doi.org/10.1089/ten.tea.2008.0526 (2009). https://doi.org/10.1038/s41598-020-67417-4 Scientific Reports \| (2020) 10:10556 \| Author contributions J.-J.H. designed the experiments and supervised the project. H.-Y.T.-M. and Y.-H.C. performed the experiments and analyzed the data. H.-Y.T.-M. and J.-J.H. drafted and wrote the manuscript. J.-J.H. edited and revised the manuscript. All authors approved the manuscript. Competing interests h p g The authors declare no competing interests. Acknowledgements g We are grateful to Dr. Shang-Rung Wu for her technical support on TEM. Financial supports from the Ministry of Science and Technology at Taiwan (MOST 105-2221-E-006-176) and the National Health Research Institute at Taiwan (NHRI-EX109-10827EI) are gratefully acknowledged. g We are grateful to Dr. Shang-Rung Wu for her technical support on TEM. Financial supports from the Ministry of Science and Technology at Taiwan (MOST 105-2221-E-006-176) and the National Health Research Institute at Taiwan (NHRI-EX109-10827EI) are gratefully acknowledged. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 20. Hoenicka, M., Lehle, K., Jacobs, V. R., Schmid, F. X. & Birnbaum, D. E. Properties of the human umbilical vein as a living scaffold for a tissue-engineered vessel graft. Tissue Eng. 13, 219–229. https://doi.org/10.1089/ten.2006.0121 (2007).l t 21. Montoya, C. V. & McFetridge, P. S. Preparation of ex vivo-based biomaterials using convective flow decellularization. Tissue Part C-Methods 15, 191–200. https://doi.org/10.1089/ten.tec.2008.0372 (2009). p g ( ) 2. Tuan-Mu, H. Y., Yu, C. H. & Hu, J. J. On the decellularization of fresh or frozen human umbilical arteries: implications for small- p g 22. Tuan-Mu, H. Y., Yu, C. H. & Hu, J. J. On the decellularization of fresh or frozen human umbilical arteries: implications for small- diameter tissue engineered vascular grafts. Ann. Biomed. Eng. 42, 1305–1318. https://doi.org/10.1007/s10439-014-1000-1 (2014). J J p diameter tissue engineered vascular grafts. Ann. Biomed. Eng. 42, 1305–1318. https://doi.org/10.1007/s10439-014-1000-1 (2014) t 23. Hu, J. J. et al. Time courses of growth and remodeling of porcine aortic media d chemical examination. J. Histochem. Cytochem. 56, 359–370 (2008). 3. Hu, J. J. et al. Time courses of growth and remodeling of porcine aortic media during hypertension: a quantitative immunohisto- chemical examination. J. Histochem. Cytochem. 56, 359–370 (2008).f y ( ) 4. Hu, J. J., Chao, W. C., Lee, P. Y. & Huang, C. H. Construction and characterization of an electrospun tubular scaffold for small- diameter tissue-engineered vascular grafts: a scaffold membrane approach. J. Mech. Behav. Biomed. Mater. 13, 140–155 (2012). g gtf pp 5. Gui, L., Chan, S. A., Breuer, C. K. & Niklason, L. E. Novel utilization of serum in tissue decellularization. Tissue Eng. Part C Methods 16, 173–184. https://doi.org/10.1089/ten.TEC.2009.0120 (2010). p g 6. Gilbert, T. W., Sellaro, T. L. & Badylak, S. F. Decellularization of tissues and organs. Biomaterials 27, 3675–3683. https://doi org/10.1016/j.biomaterials.2006.02.014 (2006). g j 27. Malone, J. M., Brendel, K., Duhamel, R. C. & Reinert, R. L. Detergent-extracted small-diameter vascular prostheses. J. Vasc. Surg. 1, 181–191 (1984).f 8. Lu, Q. J., Ganesan, K., Simionescu, D. T. & Vyavahare, N. R. Novel porous aortic elastin and collagen scaffolds for tissue engineer- ing. Biomaterials 25, 5227–5237. https://doi.org/10.1016/j.biomaterials.2003.12.019 (2004). 9. Wilshaw, S. P. et al. Development and characterization of acellular allogeneic arterial matrices. Tissue Eng. Part A 18, 471–483 https://doi.org/10.1089/ten.tea.2011.0287 (2012).h p g 30. Funamoto, S. et al. The use of high-hydrostatic pressure treatment to decellularize blood vessels. Biomaterials 31, 3590–3595 (2010).f p g . Funamoto, S. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-67417-4. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-67417-4. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-67417-4. Correspondence and requests for materials should be addressed to J.-J.H. Correspondence and requests for materials should be addressed to J.-J.H. Correspondence and requests for materials should be addressed to J.-J.H. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 Scientific Reports \| (2020) 10:10556 \| https://doi.org/10.1038/s41598-020-67417-4
https://openalex.org/W2915068360	https://www.nature.com/articles/s41598-018-37841-8.pdf	English	null	Operation mode of a step-feed anoxic/oxic process with distribution of carbon source from anaerobic zone on nutrient removal and microbial properties	Scientific reports	2,019	cc-by	10,773	Yijun Shen1,2, Dianhai Yang1, Yang Wu2, Hao Zhang1 & Xinxi Zhang2 This study investigated the operation mode of a step-feed anoxic/oxic (A/O) process with distribution of the carbon source from the anaerobic zone in terms of the treatment effects on sewage with low carbon and high nitrogen and phosphorus. After seven phases of operation, an optimal flow distribution ratio of 75%:25% was obtained from the anaerobic zone, and the concentrations of chemical oxygen demand, ammonia nitrogen, total nitrogen, and total phosphorous in the effluent were 20.8, 0.64, 14.2, and 0.89 mg/L, respectively. The presence of an internal reflux system in the deaeration zone improved the treatment. 16S rRNA high-throughput sequencing revealed that the microbial communities in aerobic zone I(O1) of the first-step A/O sludge were different from those in aerobic zone I (O2) of the second- step A/O sludge, whereas microbial communities of the seed sludge were similar to those in O2 of the second-step A/O sludge. The richness and diversity of microbial communities in O1 of the first-step A/O sludge samples were higher than those in O2 of the second-step A/O and seed sludge. At the optimal flow distribution ratio, the microbial abundance and treatment removal efficiency were the highest. Eutrophication caused by high concentrations of nitrogen and phosphorus has serious effects on the environ- ment and on human health globally. Chaohu Lake Basin, which is one of the five freshwater lakes in China, faces a serious eutrophication problem, and tail water from wastewater treatment plants (WWTPs) is one of the main sources of pollution. A previous study has indicated that nutrient pollution from effluents of wastewater plants is more serious than that from non-point sources1. Municipal wastewater in southern China is characterised by high nitrogen and phosphorus and low carbon content. Aiming at lower nitrogen and phosphorus concentrations is essential not only in the design of new plants, but also for upgrading existing wastewater treatment plants. The nitrogen and phosphorus removal efficiencies of conventional active sludge technologies are relatively low and are limited by the carbon source2. Several technologies, such as external organic carbon for post-denitrification and recirculation approaches, can be used to solve this problem. However, these countermeasures require addi- tional resources and energy and hence, increase the operation costs and environmental footprint. Therefore, it is necessary to take full advantage of carbon sources in the raw wastewater. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 23 August 2018 Accepted: 14 December 2018 Published: xx xx xxxx Operation mode of a step- feed anoxic/oxic process with distribution of carbon source from anaerobic zone on nutrient removal and microbial properties Received: 23 August 2018 Accepted: 14 December 2018 Published: xx xx xxxx Yijun Shen1,2, Dianhai Yang1, Yang Wu2, Hao Zhang1 & Xinxi Zhang2 Results and Discussionfl Effect of flow distribution ratios on COD and nutrient removal efficiency. The test was run for 120 days in 7 phases. Phase I~VI was carbon source distribution of raw influent and the ratio was calculated by the flow feeding each in turn anoxic zone accounted for the proportion of the influent flow; while phase VII was car- bon source distribution from anaerobic zone and the ratio was calculated by the flow feeding each in turn anoxic zone accounted for the proportion of the mix liquor flow (summation of the influent flow and returned activated sludge flow).The internal reflux was at the end of deaeration zone during phase I~III, whereas at end of aerobic zone I during phase IV~VII. The Flow distribution ratio was no distribution, 50%:50%, 25%:75%, 75%:25%, 75%:25%, 75%:25% and 75%:25% in each phase, respectively. Effect of flow distribution ratio on COD removal efficiency. COD removal by the pilot-scale reactor through- out the operation period is presented in Fig. 1a. The average COD concentrations in the effluents were 24.7, 21.7, 53.4, 32.9, 42.9, 28.7, and 20.8 mg/L, and the average removal efficiencies were 84.4%, 88.7%, 81.6%, 84.6%, 82.1%, 86.4%, and 88.0% for phases I to VII, respectively. The effluent concentrations in all phases except phase III met the integrated wastewater discharge standard (class A discharge standards, GB18918-2002, China; here- after abbreviated as Class A). When the ratio of raw influent distribution was 25%:75% in phase III, the COD flow and total COD amount were high in aerobic zone II, very limited in the anaerobic zone and aerobic zone I, and the COD was inadequately depleted and was discharged with the effluent. The flow distribution ratio hardly had an effect on COD removal efficiency, and the removal capacity of the pilot-scale reactor was stable and effi- cient. As shown in Supplementary Fig. S1, the COD concentration along the flow tended to decrease in general. Nevertheless, the COD concentration in head of aerobic zone I (O1) to head of anoxic zone II (AN2) rose in phase VI, because 25% of the raw wastewater was distributed into anoxic zone II, and thus, COD in O1 was higher than that that in AN2. COD removal was higher in the anaerobic and anoxic zones than in the aerobic zone in all phases. www.nature.com/scientificreports/ 14.2 mg/L and from 1.98 to 0.57 mg/L, respectively7. The treatment effect met the summer resource consent standard when the distribution ratio of a four-stage step-feed A/O reactor was set as 20%:30%:25%:25%8. A pre- vious study reported effluent concentrations of chemical oxygen demand (COD), ammonia nitrogen ( + NH4 -N), TN, and TP of 33.05, 0.58, 9.26, and 0.46 mg/L, respectively, when the distribution ratio of a pilot modified step-feed A/O technology was set as 25%:35%:35%:10%9. An A/O-like two-stage biological aerated filter process, four-stage anaerobic/anoxic/oxic/oxic (A2/O2) process, and two-stage bioaugmented A/O biofilm process have been investigated10–12. The maximum + NH4 -N removal reached 92.5%, 97.8%, and 95% when average influent concentrations were approximately 20.92, 228.2 ± 55.5, and 10–30 mg/L, respectively10–12. Based on these previous studies, which mainly addressed the distribution of raw wastewater, we developed a novel step-feed A/O process with distribution of carbon sources from the anaerobic zone. We termed the pro- cess ‘anaerobic zone carbon source distribution (ACD) step-feed A/O’. In comparison to previously reported processes, we made two important adjustments to improve process performance: internal nitrification liquid recycling, and distribution of the carbon source from the anaerobic zone.fi y g For efficient and steady operation of the ACD step-feed A/O process, it is imperative to understand the com- position of microbial population in the process and to determine the relationship between reactor function and dominant population. Metagenomics allows analysing all microbes in an environmental sample without cultiva- tion. It relies on high-throughput sequencing technology and efficient data processing software capable of man- aging large data volumes13. Metagenomics facilitates qualitative and quantitative descriptions of sub-dominant groups at relative abundances of 0.01–0.10%14. Microbial biodiversity and community structure are more accu- rately reflected by high-throughput sequencing because isolation and culture of microbes is not required15. The technology is increasingly used to precisely monitor bacterial communities in environmental samples, such as activated sludge16,17. g In this study, a pilot plant with a novel ACD step-feed A/O was utilised to treat domestic sewage and to inves- tigate the effects of distribution ratio and reflux mode on denitrification and phosphorous removal capacities. High-throughput sequencing analysis was used to elucidate variations in the microbial community structure and succession of the dominant microbial communities. Control parameters of wastewater treatment in the pilot plant were optimized, and the relationship between reactor performance and the microbial community was deter- mined. www.nature.com/scientificreports/ The results from this study are likely to contribute to our practical knowledge of and the theoretical basis for upgrading WWTPs. Yijun Shen1,2, Dianhai Yang1, Yang Wu2, Hao Zhang1 & Xinxi Zhang2 Optimal utilization of carbon sources is the main focus of step-feed technology, which optimizes carbon source distribution in the wastewater treatment process to rationally distribute limited carbon sources to denitrification and phosphorous removal reactors. Maximum utilization of carbon sources is achieved because invalid carbon oxidation in the aerobic zone is avoided3–5. Step-feed technology was first utilized in the 1990s and has since been implemented in China and other countries6.h The use of a three-stage step-feed anoxic/oxic (A/O) process for the treatment of raw wastewater resulted in reductions in the concentrations of total nitrogen (TN) and total phosphorus (TP) in the effluent from 20.8 to 1State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, P.R. China. 2Engineering Research Center of Biomembrane Water Purification and Utilization Technology, Ministry of Education, School of Civil Engineering and Architecture, Anhui University of Technology, Ma’anshan, 243032, P.R. China. Correspondence and requests for materials should be addressed to D.Y. (email: yangdianhai@tongji.edu.cn) Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 1 1 www.nature.com/scientificreports/ Results and Discussionfl Because of back mixing caused by unstable operation induced by the stirring pad- dle in phase IV, part of the flow at the beginning of anoxic zone II mixed with flow at the end of aerobic zone I, and therefore, part of the + NH4 -N from the raw wastewater was diluted. In contrast, at the beginning of aerobic zone II, + NH4 -N, which originated from the main stream of raw wastewater distribution, was not diluted; thus, the concentration of + NH4 -N in aerobic zone I was higher than that in anoxic zone II. The decrease in + NH4 -N was the most obvious in the anaerobic zone. First, because the + NH4 -N was distributed into each reaction zone according the flow distribution ratio, it was diluted. Subsequently, NH4 +-N was continuously diluted by the return sludge to result in obviously decreased concentrations. The concentration of effluent NH4 +-N was generally below 2 mg/L (Fig. 1b), the nitrification capacity of the system was sufficient as indicated by effective nitrification; therefore, the removal effect of TN was determined by denitrification. As shown in Supplementary Fig. S3, TN removal was low due to insufficient carbon source in the anoxic zone I during phases II and III and, therefore, denitrification was inefficient and resulted in substantial accumulation of nitrate nitrogen NO ( 3 −-N), which further lead to sub-optimal removal performance in subsequent reaction zone. The preferable treatment effect observed in phases VI and VII was due to the presence of sufficient carbon source in the anoxic zone I of first-step A/O. Therefore, denitrification was complete. Meanwhile, the internal reflux from aerobic zone I led to slight accumu- lation of nitrate nitrogen in anoxic zone I. The denitrification reaction was partly restrained by insufficient carbon source in anoxic zone II during phase VI. Because the carbon source was distributed from the anaerobic zone in phase VII, the denitrification reaction could proceed with more sufficient carbon source in anoxic zone II. The concentration of effluent nitrate nitrogen was approximately 9 mg/L, and TN removal was better than that during phase VII. The NO3 −-N concentration decreased in the deaeration zone during phases V and VII, which was deduced to be the effect of endogenous denitrification. Microbial substances were used as carbon source for deni- trification, which not only reduced the carbon source demand, but also reduced the sludge yield. Results and Discussionfl + NH4 -N in phase IV was different from that in the other phases. A stirring paddle was installed between the anoxic zone and aerobic zone. Because of back mixing caused by unstable operation induced by the stirring pad- dle in phase IV, part of the flow at the beginning of anoxic zone II mixed with flow at the end of aerobic zone I, and therefore, part of the + NH4 -N from the raw wastewater was diluted. In contrast, at the beginning of aerobic zone II, + NH4 -N, which originated from the main stream of raw wastewater distribution, was not diluted; thus, the concentration of + NH4 -N in aerobic zone I was higher than that in anoxic zone II. The decrease in + NH4 -N was the most obvious in the anaerobic zone. First, because the + NH4 -N was distributed into each reaction zone according the flow distribution ratio, it was diluted. Subsequently, NH4 +-N was continuously diluted by the return sludge to result in obviously decreased concentrations. The concentration of effluent NH4 +-N was generally below 2 mg/L (Fig. 1b), the nitrification capacity of the system was sufficient as indicated by effective nitrification; therefore, the removal effect of TN was determined by denitrification. As shown in Supplementary Fig. S3, TN removal was low due to insufficient carbon source in the anoxic zone I during phases II and III and, therefore, denitrification was inefficient and resulted in substantial accumulation of nitrate nitrogen NO ( 3 −-N), which further lead to sub-optimal removal performance in subsequent reaction zone. The preferable treatment effect observed in phases VI and VII was due to the presence of sufficient carbon source in the anoxic zone I of first-step A/O. Therefore, denitrification was complete. Meanwhile, the internal reflux from aerobic zone I led to slight accumu- lation of nitrate nitrogen in anoxic zone I. The denitrification reaction was partly restrained by insufficient carbon source in anoxic zone II during phase VI. Because the carbon source was distributed from the anaerobic zone in phase VII, the denitrification reaction could proceed with more sufficient carbon source in anoxic zone II. The concentration of effluent nitrate nitrogen was approximately 9 mg/L, and TN removal was better than that during phase VII. The NO3 −-N concentration decreased in the deaeration zone during phases V and VII, which was deduced to be the effect of endogenous denitrification. Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 Results and Discussionfl The decrease in + NH4 -N was the most obvious in the anaerobic zone. First, because the + NH4 -N was distributed into each reaction zone according the flow distribution ratio, it was diluted. Subsequently, NH4 +-N was continuously diluted by the return sludge to result in obviously decreased concentrations. The concentration of effluent NH4 +-N was generally below 2 mg/L (Fig. 1b), the nitrification capacity of the system was sufficient as indicated by effective nitrification; therefore, the removal effect of TN was determined by denitrification. As shown in Supplementary Fig. S3, TN removal was low due to insufficient carbon source in the anoxic zone I during phases II and III and, therefore, denitrification was inefficient and resulted in substantial accumulation of nitrate nitrogen NO ( 3 −-N), which further lead to sub-optimal removal performance in subsequent reaction zone. The preferable treatment effect observed in phases VI and VII was due to the presence of sufficient carbon source in the anoxic zone I of first-step A/O. Therefore, denitrification was complete. Meanwhile, the internal reflux from aerobic zone I led to slight accumu- lation of nitrate nitrogen in anoxic zone I. The denitrification reaction was partly restrained by insufficient carbon source in anoxic zone II during phase VI. Because the carbon source was distributed from the anaerobic zone in phase VII, the denitrification reaction could proceed with more sufficient carbon source in anoxic zone II. The concentration of effluent nitrate nitrogen was approximately 9 mg/L, and TN removal was better than that during phase VII. The NO3 −-N concentration decreased in the deaeration zone during phases V and VII, which was deduced to be the effect of endogenous denitrification. Microbial substances were used as carbon source for deni- trification, which not only reduced the carbon source demand, but also reduced the sludge yield. Figure 1. Effect of flow distribution ratios on removal efficiency for (a) COD, (b) NH4 +-N, (c) TN, (d) TP. Figure 1. Effect of flow distribution ratios on removal efficiency for (a) COD, (b) NH4 +-N, (c) TN, (d) TP. + NH4 -N in phase IV was different from that in the other phases. A stirring paddle was installed between the anoxic zone and aerobic zone. Results and Discussionfl This was because, as the raw wastewater flowed into the anaerobic and anoxic zones, the carbon source was utilised for the release of phosphorus and denitrification. Subsequently, the raw wastewater flowed into the aerobic zone, and the ineffective utilization of carbon source was averted and the competition among hetero- trophic bacteria for the organic matter was reduced. These findings highlight the advantage of the distribution of carbon source from the anaerobic zone in the novel step-feed A/O technology. Effect of flow distribution ratio on nitrogen removal efficiency. The average concentrations of effluent ammonia nitrogen (NH4 +-N) were 1.37, 0.83, 2.80, 1.91, 0.56, 0.85, and 0.64 mg/L, and the average removal efficiencies were 95.0%, 96.9%, 91.0%, 93.1%, 97.1%, 96.8, and 96.5% from phases I to VII, respectively (Fig. 1b). The effluent qual- ity of each phase satisfied the integrated wastewater discharge standard (Class A). The average concentrations of effluent TN were 21.0, 20.9, 19.3, 18.7, 15.4, 16.3, and 14.2 mg/L, and the average removal efficiencies were 37.8%, 43.1%, 47.4%, 50.8%, 50.1%, 50.1%, and 50.8% for phases I to VII, respectively (Fig. 1c). The effluent quality of phase VII satisfied the integrated wastewater discharge standard (Class A). These results indicate that the flow distribution ratio has a minimal influence on the removal of NH4 +-N, but a strong influence on TN removal. As shown in Supplementary Fig. S2, the + NH4 -N concentrations decreased along the flow; however, the trend in Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 2 www.nature.com/scientificreports/ Figure 1. Effect of flow distribution ratios on removal efficiency for (a) COD, (b) NH4 +-N, (c) TN, (d) TP. + NH4 -N in phase IV was different from that in the other phases. A stirring paddle was installed between the anoxic zone and aerobic zone. Because of back mixing caused by unstable operation induced by the stirring pad- dle in phase IV, part of the flow at the beginning of anoxic zone II mixed with flow at the end of aerobic zone I, and therefore, part of the + NH4 -N from the raw wastewater was diluted. In contrast, at the beginning of aerobic zone II, + NH4 -N, which originated from the main stream of raw wastewater distribution, was not diluted; thus, the concentration of + NH4 -N in aerobic zone I was higher than that in anoxic zone II. Results and Discussionfl Therefore, phosphorus removal is better when the SRT is short and the discharge amount of excess sludge is large. Because the SRT in the reactor process in this study was more than 20 days, phosphorus removal was not satisfactory. General trends in phosphate concentra- tions in different phases along the flow are shown in Supplementary Fig. S4. The reduction in phosphate was the most obvious in the anaerobic zone. In general, the most efficient phosphorus removal was observed during phases VI and VII, in which sufficient carbon was present to allow anaerobic phosphorus release in the anaerobic zone. The aerobic phosphorus uptake increases with an increase in anaerobic phosphorus release; therefore, the effect on TP removal could be improved20,21. In these phases, the maximum phosphorus release rate was 448%, which is in accordance with rates reported in other studies, i.e., 415%9 and 495%22. Therefore, TP removal could be improved by adjusting process parameters, such as the flow distribution ratio and the internal reflux ratio, while maintaining a lower concentration of nitrate nitrogen in the anaerobic zone and ensuring optimal TN removal. As shown in Supplementary Fig. S4, TP removal occurred to a certain extent in the anoxic zone of each phase, and it is concluded that denitrifying phosphate-accumulating organisms bacteria used nitrate nitrogen as electron acceptor for denitrification phosphorus removal, which was most efficient when the flow distribution ratio was 75%:25%. Effect of internal reflux position on pollutant removal efficiency. In this study, the internal reflux port was located at the end of deaeration zone (DZR) during phases I, II, and III and at the end of aerobic zone I (OIR) during phases IV, V, VI, and VII. The concentrations of ammonium, nitrate and phosphate along the flow were measured to investigate the effect of the position of internal reflux. The nitrification liquor containing abundant nitrate nitrogen was recycled into anoxic zone I by the internal reflux system to utilise nitrate nitrogen as an electron acceptor for denitrification in the anoxic zone. As shown in Fig. 2, the position of the internal reflux system had no significant impact on the efficiency of ammonium nitrogen removal and ammonium nitrogen concentrations along the flow. However, denitrification was better when the internal reflux system was posi- tioned at the end of the deaeration zone. Results and Discussionfl Microbial substances were used as carbon source for deni- trification, which not only reduced the carbon source demand, but also reduced the sludge yield. Effect of flow distribution ratio on phosphorus removal efficiency. The average concentrations of effluent TP were 1.58, 1.67, 1.68, 1.67, 2.18, 0.90 and 0.89 mg/L, and the average removal efficiencies were 40.7%, 48.1%, 50.4%, 56.0%, 37.7%, 67.3%, and 58.9% for phases I to VII, respectively (Fig. 1d). The amount of TP removed in the anaerobic zone during all phases except V was 34.0% (mass fraction), 21.6%, 27.6%, 28.0%, 15.7%, and 13.9%, respectively. Because the NO3 −-N concentration of the returned activated sludge was relatively high, and the deni- trifying bacteria had precedence over the polyphosphate-accumulating bacteria in utilizing the carbon source of the raw wastewater, the amount of carbon for anaerobic phosphorus release was reduced. Consequently, anaero- bic phosphorus release was suppressed, which further influenced phosphorus removal. TP removal was the most efficient in phases VI and VII, which was deduced to result from lower denitrification. The relatively low temper- ature might have been one of the factors influencing TP removal, because it might have reduced the activity of polyphosphate-accumulating bacteria. Additionally, the maximum phosphate release rate as well as the maxi- mum phosphorus uptake rate were reduced, thereby inhibiting phosphorus removal18. In previous related studies, Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 3 www.nature.com/scientificreports/ p / Figure 2. Effect of different internal reflux system position on contaminate concentration. OIR indicates that the internal reflux system was positioned at the end of aerobic zone I. DZR indicates that the internal reflux system was positioned at the end of the deaeration zone. Figure 2. Effect of different internal reflux system position on contaminate concentration. OIR indicates that the internal reflux system was positioned at the end of aerobic zone I. DZR indicates that the internal reflux system was positioned at the end of the deaeration zone. phosphorus removal was effectively improved when the sludge retention time (SRT) was reduced and was con- trolled in 8 to 12 days19. On the one hand, activated sludge had higher biological activity and the bacteria con- tained more phosphorus in the case of a shorter SRT. On the other hand, polyphosphate-accumulating organisms (PAOs) are a class of heterotrophic microorganisms working optimally under short SRT, and phosphorus is removed by discharge of the phosphorus-rich excess sludge. Results and Discussionfl A higher Shannon index represents greater diversity, whereas a higher Simpson index indicates lower diversity. As shown in Table 1, trends in these indices were quite consistent with those in ACE and Chao1. The biodiversity in aerobic zone I was higher than that in aerobic zone II and the seed sludge. Moreover, the biodiversity in aerobic zone I was nearly equal in the three different phases, and the same as that in aerobic zone II. The biodiversity in the seed sludge was nearly equal to that in aerobic zone II. Microbial community biodiversity affects system stability; a high biodiver- sity indicates that the community consists of populations with different biological and ecological features, result- ing in greater stability and resistance to volatility24. The activated sludge system is a mini ecosystem, and certain functions of the reactor are enhanced as certain microorganisms decrease and other, functional bacteria increase in systems such as nitritation25–27, phosphorus removal28, and anammox29 systems. Stabilization of the activated sludge system is based on the accumulation of process-functional bacteria, whereas has limited relevance with microbial community diversity. The accumulation of functional bacteria mainly relies on the artificial reinforce- ment in the system. Therefore, sample biodiversity did not obviously differ among different phases. Microbial community composition of sludge at various phases. Figure 3 shows the dynamic changes in community composition in terms of relative abundances of different phyla (Fig. 3a) and genera (Fig. 3b) in the three phases samples and the seed sludge. Non-metric multidimensional scaling (NMDS) (Fig. S5) could effec- tively evaluate the similarity and difference of microbial communities. Proteobacteria were predominant in the seed sludge, followed by Bacteroidetes, Chloroflexi and Planctomycetes. Firmicutes were predominant in aerobic zone I, followed by Proteobacteria, Bacteroidetes and Actinobacteria. Proteobacteria were predominant in aero- bic zone II, followed by Bacteroidetes, Chloroflexi and Planctomycetes. The relative abundances of Acidobacteria, Fusobacteria, Verrucomicrobia, Thaumarchaeota, and Tenericutes in aerobic zone I and Firmicutes, Acidobacteria, Actinobacteria, and Nitrospirae in aerobic zone II and seed sludge were higher than 1%. As shown in Fig. 3a, the microbial community of the seed sludge was similar to that of aerobic zone II, but different from that of aerobic zone I. These results are consistent with those of a previous study30. The differences among the seven samples were apparent at the genus level, especially for samples in aerobic zones I between samples in aerobic zones II and the seed sludge. Results and Discussionfl Samples of aerobic zone II of the pilot-scale reactor had a similar richness, which ranged from 40000 to 50000 (ACE) in different phases, and richness in the seed sludge ranged from 15000 to 30000 (ACE), which was similar to that in aerobic zone II. The microbial richness in aerobic zone I was higher than that in aerobic zone II and the seed sludge. It can be concluded that the step-A/O process with carbon source distributed from the anaerobic zone is more favourable for microbial richness than the A2/O technology of the local WWTP where the seed sludge was obtained from. In aerobic zone I, there was more organic matter and nutrition for the bacteria to grow. Additionally, the main biochemical reactions in aerobic zone I were nitrification and phosphorus accumulation, which resulted in a higher microbial richness this zone. Suffix named ZS1 represented samples taken from aerobic zone I(O1), and suffix named ZS2 represented samples taken from aerobic II(O2).The sludge samples were obtained from the aerobic zone because microorganisms in this zone are the most representative23. Alpha diversity indices for microbial community distribution are listed in Table 1. The coverage index indicates the percentage of individuals sampled in the microbial community. The coverage indices ranged from 71.0% to 89.2% and represented the main bacterial species in the samples, indicat- ing that the sample was representative of the microbial community in the sludge. The abundance-based coverage estimation (ACE) index and Chao1 index (Table 1) give an indication of species richness. Samples of aerobic zone II of the pilot-scale reactor had a similar richness, which ranged from 40000 to 50000 (ACE) in different phases, and richness in the seed sludge ranged from 15000 to 30000 (ACE), which was similar to that in aerobic zone II. The microbial richness in aerobic zone I was higher than that in aerobic zone II and the seed sludge. It can be concluded that the step-A/O process with carbon source distributed from the anaerobic zone is more favourable for microbial richness than the A2/O technology of the local WWTP where the seed sludge was obtained from. In aerobic zone I, there was more organic matter and nutrition for the bacteria to grow. Additionally, the main biochemical reactions in aerobic zone I were nitrification and phosphorus accumulation, which resulted in a higher microbial richness this zone. Shannon and Simpson indices give an indication of microbial community biodiversity. Results and Discussionfl The relatively poor denitrification when the internal reflux system was positioned at the end of aerobic zone I was due to insufficient nitrate nitrogen in the mixed liquor, which was the electron acceptor for denitrification. The concentration of phosphate phosphorus declined in the anoxic zone (special anoxic zone I) due to the dilution of mixed liquor and denitrifying phosphorus removal. The decline in phosphate was larger when the internal reflux system was positioned at the end of the deaeration zone than when it was positioned at the end of aerobic zone I. Therefore, placement of the internal reflux system at the end of the deaeration zone promoted phosphorus removal. Molecular biological characteristics of sludge at various phases. High-throughput sequencing was used to assess microbial diversity and community structure of the seed sludge and sludge samples taken at phases V(sample 150609ZS1,150609ZS2), VI(sample 150714ZS1,150714ZS2), and VII (sample 150803ZS1,150803ZS2). Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 4 www.nature.com/scientificreports/ Phase Sample_ID Sequences OTUs ACEa Chao1b Shannonc Simpsond Coveragee Seed sludge 150610WXY 23896 5544 27899 16027 7.26 0.00281 0.848008 V 150609ZS1 20036 7970 48117 25703 7.87 0.00250 0.710421 150609ZS2 23129 4314 14646 10028 7.08 0.00263 0.892473 VI 150714ZS1 17875 7401 42624 23752 7.91 0.00202 0.700196 150714ZS2 21806 5313 22784 14193 7.26 0.00326 0.845364 VII 150803ZS1 18482 7306 46552 23615 7.78 0.00249 0.710908 150803ZS2 22910 5685 29823 17130 7.27 0.00370 0.834745 Table 1. Alpha diversity of activated sludge samples. a,bCommunity richness (a higher number represents Table 1. Alpha diversity of activated sludge samples. a,bCommunity richness (a higher number represents greater richness). c,dCommunity diversity (a higher Shannon index represents greater diversity while a higher Simpson value represents lower diversity). eSampling depth. Suffix named ZS1 represented samples taken from aerobic zone I(O1), and suffix named ZS2 represented samples taken from aerobic II(O2).The sludge samples were obtained from the aerobic zone because microorganisms in this zone are the most representative23. Alpha diversity indices for microbial community distribution are listed in Table 1. The coverage index indicates the percentage of individuals sampled in the microbial community. The coverage indices ranged from 71.0% to 89.2% and represented the main bacterial species in the samples, indicat- ing that the sample was representative of the microbial community in the sludge. The abundance-based coverage estimation (ACE) index and Chao1 index (Table 1) give an indication of species richness. Results and Discussionfl Therefore, more than 50% the final product was lactic acid fermented by Lactobacillus which is widely used in sewage or sludge treatment31. Unclassified Saprospiraceae are abundant in enhanced biological phosphorus removal plants and in anaerobic digestion32. Anaerolineaceae are often abundant in sludge from mainland China16 and their main function is to degrade carbohydrates or cell tissues in the anaerobic system33. Dynamic changes in functional microorganisms. Nitrifying and denitrifying bacterial communities play an important role in ammonia removal in the step-A/O with carbon resource distribution from the anaerobic zone. Ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) collectively convert ammonium to nitrate during the nitrification process, and TN is removed during the denitrification process. AOB can be clas- sified into β and γ subclasses of Proteobacteria according to a previous study34. The main AOB genera are Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio35. Nitrospira, Nitrospina, Nitrobacter, and Nitrococcus are core NOB. Nitrospira belong to the phylum Nitrospirae and the other NOB have been classified into three subclasses of Proteobacteria and Chloroflexi36. In the seven sludge samples, AOB detected belonged to unclassified Nitrosomonadaceae, other AOB genera were not found. The AOB abundances in aerobic zones I and II were 0.48–0.7% and 1.05–1.89% (Fig. 4a), respectively. The AOB abundance in the seed sludge was 1.91%. The NOB genera detected in the seven samples were Nitrospira, Nitrospina, and Nitrobacter, whereas Nitrococcus was not detected. Nitrospira was the most abundant genus, with an abundance of more than 0.25% in aerobic zone I and of 0.98% in the other samples. Nitrospina and Nitrobacter were substantially less abundant than Nitrospira. Thus, it is concluded that Nitrosomonas and Nitrospira were the dominant genera in the AOB and NOB communi- ties, respectively. As shown in Fig. 4a, the AOB abundance in aerobic zone II was nearly same as that in the seed sludge, but 2–3 times that in aerobic zone I. The total abundance of AOB in the pilot-scale reactor was greater than that in the seed sludge. The NOB abundance in aerobic zone II was 3–4 times that in aerobic zone I, and the total NOB abundance in the pilot-scale reactor was a slightly lower than that in the seed sludge. Nitrification mainly occurred in aerobic zone II; therefore, the presence of an internal reflux system in the deaeration zone was benefi- cial for the removal of TN. Results and Discussionfl In total, there were 444 (150609ZS1), 391 (150609ZS2), 384 (150610WXY), 638 (150714ZS1), 399 (150714ZS2), 622 (150803ZS1), and 407 (150803ZS2) genera in the seven samples, respectively. Additionally, 22 (150609ZS1), 25 (150609ZS2), 23 (150610WXY), 26 (150714ZS1), 21 (150714ZS2), 23 (150803ZS1), and 20 (150803ZS2) genera had an abundance greater than 1% in all samples. The number of genera in aerobic zone I was nearly twice that in aerobic zone II, especially in sludge samples from the last two phases of the pilot-scale reactor. A heatmap (Fig. 3b) for the 15 most abundant genera was constructed to evaluate similarities and differ- ences among the seven samples. Based on the heatmap, the seven samples could be divided into two categories, i.e., three samples in aerobic zone I that had similar composition, and the seed sludge microbial community, which had a composition similar to that in the other three samples in aerobic zone II. This view was also con- firmed by NMDS diagrams (Fig. S5). Lactobacillus, Incertae Sedis, unclassified Ruminococcaceae, Fusobacterium and Vibrio were the five most dominant genera in aerobic zone I. Unclassified Saprospiraceae, unclassified Anaerolineaceae, Acidovorax, Azospira and unclassified Comamonadaceae were the five most dominant genera Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 5 www.nature.com/scientificreports/ Figure 3. Relative abundances of the microbial communities at (a) phylum level and (b) genus level in the three different phases and seed sludge. Less than 1% of total the bacterial community composition was classified as ‘others’. Figure 3. Relative abundances of the microbial communities at (a) phylum level and (b) genus level in the three different phases and seed sludge. Less than 1% of total the bacterial community composition was classified as ‘others’. in aerobic zone II and the seed sludge. Because the supplement sludge of phase V was taken from the seed sludge (150610WXY), the main genera of samples (150714ZS2, 150803ZS2) were similar to those in the seed sludge, especially for 150714ZS2 (phase VI). In phase VI (150610WXY, 150714ZS2), the dominant genus was unclassi- fied Saprospiraceae, whereas in the last phase, where carbon source distribution was from the anaerobic zone, the dominant genus was Acidovorax (150803ZS2). The sludge samples from before the addition of supplement sludge (150609ZS2) were different from the seed sludge in terms of composition, and unclassified Anaerolineaceae was the dominant genus. Lactobacillus is a class of acid-resistant chemotrophic bacteria that can produce lactic acid from carbohydrate. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Abundances of potential (a) nitrifying and denitrifying bacteria and (b) phosphorus-removing bacteria in the seven samples. Figure 4. Abundances of potential (a) nitrifying and denitrifying bacteria and (b) phosphorus-removing bacteria in the seven samples. which are taxonomically and phylogenetically diverse. The potential denitrifying bacteria in the seven samples were classified into 11 families (Fig. 4a). The denitrifying bacterial families in three samples from aerobic zone I had very high similarity because the denitrifying bacteria come from anoxic zone I, where not only were the car- bon source and nitrate of reflux liquid sufficient, but the environment was consistent and suitable for bacterial sustenance. The first six families were the same, i.e., Bacillaceae (3.75–4.49%), Comamonadaceae (1.01–1.62%), Cytophagaceae (0.71–0.81%), Rhizobiales Incertae Sedis (0.46–0.59%), Hyphomicrobiaceae (0.32–0.45%) and Haliangiaceae (0.2–0.32%). Bacillaceae and Comamonadaceae were the dominant denitrifying bacteria and their combined abundance was approximately 60% of the total abundance of denitrifying bacteria in aerobic zone I, with Bacillaceae accounting for 45% of the total abundance. The denitrifying bacterial families in three samples from aerobic zone II were different each other in the three phases. Only the most abundant denitrifying bacteria, Comamonadaceae, which accounted for 40–50% of the total denitrifying bacterial abundance in aerobic zone II, was similar. The concentration of nitrate was not very high in anoxic zone II of the second step of A/O and the carbon resource was relatively insufficient; therefore, the variation in the denitrifying bacterial community could be attributed to substrate diversification. The bacterial families in aerobic zone II during phase VI were quite sim- ilar to those in the seed sludge, whereas families in phase VII were different from those in the seed sludge. The total abundance of denitrifying bacteria was higher in phase VII than in phases VI and V, which is consistent with the fact that the TN removal efficiency was the highest during phase VII (Fig. 1c). Comamonadaceae are typical anoxic denitrifying bacteria and have also been reported by Spring et al.37 and Jahan et al.38. Bacillus (family Bacillaceae) are aerobic denitrifying bacteria that can convert NO3 −-N to N2 in an aerobic environment39. In general, both anoxic denitrifying bacteria and aerobic denitrifying bacteria are responsible for TN removal. The dominant microorganisms associated with phosphorus removal are shown in Fig. 4b. Beijerinckiaceae, Bacillaceae, Sphingomonadaceae, Rhizobiales Incertae Sedis, Rhodospirillaceae, and Rhodocyclaceae were the six families con- stituting the core PAOs. Results and Discussionfl The total AOB abundance in aerobic zones I and II increased with each reaction phase; however, the total NOB abundance in these zones remained quite constant. It is inferred from Figs 1c and 4a that the 75%:25% distribution from the anaerobic zone was favourable for nitrification. Denitrification capacity is widely found in bacteria, archaea, and some eukaryotes (e.g., fungi), and bacteria are primarily responsible for nitrate reduction in natural and engineered ecosystems17. Denitrification capacity is widespread among bacteria, Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 6 www.nature.com/scientificreports/ The PAO community in the first and second step of A/O was quite similar in the three phases, and the PAO community in the seed sludge was highly similar to that in the second step of A/O of the pilot-scale reactor. Bacillaceae was the most abundant family in the first-step A/O, and its abundance in each phase which are taxonomically and phylogenetically diverse. The potential denitrifying bacteria in the seven samples were classified into 11 families (Fig. 4a). The denitrifying bacterial families in three samples from aerobic zone I had very high similarity because the denitrifying bacteria come from anoxic zone I, where not only were the car- bon source and nitrate of reflux liquid sufficient, but the environment was consistent and suitable for bacterial sustenance. The first six families were the same, i.e., Bacillaceae (3.75–4.49%), Comamonadaceae (1.01–1.62%), Cytophagaceae (0.71–0.81%), Rhizobiales Incertae Sedis (0.46–0.59%), Hyphomicrobiaceae (0.32–0.45%) and Haliangiaceae (0.2–0.32%). Bacillaceae and Comamonadaceae were the dominant denitrifying bacteria and their combined abundance was approximately 60% of the total abundance of denitrifying bacteria in aerobic zone I, with Bacillaceae accounting for 45% of the total abundance. The denitrifying bacterial families in three samples from aerobic zone II were different each other in the three phases. Only the most abundant denitrifying bacteria, Comamonadaceae, which accounted for 40–50% of the total denitrifying bacterial abundance in aerobic zone II, was similar. The concentration of nitrate was not very high in anoxic zone II of the second step of A/O and the carbon resource was relatively insufficient; therefore, the variation in the denitrifying bacterial community could be attributed to substrate diversification. The bacterial families in aerobic zone II during phase VI were quite sim- ilar to those in the seed sludge, whereas families in phase VII were different from those in the seed sludge. The total abundance of denitrifying bacteria was higher in phase VII than in phases VI and V, which is consistent with the fact that the TN removal efficiency was the highest during phase VII (Fig. 1c). Comamonadaceae are typical anoxic denitrifying bacteria and have also been reported by Spring et al.37 and Jahan et al.38. Bacillus (family Bacillaceae) are aerobic denitrifying bacteria that can convert NO3 −-N to N2 in an aerobic environment39. In general, both anoxic denitrifying bacteria and aerobic denitrifying bacteria are responsible for TN removal. The dominant microorganisms associated with phosphorus removal are shown in Fig. 4b. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Schematic of the ACD Step-feed A/O. 1–6 indicate the measure point positions of variation along the flow. (1) anaerobic zone (ANA), (2) head of anoxic zone I (AN1), (3) head of aerobic zone I (O1), (4) head of anoxic zone II (AN2), (5) head of aerobic zone II (O2), (6) deaeration zone (DZ). (A) Distribution of raw influent; (B) Distribution from the anaerobic zone; (C) Internal reflux position in phases I, II, III; (D) Internal reflux position in phases IV, V, VI, VII. The distribution of raw influent was not simultaneous with the distribution from the anaerobic zone. Figure 5. Schematic of the ACD Step-feed A/O. 1–6 indicate the measure point positions of variation along the flow. (1) anaerobic zone (ANA), (2) head of anoxic zone I (AN1), (3) head of aerobic zone I (O1), (4) head of anoxic zone II (AN2), (5) head of aerobic zone II (O2), (6) deaeration zone (DZ). (A) Distribution of raw influent; (B) Distribution from the anaerobic zone; (C) Internal reflux position in phases I, II, III; (D) Internal reflux position in phases IV, V, VI, VII. The distribution of raw influent was not simultaneous with the distribution from the anaerobic zone. Content Range Average COD (mg/L) 52–352 205 NH4 +-N (mg/L) 7.2–39.8 27.1 − NO2 -N (mg/L) 0.0534–0.550 0.156 − NO3 -N (mg/L) 0.0–17.3 1.0 TN (mg/L) 14.2–49.0 34.5 − PO4 3 -P (mg/L) 0.6–7.8 2.5 TP (mg/L) 1.04–4.93 3.06 Table 2. Quality of the raw water. Content Range Average COD (mg/L) 52–352 205 NH4 +-N (mg/L) 7.2–39.8 27.1 − NO2 -N (mg/L) 0.0534–0.550 0.156 − NO3 -N (mg/L) 0.0–17.3 1.0 TN (mg/L) 14.2–49.0 34.5 − PO4 3 -P (mg/L) 0.6–7.8 2.5 TP (mg/L) 1.04–4.93 3.06 Table 2. Quality of the raw water. Content Range Average COD (mg/L) 52–352 205 NH4 +-N (mg/L) 7.2–39.8 27.1 − NO2 -N (mg/L) 0.0534–0.550 0.156 − NO3 -N (mg/L) 0.0–17.3 1.0 TN (mg/L) 14.2–49.0 34.5 − PO4 3 -P (mg/L) 0.6–7.8 2.5 TP (mg/L) 1.04–4.93 3.06 Table 2. Quality of the raw water. was 3.75%, 3.93%, and 4.49%, respectively. Rhodocyclaceae was the most abundant family in the second-step A/O, and its abundance in each phase and in the seed sludge was 6.64%, 7.32%, 6.4%, and 5.92%, respectively. Previous studies have found uncultured Rhodocyclaceae to be dominant in the active sludge of the enhanced biological phosphorus removal reactors40,41. www.nature.com/scientificreports/ Rhodospirillaceae have been reported to be PAOs42; however, another study argued that Rhodospirillaceae are glycogen-accumulating organisms43. As shown in Fig. S4, denitrifying phospho- rus removal was observed, which is consistent with other studies that have found Bacillaceae, Rhodocyclaceae, and Hyphomicrobiaceae to be typical denitrifying PAOs44. The total abundance of PAOs was the highest in phase VII among the three phases; therefore, the efficiency of phosphorus removal was also the highest in this phase. Disscusion In this study, the ACD step-feed A/O process was applied to treat sewage with low carbon source and high nitro- gen and phosphorus. The optimal flow distribution ratio obtained from the anaerobic zone was 75%:25% and the effluent concentration was consistent with the class A discharge standards. The distribution ratio had negligible influence on the removal of COD and + NH4 -N but had a large influence on the removal of TN and TP. The inter- nal reflux position in the deaeration zone improved the removal of TN and TP. High-throughput sequencing revealed the pollutant removal mechanisms, microbial communities and functional microbial characteristics in the seven sludge samples. The microbial communities in O1 sludge was highly different from those in O2 while microbial communities of seed sludge was very similar to those of O2 sludge in the pilot plant. The abundances of corresponding major functional species were the highest in the pilot-scale plant when the operation mode was optimal and Nitrosomonas, Nitrospira, Bacillaceae and Rhodocyclaceae were the dominant nitrogen and phospho- rus removal communities, respectively. In general, this process offers several advantages proved by the test. First, when the flow distribution is from the anaerobic zone, the carbon sources in raw wastewater can be preferentially utilized by phosphorus-accumulating organisms to enhance phosphorus removal. Second, slow degradation of COD can be converted into the solution COD in the anaerobic zone, therefore, the quality of carbon sources can be improved; finally, the sludge from the anaerobic zone can also be used as carbon sources by the microbes. www.nature.com/scientificreports/ Beijerinckiaceae, Bacillaceae, Sphingomonadaceae, Rhizobiales Incertae Sedis, Rhodospirillaceae, and Rhodocyclaceae were the six families con- stituting the core PAOs. The PAO community in the first and second step of A/O was quite similar in the three phases, and the PAO community in the seed sludge was highly similar to that in the second step of A/O of the pilot-scale reactor. Bacillaceae was the most abundant family in the first-step A/O, and its abundance in each phase Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 7 Methods ACD t f The influent flow, internal reflux ratio, and returned activated sludge ratio were set as 2 m3·h−1, 200%, and 50%, respectively. The concentration of MLSS was 3000–4500 mg/L and was controlled by discharging excess sludge from the bottom of the secondary clarifier. A DO probe was placed in the aerobic zones where air was pumped from the disc aerator at the bottom of the reactor, and DO concentrations were automatically controlled at 2.0 mg/L. The test procedure was divided into seven phases, and the pollutant removal rate, which was affected by the flow distribution ratio, was analysed during different phases, while other reaction conditions were kept constant. Running modes and control parameters in the different phases are shown in Table 3. Sludge was supplied to the pilot-scale reactor in phases V, VI, and VII to maintain the MLSS concentration. The effect of pollution variation along the flow was tested in the last few days of each stable operation phase. Microbial community analysis. DNA extraction and PCR amplification. Sludge sample 150610WXY was taken from the seed sludge, while samples 150609ZS1, 150714ZS1, and 150803ZS1 were taken from aer- obic zone I of the pilot reactor. Samples 150609ZS2, 150714ZS2, and 150803ZS2 were taken from aerobic zone II. 150609ZS1, 150609ZS2 represented phase V; 150714ZS1, 150714ZS2 represented phase VI and 150803ZS1, 150803ZS2 represented phase VII. The Samples from the pilot reactor was taken at the end of the each phase. Total active sludge DNA was extracted using the E.Z.N.A.® Soil DNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA), and the DNA quality was assessed by 2% (w/v) agarose gel electrophoresis47. The V3–V4 regions of the bac- terial 16S ribosomal RNA gene were amplified by PCR (BIO-RAD T100TM Thermal Cycler, USA) with universal primers 341 F (5′-CCTACGGGNGGCWGCAG-3′) and 805 R (5′-GACTACHVGGGTATCTAATCC-3′). Details of the PCR procedure are available in the Supplementary Methods. Illumina MiSeq sequencing. The DNA samples were paired-end sequenced (2 × 200 bp) on an Illumina MiSeq platform (Sangon Biotech, Shanghai, China) according to standard protocols. Primer sequences, reads shorter than 50 bp, and sequences with low complexity and quality were removed using the Prinseq software (Vision 0.20.4). Read quality met the basic analysis requirement because the length of most sequences was within 400– 600 bp, the average length of sequences was 440 bp, and the number of sequences was more than 500 after quality control. Methods ACD t f Phase Time (days) Flow distribution ratioa Sludge retention time (days) Position of the internal reflux I 1–13 no distribution of the raw influent 15–20 end of deaeration zone II 14–33 ID 50%:50% 15–20 end of deaeration zone III 34–53 ID 25%:75% 15–20 end of deaeration zone IV 54–67 ID 75%:25% no sludge discharge end of aerobic zone I Vb 68–84 ID 75%:25% no sludge discharge end of aerobic zone I VI 85–101 ID 75%:25% 25–30 end of aerobic zone I VII 102–120 AD 75%:25% 25–30 end of aerobic zone I Table 3. Operation modes and control parameters in different phases. aID was carbon resource distribution of raw influent, and the ratio was calculated by the flow feeding each in turn anoxic zone accounted for the proportion of the influent flow; AD was carbon resource distribution from the anaerobic zone, and the ratio was calculated by the flow feeding each in turn anoxic zone accounted for the proportion of the mix liquor flow (summation of the influent flow and returned activated sludge flow). bThe pollution variation along the flow was not measured during phase V because the sludge of this phase was cultured after the supplemental sludge was added in the pilot-scale reactor. Table 3. Operation modes and control parameters in different phases. aID was carbon resource distribution of raw influent, and the ratio was calculated by the flow feeding each in turn anoxic zone accounted for the proportion of the influent flow; AD was carbon resource distribution from the anaerobic zone, and the ratio was calculated by the flow feeding each in turn anoxic zone accounted for the proportion of the mix liquor flow (summation of the influent flow and returned activated sludge flow). bThe pollution variation along the flow was not measured during phase V because the sludge of this phase was cultured after the supplemental sludge was added in the pilot-scale reactor. Parameters tested and analytical methods. COD, TP, phosphate (PO4 3−-P), + NH4 -N, nitrite (NO2 −-N), NO3 −-N, TN, mixed liquor suspended solid (MLSS), and mixed liquor volatile suspended solid (MLVSS) were measured using standard methods46. Dissolved oxygen (DO), oxidation-reduction potential (ORP), and temper- ature were measured continuously by on-line probes (HACH, USA), and the pH was measured with a HQ40d portal multi meter (HACH). Experimental procedure. The pilot-scale reactor test lasted for 120 days and was conducted at a tempera- ture of 10–25 °C. Methods ACD t f Operational taxonomic units (OTUs) were clustered using a 97% similarity cut-off via Uclust (vision 1.1.579), and chimeric sequences were identified and removed using chimera.uchime (Mothur, Vision 1.30.1) based on alignment against the SILVA database (http://www.arb-silva.de/). The taxonomy of each 16S rRNA gene sequence was analysed with RDP Classifier (http://rdp.cme.msu.edu/) against the SILVA 16S rRNA database, using a confidence threshold of 80%. Sequence richness was calculated for each sample and species taxonomic units were determined to construct richness arrays of samples and species taxonomic units. Species diversity and community differences were determined by alpha diversity analysis. Richness and diversity of microbial commu- nities were evaluated on the basis of ACE, Chao 1, Simpson, and Shannon indices using Mothur (v.1.30.1, http:// www.mothur.org/). Methods ACD t f ACD step-feed A/O pilot reactor. In this study, domestic wastewater was treated in a novel step-feed A/O with distribution of carbon sources from the anaerobic zone of the pilot plant. A schematic is presented in Fig. 5. The volume ratio of the pre-anoxic, anaerobic, anoxic, aerobic and deaeration zones was 1.0:4.0:4.8:6.5:2.6, and the total hydraulic retention time was 17 h. Seed sludge and wastewater. Raw wastewater was obtained from the effluent of a vortex-type grit cham- ber of a WWTP in Hefei, China. The average C/N (mass) and C/P (mass) ratios were 6.2 and 70, respectively (Table 2), thus, the organic carbon source was typically limiting45. Seed sludge was also obtained from the second- ary clarifier of the WWTP. The performance of the sludge was satisfactory after being domesticated and cultured for 15 days. 8 Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 www.nature.com/scientificreports/ Phase Time (days) Flow distribution ratioa Sludge retention time (days) Position of the internal reflux I 1–13 no distribution of the raw influent 15–20 end of deaeration zone II 14–33 ID 50%:50% 15–20 end of deaeration zone III 34–53 ID 25%:75% 15–20 end of deaeration zone IV 54–67 ID 75%:25% no sludge discharge end of aerobic zone I Vb 68–84 ID 75%:25% no sludge discharge end of aerobic zone I VI 85–101 ID 75%:25% 25–30 end of aerobic zone I VII 102–120 AD 75%:25% 25–30 end of aerobic zone I Table 3. Operation modes and control parameters in different phases. aID was carbon resource distribution of raw influent, and the ratio was calculated by the flow feeding each in turn anoxic zone accounted for the proportion of the influent flow; AD was carbon resource distribution from the anaerobic zone, and the ratio was calculated by the flow feeding each in turn anoxic zone accounted for the proportion of the mix liquor flow (summation of the influent flow and returned activated sludge flow). bThe pollution variation along the flow was not measured during phase V because the sludge of this phase was cultured after the supplemental sludge was added in the pilot-scale reactor. References 1. Carey, R. O. & Migliaccio, K. W. Contribution of wastewater treatment plant effluents to nutrient dynamics in aquatic systems: a review. Environ. Manage. 44, 205–217 (2009).f g 2. Liu, W. et al. Effect of return sludge pre-concentration on biological phosphorus removal in a novel oxidation ditch. Chin. J. Chem Eng. 20, 747–753 (2012). 2. Liu, W. et al. Effect of return sludge pre-concentration on biological phosphorus removal in a novel oxidation ditch. Chin. J. Chem. Eng. 20, 747–753 (2012). 3 Zh G B P Y Z Zh i L M W Y & W S Y P f d ti i ti f bi l i l it l f g p g p p Eng. 20, 747–753 (2012). 3. Zhu, G. B., Peng, Y. Z., Zhai, L. M., Wang, Y. & Wang, S. Y. Performance and optimization of biological nitrogen removal process g 3. Zhu, G. B., Peng, Y. Z., Zhai, L. M., Wang, Y. & Wang, S. Y. Performance and optimization of biological nitrogen removal proces enhanced by anoxic/oxic step feeding. Biochem. Eng. J. 43, 280–287 (2009). y p g g 4. Fillos, J. et al. Full-scale evaluation of step-feed BNR process at a New York City water pollution control plant. Proc. Water Environ. Fed. 2002, 100–109 (2002).h 5. Crawford, G. et al. The step Bio-P process at Lethbridge—over one full year of operation. Proc. Water Environ. Fed. 2000, 872–887 (2000). ( ) 6. Johnson, B. R., Goodwin, S., Daigger, G. T. & Crawford, G. V. A comparison between the theory and reality of full-scale step-feed nutrient removal systems. Water Sci. Technol. 52, 587–596 (2005). y 7. Ge, S., Zhu, Y., Lu, C., Wang, S. & Peng, Y. Full-scale demonstration of step feed concept for improving an anaerobic/anoxic/aerobic nutrient removal process. Bioresour. Technol. 120, 305–313 (2012).i nutrient removal process. Bioresour. Technol. 120, 305–313 (2012 p 8. Boyle, C. A., McKenzie, C. J. & Morgan, S. Internal recycle to improve denitrification in a step feed anoxic/aerobic activated sludge system. Water Sci. Technol. 60, 1661–1668 (2009).i y 9. Cao, G., Wang, S., Peng, Y. & Miao, Z. Biological nutrient removal by applying modified four step-feed technology to treat weak wastewater. Bioresour. Technol. 128, 604–611 (2013).i 0. Guo, J. et al. Start-up of a two-stage bioaugmented anoxic-oxic (A/O) biofilm process treating petrochemical wastewater under different DO concentrations. Bioresour. Technol. 100, 3483–3488 (2009). f 1. References Comparison of the bacterial communities in anaerobic, anoxic, and of a pilot A(2)O process using pyrosequencing analysis. Curr. Microbiol. 66, 555–565 (2013). g y g y es, A. R. & Carpenter, S. R. Stability and diversity of ecosystems. Science. 317, 58–62 (2007). 24. Ives, A. R. & Carpenter, S. R. Stability and diversity of ecosystems. Science. 317, 58–62 (2007). 24. Ives, A. R. & Carpenter, S. R. Stability and diversity of ecosystems. Science. 317, 58–62 (2007). 2 Sh L W Y Y & M F G R f d i bi l l f i i i b bi J M b 5. Shen, L. W., Yao, Y. & Meng, F. G. Reactor performance and microbial ecology of a nitritation membrane bioreactor. J. Membrane Sci. 462, 139–146 (2014).fi 6. Guo, J. H. et al. Effective and robust partial nitrification to nitrite by real-time aeration duration control in an SBR treating domestic wastewater. Process Biochem. 44, 979–985 (2009). 7. Zeng, T. et al. Analysis of microbial population dynamics in a partial nitrifying SBR at ambient temperature. Curr. Microbiol. 66 614–620 (2013).hf 614–620 (2013). 28. Miao, Z., Peng, Y., Xue, G., Wang, S. & Weng, D. The long-term effect of carbon source on the microbial community in the EBPR system and stoichiometry. IPCBEE. 51, 6–10 (2013). 8. Miao, Z., Peng, Y., Xue, G., Wang, S. & Weng, D. The long-term effect of carbon source on the microbial community in the EBPR system and stoichiometry. IPCBEE. 51, 6–10 (2013).i y y 29. Persson, F. et al. Community structure of partial nitritation-anammox biofilms at decreasing substrate concentrations and low temperature. Microb. Biotechnol. 10, 761–772 (2017). p 0. Yang, C. et al. Phylogenetic diversity and metabolic potential of activated sludge microbial communities in full-scale wastewater treatment plants. Environ. Sci. Technol. 45, 7408–7415 (2011). 30. Yang, C. et al. Phylogenetic diversity and metabolic potential of activated sludge microbia treatment plants. Environ. Sci. Technol. 45, 7408–7415 (2011). 30. Yang, C. et al. Phylogenetic diversity and metabolic potentia 30. Yang, C. et al. Phylogenetic diversity and metabolic potential o treatment plants. Environ. Sci. Technol. 45, 7408–7415 (2011). 31. Zhang, Y. & Vadlani, P. V. Lactic acid production from biomass-derived sugars via co-fermentation of Lactobacillus brevis and Lactobacillus plantarum. J Biosci Bioeng. 119, 694–699 (2015). Lactobacillus plantarum. J Biosci Bioeng. 119, 694–699 (2015). p g 32. Jin, Z. et al. References Peng, Y., Wang, X., Wu, W., Li, J. & Fan, J. Optimisation of anaerobic/anoxic/oxic process to improve performance and reduce operating costs. J. Chem. Technol. Biotechnol. 81, 1391–1397 (2006).i f . Peng, Y., Wang, X., Wu, W., Li, J. & Fan, J. Optimisation of ana p g 12. Liu, B. et al. Feasibility of a two-stage biological aerated filter for depth processing of electroplating-wastewater. Bioresour. Technol. 100, 3891–3896 (2009). 13. Yuan, Y. et al. Microbial community characteristics of a completely autotrophic nitrogen removal over nitrite (CANON) system based on high-throughput sequencing technology. J. Beijing Univ. Technol. 41, 1485–1492, https://doi.org/10.11936/ bjutxb2015010005 (2015).i j 14. Guo, F. & Zhang, T. Profiling bulking and foaming bacteria in activated sludge by high throughput sequencing. Water Res. 46 2772–2782 (2012). 15. Tringe, S. G. & Rubin, E. M. Metagenomics: DNA sequencing of environmental samples. Nat. Rev. Genet. 6, 805–814 (2005). 16. Zhang, T., Shao, M. F. & Ye, L. 454 pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants. ISME J. 6, 1137–1147 (2012).i 7. Lu, H., Chandran, K. & Stensel, D. Microbial ecology of denitrification in biological wastewater treatment. Water Res. 64, 237–254 (2014).f 18. Helmer, C. & Kunst, S. Low temperature effects on phosphorus release and uptake by microorganisms in EBPR plants. Water Sci. Technol. 37, 531–539 (1998). 19. Kuba, T., VanLoosdrecht, M. C. M., Brandse, F. A. & Heijnen, J. J. Occurrence of denitrifying phosphorus removing bacteria in modified UCT-type wastewater treatment plants. Water Res. 31, 777–786 (1997). i y 20. Carucci, A., Majone, M., Ramadori, R. & Rossetti, S. Biological phosphorus removal with different organic substrates anaerobic/aerobic sequencing batch reactor. Water Sci. Technol. 35, 161–168 (1997).f g 1. Mulkerrins, D., Dobson, A. D. & Colleran, E. Parameters affecting biological phosphate removal from wastewaters. Environ. Int. 30 249–259 (2004). 22. Ge, S. et al. Enhanced nutrient removal in a modified step feed process treating municipal wastewater with different inflow distribution ratios and nutrient ratios. Bioresour. Technol. 101, 9012–9019 (2010). 23. Kim, B. C., Kim, S., Shin, T., Kim, H. & Sang, B. I. Comparison of the bacterial communities in anaerobic, anoxic, and oxic cham f il t A(2)O i i l i C Mi bi l 66 555 565 (2013) 23. Kim, B. C., Kim, S., Shin, T., Kim, H. & Sang, B. I. Data Availability y All data generated or analysed in this study are included in this published article and in the Supplementary In- formation files. 9 Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 www.nature.com/scientificreports/ Acknowledgementsh g This work was supported by Major Science and Technology Program for Water Pollution Control and Treatment in China (Grant No. 2011ZX07303-002). g This work was supported by Major Science and Technology Program for Water Pollution Control and Treatment in China (Grant No. 2011ZX07303-002). Author Contributions D.Y. and Y.S. conceived the research idea and obtained funding. Y.S. and Y.W. designed the experiment. Y.W. and H.Z. carried out the experiment. X.Z. gave important advice on the experimental design. Y.S. and Y.W. led manuscript preparation, with substantial inputs on data interpretation from Y.W. and H.Z. All authors have reviewed the manuscript before submission. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 1. Tsuneda, S., Miyauchi, R., Ohno, T. & Hirata, A. Characterization of denitrifying polyphosphate-accumulating organisms in activated sludge based on nitrite reductase gene. J. Biosci. Bioeng. 99, 403–407 (2005). g g g 42. Zhang, G., Jia, X., Chen, H. & Luo, W. A new phenomenon in the anaerobic process with simultaneous denitrification and phosphorus removal. Acta Sci. Circumst. 32, 555–567 (2012).f p p 43. Shen, N., Chen, Y. & Zhou, Y. Multi-cycle operation of enhanced biological phosphorus removal (EBPR) with different carbon sources under high temperature. Water Res. 114, 308–315 (2017).i sources under high temperature. Water Res. 114, 308–315 (2017) 44. Kim, J. M., Lee, H. J., Lee, D. S. & Jeon, C. O. Characterization of the denitrification-associated phosphorus uptake properties of “Candidatus Accumulibacter phosphatis” clades in sludge subjected to enhanced biological phosphorus removal. Appl. Environ. Microbiol. 79, 1969–1979 (2013).f , ( ) 5. Sun, S. P. et al. Effective biological nitrogen removal treatment processes for domestic wastewaters with low C/N ratios: A review Environ. Eng. Sci. 27, 111–126 (2010). g , ( ) 6. Federation, W. E. & Association, A. P. H. Standard methods for the examination of water and wastewater. APHA: Washington, DC USA (2005). ( ) 47. Ma, J., Wang, Z., He, D., Li, Y. & Wu, Z. Long-term investigation of a novel electrochemical membrane bioreactor for low-strength municipal wastewater treatment. Water Res. 78, 98–110 (2015). References Microbial and metabolic characterization of a denitrifying phosphorus-uptake/side stream phosphorus removal system for treating domestic sewage. Biodegradation. 25, 777–786 (2014).il g g g ( ) 3. Yamada, T. et al. Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum in mesophilic and thermophilic methanogenic sludge granules. Appl. Environ. Microbiol. 71, 7493–7503 (2005). 34. Hayatsu, M., Tago, K. & Saito, M. Various players in the nitrogen cycle: Diversity and functions of the microorganisms involved in nitrification and denitrification. Soil Sci. Plant Nutr. 54, 33–45 (2008). ii 35. Duan, L., Song, Y. H., Xia, S. Q. & Hermanowicz, S. W. Characterization of nitrifying microbial community in a submerged membrane bioreactor at short solids retention times. Bioresour. Technol. 149, 200–207 (2013). ( ) 6. Rani, S., Koh, H. W., Rhee, S. K., Fujitani, H. & Park, S. J. Detection and diversity of the nitrite oxidoreductase alpha subunit (nxrA gene of Nitrospina in marine sediments. Microb. Ecol. 73, 111–122 (2017). g p 37. Spring, S., Jackel, U., Wagner, M. & Kampfer, P. Ottowia thiooxydans gen. nov., sp. nov., a novel facultatively anaerobic, N2O- producing bacterium isolated from activated sludge, and transfer of Aquaspirillum gracile to Hylemonella gracilis gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 54, 99–106 (2004). 38. Jahan, K., Hoque, S. & Ahmed, T. Activated sludge and other suspended culture processes. Water Environ. Res. 84, 1029–1080 (2012).ii ii y p 40. Oehmen, A. et al. Advances in enhanced biological phosphorus removal: from micro to macro scale. Water Res. 41, 2271–2300 (2007). ii y p 40. Oehmen, A. et al. Advances in enhanced biological phosphorus removal: from micro to macro scale. Water Res. 41, 2271–2300 (2007). Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 10 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-37841-8. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-37841-8.h Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ublisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports \| (2019) 9:1153 \| https://doi.org/10.1038/s41598-018-37841-8 11
https://openalex.org/W2783016836	https://europepmc.org/articles/pmc5770416?pdf=render	English	null	Integrated miRNA-mRNA spatial signature for oral squamous cell carcinoma: a prospective profiling study of Narrow Band Imaging guided resection	Scientific reports	2,018	cc-by	10,494	Integrated miRNA-mRNA spatial signature for oral squamous cell carcinoma: a prospective profiling study of Narrow Band Imaging guided resection Received: 13 October 2017 Accepted: 29 December 2017 Published: xx xx xxxx Received: 13 October 2017 Accepted: 29 December 2017 Published: xx xx xxxx Camile S. Farah1,2, Simon A. Fox 2 & Andrew J. Dalley1 Oral squamous cell carcinoma (OSCC) is a common malignancy for which there is poor prognosis and limited therapeutic options. The objective was to identify mRNA targets of dysregulated miRNAs in OSCC using integrated analysis and understand molecular abnormality in surgical margins. We used biopsies along the spatial axis from normal tissue defined by narrow band imaging (NBI) through conventional white light (WL) margins to tumour from 18 patients undergoing surgical resection for OSCC. Overall 119 miRNA and 4794 mRNA were differentially expressed along the adjacent normal tissue to tumour axis. Analysis of miRNA profiles demonstrated the NBI margins were molecularly distinct from both the tumour and WL margin. Integrated analysis identified 193 miRNA-mRNA interactions correlated to the spatial axis of NBI-WL-T. We used cross-validation analysis to derive a spatial interactome signature of OSCC comprising 100 putative miRNA-mRNA interactions between 40 miRNA and 96 mRNA. Bioinformatic analysis suggests that miRNA dysregulation in OSCC may contribute to activation of the oncostatin M, BDNF and TGF-β pathways. Our data demonstrates that surgical margins defined by NBI leave less potentially malignant residual tissue. The miRNA-mRNA interactome provides insight into dysregulated miRNA signalling in OSCC and supports molecular definition of tumour margins. The success of tumour resection lies in balancing disease removal with healthy tissue retention1. Locoregional relapse continues to challenge the surgical management of oral squamous cell carcinoma (OSCC), occurring in 16–20% of cases and stalling survival rate improvement2–4. Despite surgeons’ best efforts to define surgical margins away from carcinoma and dysplasia, and confirmation of this by retrospective histopathology, unde- tected minimal residual disease (MRD) can be left in-situ, precipitating relapse1,3,5. Yet non-invasive techniques are available that identify potentially malignant perilesional tissue with improved sensitivity and specificity6. Recently we reported that use of Narrow Band Imaging for pre-operative OSCC assessment led surgeons to resect visually normal tissue that harboured significant molecular abnormalities without increasing the surgical field unnecessarily, and significantly decreased locoregional recurrence7. Similarly, molecular changes indicative of early tumour development have been demonstrated in histologically ‘normal’ surgical margins of tumours from the larynx, pharynx, and oral cavity8–10. y p y y MicroRNAs (miRNA or MIR) typically comprise 22 nucleotides with partial sequence homology to regions within mRNA molecules. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 13 October 2017 Accepted: 29 December 2017 Published: xx xx xxxx Results Cli i l Clinical observations. Full patient demographic and clinical details have already been published in tabular form together with photographs that describe the positioning of sample biopsies7. Briefly, patient age, sex and patterns of environmental risk factor exposure (tobacco and alcohol intake) were typical for the RBWH Head and Neck Cancer clinic. All tumour biopsies were negative for human papilloma virus (HPV)7. Tumour histology was predominantly moderate (n = 10) to well (n = 7) differentiated OSCC, with 1 case of verrucous carcinoma7. The most current follow-up data (up to 7 years post-surgery) shows that of the 18 patients, 1 patient (5.55%) declined follow-up. At 5 years follow-up, 14/17 patients (82.35%) were alive with no local recurrence, 1/17 patient (5.88%) had died from their disease, and 2 patients (11.76%) had died disease-free from other causes. In total, 16/17 patients (94.11%) who were followed for a minimum of 5 years were still alive and had not developed local recurrence. Processing of miRNA and mRNA microarray data. GeneChip® Human Genome U133 Plus 2.0 Arrays provided 54675 mRNA probe sets, while SurePrint® G3 Human miRNA Microarrays (Release 16) provided 1205 human miRNA probes and 144 human-viral miRNA probes (not reported here). Whole genome mRNA and miRNA expression data from 54 Affymetrix mRNA arrays and 54 Agilent miRNA arrays were processed. Each dataset comprised 3 tissue samples, T, WL and NBI, from each of 18 patients. Quality control procedures elimi- nated data from 5 mRNA arrays and 10 miRNA arrays (Table 1). After normalisation and preliminary filtering, 38989 mRNA probes and 458 miRNA probes entered the bioinformatics pipeline (Fig. 1). miRNA expression profiles in OSCC margins. We investigated the miRNA expression pattern by com- parisons across the tumour, WL and NBI defined margins. Specifically, we were interested in differences in molec- ular dysregulation across the tumour borders as defined by either WL or NBI and whether there was molecular distinction between the margins. We observed 137 miRNA probes (corresponding to 119 miRNA genes) were differentially expressed in at least one sample site (Fig. 2A). Overall the largest number of statistically significant differentially expressed miRNA were found in the T vs NBI comparison. Although the majority of these miRNA were common to both the T vs WL and T vs NBI comparison, there were still 34 which were unique to T vs NBI. Integrated miRNA-mRNA spatial signature for oral squamous cell carcinoma: a prospective profiling study of Narrow Band Imaging guided resection They evoke post-transcriptional regulation of gene expression by promoting mRNA degredation and function as broad-acting regulatory hubs that may be impaired by mutation11. MicroRNA have huge influence on cellular metabolism, proliferation and differentiation, and are key to our understanding of tumorigenesis. Their link to cancer is strengthened by the clustering of miRNA loci within the genome, often 1UQ Centre for Clinical Research, The University of Queensland, Herston Qld, 4029, Australia. 2Australian Centre for Oral Oncology Research & Education, UWA Dental School, University of Western Australia, Nedlands, WA 6009, Australia. Camile S. Farah, Simon A. Fox and Andrew J. Dalley contributed equally to this work. Correspondence and requests for materials should be addressed to C.S.F. (email: camile.farah@uwa.edu.au) or S.A.F. (email: simon.fox@ uwa.edu.au) or A.J.D. (email: a.dalley@uq.edu.au) Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 1 www.nature.com/scientificreports/ Biopsy Patient Number* Site 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 mRNA T 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 WL 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 NBI 1 1 1 1 0 0 0 1 1 1 1 0 0 1 1 1 1 1 miRNA T 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 WL 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 NBI 1 1 1 1 1 0 0 1 0 1 0 1 1 1 0 0 0 0 Table 1. Quality control filtering and omission of mRNA or miRNA array data. 1 = data included, 0 = data excluded. Corresponds to Farah et al.7. Table 1. Quality control filtering and omission of mRNA or miRNA array data. 1 = data included, 0 = data excluded. Corresponds to Farah et al.7. at regions that have mutational association with cancer12. Although other studies have reported the importance of miRNA in head and neck SCC13–21, none have undertaken genome wide integration of microRNA and their target genes’ mRNA expression. Integrated miRNA-mRNA spatial signature for oral squamous cell carcinoma: a prospective profiling study of Narrow Band Imaging guided resection The involvement of perilesional molecular abnormalities in MRD can now be studied through detailed assessment of the miRNA-mRNA interactions found at the tumour site compared to molecularly normal surgical margins, taking advantage of NBI to better determine clean surgical margins in situ. This coupling of research provides opportunity for more rigorous investigations into the role of miRNA in oral tumorigenesis as drivers of neoplastic transformation.h at regions that have mutational association with cancer12. Although other studies have reported the importance of miRNA in head and neck SCC13–21, none have undertaken genome wide integration of microRNA and their target genes’ mRNA expression. The involvement of perilesional molecular abnormalities in MRD can now be studied through detailed assessment of the miRNA-mRNA interactions found at the tumour site compared to molecularly normal surgical margins, taking advantage of NBI to better determine clean surgical margins in situ. This coupling of research provides opportunity for more rigorous investigations into the role of miRNA in oral tumorigenesis as drivers of neoplastic transformation. The broad aim of our study was to characterise and integrate microRNA and mRNA expression patterns in OSCC using tissue samples along a spatial axis extending from the tumour to disease-free normal surgical margins. We prospectively sampled 18 patients with OSCC using biopsies of tumour, adjacent perilesional tis- sue (conventional surgical margin), and distant (disease-free) tissue determined by NBI. Firstly, we sought to use molecular analysis of miRNA and mRNA to confirm and extend our previous findings that conventionally determined surgical margins can harbour molecular abnormality. We characterised this spatial gradient of dys- regulation at the molecular level and through pathway and gene ontology analysis. As a consequence we aimed to create a miRNA profile of OSCC based upon improved definition of adjacent normal (non-diseased) tissue controls using NBI defined margins as opposed to the currently accepted delineation under white light. The other major objective of our study was to integrate the miRNA and mRNA data in order to select and validate an miRNA-mRNA signature for OSCC. Through this and analysis of ontology and pathways we aimed to derive biological insight into the role of miRNA in the perturbation of signalling in oral tumorigenesis. Results Cli i l The normalised expression profile of the most differentially up and down regulated miRNAs in the T vs NBI com- parison are shown in Fig. 2B. To further investigate whether miRNA expression profiles could differentiate the tumour margins we used principal component analysis (PCA) to identify a discriminatory component that sepa- rated tumour from the WL and NBI samples (Fig. 2C). Whilst PCA did not fully segregate WL from NBI samples, the PCA plot placed most WL samples between NBI and tumour samples (Fig. 2C), therefore the discriminatory component segregated tumour, WL and NBI tissue biopsies appropriately along the adjacent normal tissue to tumour axis based solely upon their miRNA profiles, in a similar pattern to that described by us previously for Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 2 www.nature.com/scientificreports/ Figure 1. Bioinformatics pipeline. Figure 1. Bioinformatics pipeline. mRNA7. Hierarchical clustering of the miRNA data was consistent with this finding with some WL samples in the same major cluster as the T samples (Fig. 3A). Given dysregulation of miRNA across the OSCC margins, we used statistical set enrichment analysis to understand the pathways which the dysregulated miRNAs participate using miEAA. We independently analysed the T vs WL and T vs NBI differentially expressed microRNAs for enrichment of miRNAs associated with specific pathways. Overall there were 28 pathways for T vs WL and 127 for T vs NBI which we calculated were significantly enriched (FDR < 0.05), the top 20 (ranked by FDR) are shown in Fig. 3B. Generally, the significantly enriched pathways comprised those associated with cancer and cancer associated signalling. Consistent with our other analysis, there was a greater level of pathway dysregulation across the spatial axis NBI-T than for WL-T (Fig. 3B). mRNA expression profiles in OSCC margins. We have previously reported our initial analysis of the differentially expressed mRNA dataset for OSCC margins by hierarchical clustering and PCA analysis to demon- strate molecular divergence between tumour, WL and NBI defined margins7. Consistent with the microRNA analysis, the largest number of statistically significant differentially expressed genes were found in the T vs NBI comparison (Fig. 4A) and there were very few genes identified by this analysis as significantly differentially expressed (DE) between WL and NBI. While the majority of the DE genes were common to both the T vs WL and T vs NBI comparisons, there were substantial numbers of genes unique to the individual comparisons (Fig. 4A). Results Cli i l The normalised expression profile of the most up and down regulated mRNAs in the T vs NBI comparison are shown in Fig. 4B. To predict the biological implications of these DE genes and further elucidate the molecular differences between the tumour margins, we performed pathway analysis using the GSEA platform for path- way enrichment. We interrogated the KEGG pathway gene sets curated in MSigDB using ranked DE gene lists from the T vs NBI and T vs WL comparisons (Fig. 4C). Our analysis found that there was a preponderance of upregulated pathways in comparison to down regulated pathways (Fig. 4C). Furthermore, 5 of the top 15 signifi- cantly enriched upregulated pathways in the T vs NBI comparison were not significant for T vs WL. Interestingly, although there were few significantly DE genes in the WL vs NBI comparison, gene ontology analysis showed they were highly enriched for genes involved in oxygen transport (GO:0015671 Biological Process Padj = 1.6E- 09). The dysregulation of mRNA expression along the NBI to T spatial axis is also demonstrated by the normal- ised expression profile of the most up and down regulated mRNAs in the T vs NBI comparison (Fig. 4B). Integration of miRNA-mRNA datasets. In order to integrate miRNA and mRNA expression profiles we first used significantly DE miRNA then combined this data with experimentally verified interactions from miR- TarBase22. Of the 119 differentially expressed miRNA in at least one pairwise comparison, 92 had experimentally verified targets on the miRTarBase database22. Sparse Partial Least-Square (sPLS) analysis of median expression values across the biopsy sites (T, WL and NBI) identified 45 miRNA that were supported by a total of 193 target (mRNA) to effector (miRNA) correlations that were statistically significant (FDR < 0.05). The WL vs NBI paired comparison did not generate miRNA-mRNA associations (data not shown).hf The differential expression data (fold-change and adjusted p-value) for the pair-wise comparisons, T vs WL and T vs NBI, were incorporated with the integrated miRNA-mRNA data. We used Circos plots to integrate the data from the miRNA and mRNA differential expression datasets, the miRNA-mRNA interactions and chromo- somal mapping of individual targets as shown in Fig. 5A and B. Greater molecular divergence was evident in the T vs NBI comparison than the T vs WL comparison (Fig. 5A and B respectively). Results Cli i l Analysis was performed separately for the T vs NBI and T vs WL comparisons and the level of significance is indicated by different colours. pairwise DE comparisons but were identified by spatial correlation along the tumour to normal tissue axis. This spatial integration included two miRNAs (miR-181a and miR-204) and six additional mRNA (HMGA1, KDM4A, MRPL38, RNF24, SLAIN1, PLEKHA8) that were not identified by individual paired comparisons (T vs WL; T vs NBI). To better understand which biological functions and pathway perturbations are associated with dysregu- lated miRNA-mRNA interactions in OSCC we applied functional and pathway annotations using overrepresenta- tion analysis with the EnrichR analysis tool (Fig. 5C). The list of target mRNAs from the correlated dataset was used to explore for GO annotation terms (from GO consortium) and biological pathways based upon the curated Wikipathways database. There were 63 GO biological process (BP) terms significantly (Padj < 0.05) associated with the dataset. These terms were summarised using REVIGO (similarity = 0.5) to reduce redundancy and the top 10 BP terms are shown in Fig. 5C with transcription regulation, cellular movement and proliferative signalling predominating. There were 16 GO molecular function (MF) terms associated with the dataset (Padj < 0.05) with the top 10 following consolidation using REVIGO shown in Fig. 5C with terms associated with ubiquitination and transcription regulation enriched. As shown in Fig. 5C overrepresentation analysis of pathways from the Wikipathways database in the dataset was consistent with perturbation of pathways implicated in tumorigenesis. In order to further explore and illustrate dysregulated miRNA-mRNA signalling we imported the highest ranked Oncostatin M signalling pathway into Cytoscape using the Wikipathways app, visualised it as a network and mapped the differentially expressed mRNAs, the correlated miRNAs and their differential expression (Fig. 5D). This network included the miR-204/SERPINE1 interaction identified through spatial correlation. Correlated miRNA-mRNA interactions in this pathway involved key molecules implicated in carcinogenesis such as JAK/ STAT pathway members, MMP13 and the protein tyrosine phosphatase PTPN11. Development and validation of an integrated miRNA-mRNA signature of OSCC margins. We next undertook additional analysis to develop and validate an integrated molecular signature of OSCC margins derived from the correlated miRNA-mRNA interactome shown in Fig. 5A and B. We took the integrated dataset which was modelled against the adjacent normal tissue to tumour spatial axis given by the three biopsy sites (NBI-WL-T) and applied a statistical cross validation test. Results Cli i l In total there were 139 putative miRNA-mRNA linkages within the T vs NBI paired comparison (86 common and 53 unique), in contrast there were 117 putative miRNA-mRNA linkages in the T vs WL paired comparison (86 common and 31 unique). There were 19 statistically significant additional miRNA-mRNA correlations which did not map to the individual Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 3 www.nature.com/scientificreports/ Figure 2. Differential miRNA expression in OSCC margins. (A) Venn diagram illustrating the relationship between differentially expressed miRNAs in the three discrete comparisons: T vs WL (Blue Circle), T vs NBI (Red Circle), WL vs NBI (Green Circle). For each group, upregulated miRNAs (red) and downregulated miRNAs (green) are tabulated. (B) Boxplots showing the expression profiles across the tumour margins of the 3 most upregulated and downregulated miRNA derived from the T vs NBI comparison. (C) Principal Component Analysis (PCA) plot of miRNA differential expression data showing spatial segregation according to biopsy location: T- tumour; WL – white light; NBI – narrow band imaging. Figure 2. Differential miRNA expression in OSCC margins. (A) Venn diagram illustrating the relationship between differentially expressed miRNAs in the three discrete comparisons: T vs WL (Blue Circle), T vs NBI (Red Circle), WL vs NBI (Green Circle). For each group, upregulated miRNAs (red) and downregulated miRNAs (green) are tabulated. (B) Boxplots showing the expression profiles across the tumour margins of the 3 most upregulated and downregulated miRNA derived from the T vs NBI comparison. (C) Principal Component Analysis (PCA) plot of miRNA differential expression data showing spatial segregation according to biopsy location: T- tumour; WL – white light; NBI – narrow band imaging. Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. (A) Unsupervised hierarchical clustering of differentially expressed miRNA and abridged heatmap. Annotations are according to patient number and biopsy site. (B) Pathway analysis by gene set enrichment of dysregulated miRNAs in OSCC margins. Pathways were derived from the KEGG (hsa) and Wikipathways (WP) databases. Analysis was performed separately for the T vs NBI and T vs WL comparisons and the level of significance is indicated by different colours. Figure 3. (A) Unsupervised hierarchical clustering of differentially expressed miRNA and abridged heatmap. Annotations are according to patient number and biopsy site. (B) Pathway analysis by gene set enrichment of dysregulated miRNAs in OSCC margins. Pathways were derived from the KEGG (hsa) and Wikipathways (WP) databases. Results Cli i l As described above we were able to capture inter- actions which were not significant within the context of pairwise comparison but might be significant when considered along the entire spatial axis. The statistical robustness of each miRNA-mRNA pairing was assessed by cross validation (LOOCV) and filtering by threshold of >0.5 (>50% of data supportive) prior to inclusion in the signature. From a total of 190 statistically significant miRNA-mRNA interactions identified by the ini- tial sPLS and Pearson’s correlation analysis, 100 were subsequently validated by LOOCV (stability) involving 40 miRNAs and 96 genes. The overall spatially integrated signature following LOOCV filtering is shown in Fig. 6 for miRNA-mRNA interactions grouped according to reciprocal (Fig. 6A) and non-reciprocal (Fig. 6B) correlations (Additional file 1). Of the 100 interactions, 13 were the additional interactions derived uniquely by spatial corre- lation and absent from the pairwise comparisons (Additional File 1). Two mRNA, VEGFA (miR-126, miR-145, Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ scientificreports/ and miR29c) and SERPINE1 (miR-145 and miR-204) were subject to regulation by more than one miRNA. The integrated miRNA-mRNA signature included 5 unique miRNA (miR-744, miR-18b, miR-98, miR-221 and let-7b) not previously reported in OSCC microRNA profiling23 Figure 4. Differential mRNA expression in OSCC margins (A) Venn diagram illustrating the relationship between differentially expressed mRNAs in the three discrete comparisons: T vs WL (Blue Circle), T vs NBI (Red Circle), WL vs NBI (Green Circle). (B) Boxplots showing the expression profiles across the tumour margins of the 3 most upregulated and downregulated mRNA derived from the T vs NBI comparison. (C) Pathway analysis of dysregulated genes (mRNA) between the OSCC margins. Up and down regulated pathways were determined using GSEA and the MsigDb curated KEGG database genesets. Pathways are ranked according to normalised enrichment score generated by GSEA. Threshold of significance is shown by the dashed line. Figure 4. Differential mRNA expression in OSCC margins (A) Venn diagram illustrating the relationship between differentially expressed mRNAs in the three discrete comparisons: T vs WL (Blue Circle), T vs NBI (Red Circle), WL vs NBI (Green Circle). (B) Boxplots showing the expression profiles across the tumour margins of the 3 most upregulated and downregulated mRNA derived from the T vs NBI comparison. (C) Pathway analysis of dysregulated genes (mRNA) between the OSCC margins. Up and down regulated pathways were determined using GSEA and the MsigDb curated KEGG database genesets. and miR29c) and SERPINE1 (miR-145 and miR-204) were subject to regulation by more than one miRNA. The integrated miRNA-mRNA signature included 5 unique miRNA (miR-744, miR-18b, miR-98, miR-221 and let-7b) not previously reported in OSCC microRNA profiling23. Results Cli i l Pathways are ranked according to normalised enrichment score generated by GSEA. Threshold of significance is shown by the dashed line. Figure 4. Differential mRNA expression in OSCC margins (A) Venn diagram illustrating the relationship between differentially expressed mRNAs in the three discrete comparisons: T vs WL (Blue Circle), T vs NBI (Red Circle), WL vs NBI (Green Circle). (B) Boxplots showing the expression profiles across the tumour margins of the 3 most upregulated and downregulated mRNA derived from the T vs NBI comparison. (C) Pathway analysis of dysregulated genes (mRNA) between the OSCC margins. Up and down regulated pathways were determined using GSEA and the MsigDb curated KEGG database genesets. Pathways are ranked according to normalised enrichment score generated by GSEA. Threshold of significance is shown by the dashed line. and miR29c) and SERPINE1 (miR-145 and miR-204) were subject to regulation by more than one miRNA. The integrated miRNA-mRNA signature included 5 unique miRNA (miR-744, miR-18b, miR-98, miR-221 and let-7b) not previously reported in OSCC microRNA profiling23. and miR29c) and SERPINE1 (miR-145 and miR-204) were subject to regulation by more than one miRNA. The integrated miRNA-mRNA signature included 5 unique miRNA (miR-744, miR-18b, miR-98, miR-221 and let-7b) not previously reported in OSCC microRNA profiling23. Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 6 entificreports/ Figure 5. Circos plots of gene and miRNA differential expression and potential regulatory linkages for (A) tumour vs NBI and (B) tumour vs WL. Outermost track is chromosome mapping. Histogram plot tracks show differential gene (mRNA) expression as up (red) and down (blue) regulated (−Log(10) P-value). Dot plot tracks show differential microRNA (miRNA) expression as up (red) and down (blue) regulated (−Log(10) P-value). Innermost track (centre) shows correlated miRNA-mRNA linkages as reciprocal (purple) and non-reciprocal (orange). (C) Functional enrichment of the correlated miRNA-mRNA dataset by overrepresentation analysis of pathways and gene ontology. The target correlated mRNA were used with the EnrichR analysis tool to derive significantly perturbed pathways and gene ontology associations. The top 10 pathways/ontology terms for each analysis are shown ranked by the EnrichR combined score parameter. (D) Functional relationships of candidate miRNA-mRNA interactions mapped onto the Oncostatin M signalling network using Cytoscape. mRNA nodes are oval and miRNA nodes are rectangular. For both mRNA and miRNA upregulated nodes are red and www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Discussionh This study of 18 patients with intra-oral OSCC used both miRNA and mRNA expression profiling and bioinfor- matic analysis to evaluate the molecular divergence between tumour core and adjacent tissue judged negative for tumour using either conventional white light or Narrow Band Imaging. The miRNA expression profile alone provided unambiguous evidence that the surgical margins determined by NBI possess fewer molecular abnor- malities than the more conservative surgical margins determined by WL examination. Pathway analysis of both the mRNA and miRNA dysregulated datasets also demonstrated greater level of biological distinction between NBI and tumour than for WL and tumour. These observations concur with our prior publication which examined mRNA expression profiles alone in the same patient cohort7, and strongly supports our premise that resection to surgical margins determined by NBI rather than by WL examination leave less potentially malignant residual tissue and thereby increase surgical success and decrease locoregional recurrence. y g g Microscopic tumour at surgical margins has been shown to increase 5-year mortality rates by 90% in a cohort of 707 intra-oral OSCC patients for whom 14.6% of surgical resections produced involved margins24. The reported accu- racy of post-operative histopathology exceeds 95%25, yet around 30% of patients with histologically negative margins experience relapse, prompting concerns about its prognostic sensitivity5,26–28. Underlying local relapse in patients with clear surgical margins are two issues: minimal residual disease (MRD) and second primary tumour development from a field of pre-neoplastic cells1. By design, this study provides insight into both of these disease processes, but is primarily focused on the avoidance of MRD. Defining the dysregulation of biological signalling networks across the tumour margins enables the development of a more precise molecular definition of an effective margin.h g p pif g The three biopsy sites studied (NBI, WL, T) comprise a spatial axis from disease-free histologically normal tissue, through non-cancerous tissue bearing evidence of abnormality, to tumour core. Initially, all of the miRNA and mRNA differential expression data was modelled collectively against this spatial axis and combined to form a putative miRNA-mRNA interactome. We strengthened our analysis by considering only experimentally vali- dated miRNA-mRNA interactions in order to develop a more robust and less complex integrated signature but this approach is necessarily limited since many putative interactions are as yet unverified. Results Cli i l Circos plots of gene and miRNA differential expression and potential regulatory linkages for (A) tumour vs NBI and (B) tumour vs WL. Outermost track is chromosome mapping. Histogram plot tracks show differential gene (mRNA) expression as up (red) and down (blue) regulated (−Log(10) P-value). Dot plot tracks show differential microRNA (miRNA) expression as up (red) and down (blue) regulated (−Log(10) P-value). Innermost track (centre) shows correlated miRNA-mRNA linkages as reciprocal (purple) and non-reciprocal (orange). (C) Functional enrichment of the correlated miRNA-mRNA dataset by overrepresentation analysis of pathways and gene ontology. The target correlated mRNA were used with the EnrichR analysis tool to derive significantly perturbed pathways and gene ontology associations. The top 10 pathways/ontology terms for each analysis are shown ranked by the EnrichR combined score parameter. (D) Functional relationships of candidate miRNA-mRNA interactions mapped onto the Oncostatin M signalling network using Cytoscape. mRNA nodes are oval and miRNA nodes are rectangular. For both mRNA and miRNA upregulated nodes are red and downregulated are blue. Figure 5. Circos plots of gene and miRNA differential expression and potential regulatory linkages for (A) tumour vs NBI and (B) tumour vs WL. Outermost track is chromosome mapping. Histogram plot tracks show differential gene (mRNA) expression as up (red) and down (blue) regulated (−Log(10) P-value). Dot plot tracks show differential microRNA (miRNA) expression as up (red) and down (blue) regulated (−Log(10) P-value). Innermost track (centre) shows correlated miRNA-mRNA linkages as reciprocal (purple) and non-reciprocal (orange). (C) Functional enrichment of the correlated miRNA-mRNA dataset by overrepresentation analysis of pathways and gene ontology. The target correlated mRNA were used with the EnrichR analysis tool to derive significantly perturbed pathways and gene ontology associations. The top 10 pathways/ontology terms for each analysis are shown ranked by the EnrichR combined score parameter. (D) Functional relationships of candidate miRNA-mRNA interactions mapped onto the Oncostatin M signalling network using Cytoscape. mRNA nodes are oval and miRNA nodes are rectangular. For both mRNA and miRNA upregulated nodes are red and downregulated are blue. 7 Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x www.nature.com/scientificreports/ Figure 6. Circos plots of the integrated miRNA-mRNA spatial signature showing (A) reciprocal and (B) non-reciprocal miRNA-mRNA linkages. Data includes all interactions which were statistically robust (cross validation stability >0.5) and spatially correlated with the normal tissue to tumour axis (Pearson’s correlation FDR adjusted p < 0.05). Ribbon colour indicates the originating miRNA. Results Cli i l Ribbon thickness is proportional to the stability of the interaction in the cross-validation analysis (0.5–1). The colour of the tabs adjacent to the gene names indicates up (red) or down (blue) mRNA expression in the tumour. Figure 6. Circos plots of the integrated miRNA-mRNA spatial signature showing (A) reciprocal and (B) non-reciprocal miRNA-mRNA linkages. Data includes all interactions which were statistically robust (cross validation stability >0.5) and spatially correlated with the normal tissue to tumour axis (Pearson’s correlation FDR adjusted p < 0.05). Ribbon colour indicates the originating miRNA. Ribbon thickness is proportional to the stability of the interaction in the cross-validation analysis (0.5–1). The colour of the tabs adjacent to the gene names indicates up (red) or down (blue) mRNA expression in the tumour. Figure 6. Circos plots of the integrated miRNA-mRNA spatial signature showing (A) reciprocal and (B) non-reciprocal miRNA-mRNA linkages. Data includes all interactions which were statistically robust (cross validation stability >0.5) and spatially correlated with the normal tissue to tumour axis (Pearson’s correlation FDR adjusted p < 0.05). Ribbon colour indicates the originating miRNA. Ribbon thickness is proportional to the stability of the interaction in the cross-validation analysis (0.5–1). The colour of the tabs adjacent to the gene names indicates up (red) or down (blue) mRNA expression in the tumour. Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x Discussionh This spatially refer- enced interactome is a “proper superset” of the three discrete paired comparisons (T vs NBI, T vs WL, WL vs NBI), meaning that it includes miRNA-mRNA interactions that do not occur in any of the paired comparisons. Subsequent analysis identified which of the statistically robust miRNA-mRNA interactions were operative in each paired comparison, and could be correlated with the normal tissue – tumour spatial axis. p p p Integration of the putative miRNA-mRNA interactome with the paired comparisons of T vs WL and T vs NBI generated a complicated picture of miRNA-mRNA interaction (Fig. 5A and B) which was further refined by cross validation. Throughout this analysis we consistently found more miRNA-mRNA interactions in the TvsNBI comparison than the TvsWL comparison and a gradient of miRNA-mRNA dysregulation along the spa- tial axis. Together with the ideographic representations (Figs 5 and 6), the present study is the most comprehen- sive analysis of putative miRNA-mRNA interactions within OSCC published to date. Furthermore, these findings have broader implications since there is increasing interest in molecular heterogeneity within tumours and their boundaries29,30 although few studies have examined tumour margins spatially to the extent of integrated molec- ular detail used here. Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 8 www.nature.com/scientificreports/ As described, our integrated miRNA-mRNA signature included 5 unique miRNA (miR-744, miR-18b, miR- 98, miR-221, let-7a and let-7b) not previously reported in OSCC or head and neck squamous cell carcinoma (HNSCC) microRNA profiling23. Based upon recent comprehensive review of aberrant microRNAs in HNSCC23 of the 40 microRNA found in the integrated signature reported here 28 miRNA had been previously reported as dysregulated in OSCC31–34 and 7 miRNA had been reported in HNSCC23. The direction of dysregulation found in our own study was consistent with those reported for these miRNAs elsewhere. Thus, the miRNA profile we have developed through integrated analysis is both a consolidation and extension of these previous studies. p g g y p Enrichment analysis of dysregulation in the individual miRNA and mRNA data as well as the integrated miRNA-mRNA dataset provided further insight into the mechanisms and downstream consequences of this dysregulated signalling. Broadly, pathway enrichment of the miRNA and mRNA DE data was consistent with the findings of our study supporting increasing molecular dysregulation along the NBI to tumour axis. Discussionh Focal adhe- sion was amongst the top 10 dysregulated pathways in both the miRNA and mRNA DE datasets (Figs 3B and 4C) but was not identified in the integrated data. In the integrated data GO analysis demonstrated a preponderance of dysregulation in gene regulatory molecular function, a well-recognised feature of miRNA in preferentially target- ing nuclear signalling mechanisms35. Key oncogenic pathways were identified as perturbed across OSCC margins including oncostatin M, brain derived neurotrophic factor (BDNF) and TGF-β signalling pathways (Fig. 5C). Oncostatin M signalling has been previously demonstrated to contribute to tumour progression and metastases through JAK-STAT signalling36,37. Aberrant expression of oncostatin M has been described in OSCC and cervical SCC as associated with poor clinical outcomes38,39. In oral and cervical SCC tissue transglutaminase (TGM2) has been identified as an important downstream mediator of Oncostatin M signalling40, and in our own data we found that TGM2 was upregulated in both T vs NBI and T vs WL. The oncostatin M, BDNF and TGF-β pathways have been demonstrated to co-operate to drive EMT and cancer stem cell expression signatures in other cancers. Recent evidence indicates that oncostatin M cooperates with TGF-β signalling to activate EMT via SMAD341. In our own data SMAD3 ranked highest when we used Enrichr to interrogate the database of transcription fac- tor targets (TRANSFAC) with the integrated dataset (results not shown). Similarly in non-small cell lung can- cer, STAT3 and BDNF signalling have been shown to cooperatively activate proliferative signalling42. While the contribution of STAT3 signalling to HNSCC progression are well recognised, the mechanisms underlying this activation are poorly understood37. Our data support a contribution by miRNA and their targets to this dysreg- ulation with downstream effects upon multiple interacting oncogenic pathways. The ultimately derived putative miRNA-mRNA signature included interactions that had statistically supported correlation to the normal tissue– tumour spatial axis, as well as excluding all non-statistically robust interactions. Thus, the spatially correlated interactome remains a “proper superset” of the three discrete paired comparisons. Out of 100 interactions, it con- tains 13 which were exclusively identified by spatial integration including two miRNAs (miR-181a and miR-204) and five additional mRNA (HMGA1, KDM4A, RNF24, SLAIN1, PLEKHA8) that were not represented within the individual paired comparisons (T vs WL; T vs NBI). Discussionh These miRNA-mRNA interactions require further inves- tigation but this supports the validity of our underlying premise that we could exploit the availability of the three biopsy locations to derive greater information regarding miRNA and mRNA dysregulation using spatial analysis. In this study we observed a large number of non-reciprocal miRNA-mRNA correlations which is intuitively inconsistent with our current understanding of miRNA mechanisms of regulation11, but is a common finding in integrated analysis of miRNA-mRNA expression profiles43,44. Recent evidence suggests that such positive corre-l p y g g g y g g p y In this study we observed a large number of non-reciprocal miRNA-mRNA correlations which is intuitively inconsistent with our current understanding of miRNA mechanisms of regulation11, but is a common finding in integrated analysis of miRNA-mRNA expression profiles43,44. Recent evidence suggests that such positive corre- lations may be a consequence of an adaptive response to changes in mRNA expression and reflect homeostatic mechanisms regulating miRNA expression44. Furthermore, we included non-reciprocal correlations in our net- work analysis because miRNA interactions with their targets may depend upon the topology of the signalling pathway45. For this reason, while we focussed upon negative correlations since they reflect direct miRNA regula- tory networks, we did not entirely exclude non-reciprocal correlations from our analysis. Conclusionsh This study has shown that miRNA differential expression, putative miRNA-mRNA interactions and dysregulated biological signalling are greater in T vs NBI than in T vs WL. Our analysis across the three biopsy sites strongly supports our premise that resection of OSCC to surgical margins that are determined by NBI rather than by WL will leave less potentially malignant residual tissue and thereby increase the likelihood of surgical success. This is significant for surgical management of oral cancer but also has implications for molecular analyses of cancer genetic dysregulation using conventionally defined “normal” tissue observed under white light visualization. By stringent analysis we identified the specific miRNA-mRNA interactions that correlated with the normal tissue to tumour spatial axis. This signature is robust, statistically validated and is not conflicted by control “normal” tissue which may harbour molecular abnormality. These particular interactions are likely to include miRNA-mRNA interactions that accompany oral neoplastic transformation. Our integrated bioinformatic analysis suggests that miRNA dysregulation in OSCC could be a mechanism contributing to activation of the oncostatin M, BDNF and TGF-β pathways with signalling via STAT3 and SMAD3. Finally, our statistically robust miRNA signature in OSCC is a foundation for translational development of molecular assays which can be applied to the definition of surgical margins with greater fidelity than current methods. Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x Methods i Patient group. Eighteen patients with intra-oral squamous cell carcinoma (excluding lip, pharynx and hypopharynx) who were scheduled for surgical resection were enrolled prospectively. Patient demographics, tumour characteristics and surgery have been described by us previously7. This study was run in strict accord- ance with the principles outlined in the Declaration of Helsinki (2008) after ethical review and approval from the Royal Brisbane and Women’s Hospital Human Research Ethics Committee (HREC/08/QRBW20 and HREC/10/ QRBW336). All subjects provided written informed consent to participation in the study. Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 9 www.nature.com/scientificreports/ Sample collection and characteristics. Prior to surgery, primary OSCC sites were visualised under White Light (WL) and then by Narrow Band Imaging (NBI) using an Olympus NBI ENF-VQ nasendoscope with CLV-180 light source and processor (Olympus Medical Systems Corp., Tokyo, Japan). Upon completion of the OSCC resection which was taken to ≥5 mm beyond the NBI defined surgical margin, a 4 mm punch biopsy was taken for analysis from within the following zones as reported by us previously7. 1. The NBI margin – 5 mm beyond the limit of tissue abnormality visible by NBI.h 1. The NBI margin – 5 mm beyond the limit of tissue abnormality visible by NBI.h h g y y y 2. The White Light (WL) margin –5 mm beyond the limit of tissue abnormality visible by White Light Th f h ( ) h g y y y 2. The White Light (WL) margin –5 mm beyond the limit of tissue abnormality visible by White Light. 3 Th f h i (T) h 3. The core of the primary tumour (T). The biopsies were immediately immersed in RNAlater (Ambion, Life Technologies, Carlsbad, USA) and sub- sequently stored at −80 °C. The NBI margin sample is histologically normal disease-free tissue adjacent tumour as previously reported by us7. RNA isolation. Frozen biopsies were ground in liquid nitrogen then incubated in 500 μL of lysis buffer (Buffer RLT; Qiagen, Hilden, Germany), with 200 ng of Proteinase K (Invitrogen, Life Technologies, Carlsbad, USA) overnight with mixing at 37 °C. After incubation, 200 μL of lysate was used for RNA isolation with TRIzol Reagent (Invitrogen, Life Technologies, Carlsbad, USA) following the manufacturer’s protocol that was optimised to increase nucleic acid recovery by addition of 10 μg of RNase-free glycogen as a carrier and an overnight incu- bation at −20 °C. Methods i After DNase treatment with the TURBO DNA-free Kit (Ambion, Life Technologies, Carlsbad, USA), RNA was purified by sodium acetate precipitation (Ambion, Life Technologies, Carlsbad, USA); quality and quantity assessments used a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and Qubit fluorometer (Invitrogen, Life Technologies, Carlsbad, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer and RNA 6000 Nano kit (Agilent Technologies, Santa Clara, USA). Gene expression profiling and bioinformatic quality control. This study utilised GeneChip® Human Genome U133 Plus 2.0 Arrays (Affymetrix, Santa Clara, USA) and SurePrint® G3 Human miRNA Microarrays Release 16, 8 × 60 K (Agilent Technologies, Santa Clara, USA) to derive whole genome mRNA and miRNA expres- sion data from three samples (NBI, WL, and T) for each of 18 patients, giving a total of 108 expression profiles. 100 ng of total RNA was the input for target preparation for both mRNA and miRNA hybridisations using the GeneChip 3′ IVT Express Kit (Affymetrix, Santa Clara, USA) and the miRNA Microarray System with miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) respectively. Manufacturers’ protocols, reagents and spike-in controls were used throughout. The resultant labelled and amplified RNA (aRNA) was subject to quality control (QC) assessment of size distribution and yield using an Agilent 2100 Bioanalyzer prior to array hybridisation. Bioinformatic quality control and normalisation of array data. An overview of the bioinformat- ics pipeline is shown in Fig. 1. Expression data from 54 mRNA and 54 miRNA arrays were processed through discrete QC pipelines: mRNA via affyAnalysisQC46 and simpleaffy47; miRNA via the AgiMicroRNA package48. Good library quality and consistent hybridization quality were evident, however some data from both mRNA and miRNA arrays were identified as outliers and excluded from further processing (Table 1). The remaining mRNA array data (49 samples) were normalised using the GeneChip robust multiarray average (GCRMA) normalisa- tion method49, while the remaining miRNA array data (44 samples) were normalised by the Robust Multi-array Average (RMA) method without correction followed by quantile normalisation48. Bioinformatic data analysis was performed at the Queensland Facility for Advanced Bioinformatics (QFAB). MicroRNA and gene expression profiling and clustering. After normalisation, preliminary filtering removed probes with coefficients of variation <0.1 across all mRNA or miRNA arrays. Next, differential expres- sion of genes was tested using the Limma R package50 via 3 pair-wise comparisons: T vs WL; T vs NBI; WL vs NBI. Methods i Linear models were applied to the (log-transformed) mRNA and miRNA expression data, then analysed by paired t-tests adjusted for multiple comparisons via the Benjamini and Hochberg (BH) procedure51. The datasets were again filtered to remove probes that were not differentially expressed in at least one group (Padj < 0.01). gif y g j Clustering of differentially-expressed genes was achieved by Principal Component Analysis (PCA) using the mixOmics software package52. The PCA analysis used normalised expression data subject only to preliminary filtering that removed probes with coefficients of variation <0.1 across all arrays. Hierarchical clustering was performed using Cluster 3.053. Integration of differentially expressed miRNA-mRNA. Micro-RNA sequences, annotations and chro- mosome positions and clustering were identified using the miRBase database (version 16)54. All cross-mapping of mRNA targets to miRNA effectors used the experimentally validated interactions curated in miRTarBase database (version 4.5)22. Our analysis sought generalised correlation models and therefore used median cohort mRNA and miRNA expression values for T, WL and NBI. MicroRNA to mRNA correlations were established using sparse partial least-square (sPLS) analysis55 with concurrent parameter tuning to optimise the number of mRNA retained for each miRNA. Only data for biopsies where there were both mRNA and miRNA expression data were used for integrated analysis. The statistical significance of each correlation was tested using Pearson’s product-moment correlation coefficients corrected for multiple testing using the BH procedure51 to calculate adjusted p-values to estimate FDR. Circos plots for visualisation of mRNA and miRNA differential expression, and correlated interactions were generated using Circa (OMGenomics.com). Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 10 www.nature.com/scientificreports/ Cross validation of mRNA to miRNA correlations. Rotation estimation for each miRNA-mRNA cor- relation was achieved by leave one out cross validation (LOOCV) in order to establish the robustness of each putative interaction. For this, each patient’s data was omitted sequentially prior to repeating the mRNA to miRNA sPLS analysis. The number of times an mRNA target was selected (out of 18) is provided as Stability Rate, whereby a value of 1 implies perfect stability (mRNA selected 18 out of 18 times) whereas a value of 0.33 would indicate poor stability (mRNA selected 6 out of 18 times). The arbitrary threshold used here for inclusion in the final miRNA-mRNA signature was stability ≥0.5 (i.e. 50%). Circos plots for visualisation of the integrated miRNA-mRNA signature were generated using Circos Table Viewer v0.63–956. Methods i Gene set enrichment analysis for biological pathways and gene ontology terms. Gene set enrich- ment analysis of biological pathways associated with differentially expressed miRNAs was performed using the miEAA tool (https://ccb-compute2.cs.uni-saarland.de/mieaa_tool/)57. For pathway analysis of the differentially expressed mRNA genesets we used GSEA hosted at the Broad Institute (https://genepattern.broadinstitute.org/gp/)58. The KEGG (Kyoto Encyclopedia of Genes and Genomes pathway) and Reactome curated pathway genesets from Molecular Signature Database (MsigDB, http://www.broadinstitute.org/gsea/msigdb/) were used. For GSEA gene differentially expressed gene lists were pre-ranked by logarithm transformation of p-value (base 10) and application of the sign of the fold-change (i.e. upregulated genes given positive values). Overrepresentation analysis (ORA) of differentially expressed genes derived from the integrated miRNA-mRNA data for pathways and gene ontology terms was performed using the web based platform EnrichR (http://amp.pharm.mssm.edu/Enrichr)59 which permits inter- rogation of multiple databases. Up and down regulated gene lists were evaluated for significant enrichment against the following gene set libraries: GO Biological Process, GO Cellular Component, GO Molecular Function (all from http://www.geneontology.org), the Wikipathways database (http://www.wikipathways.org/) and KEGG (http://www. kegg.jp/kegg) (all current as of May 2017). Enriched annotations/pathways were selected/ranked based upon com- bined score which was calculated by the EnrichR platform following Z-score permutation background correction on the Fischer Exact Test p-value60. In order to facilitate the interpretation of GO analysis the selected output annotation lists were further consolidated and summarised using REVIGO61, a tool that removes redundant terms based upon similarity. Network analysis was performed using Cytoscape 3.5.162 and the Wikipathways plugin63. References Improved surgical margin definition by narrow band imaging for resection of oral squamous prospecti e gene e pression profiling stud Head Neck 38 832 839 https //doi org/10 1002/hed 23989 (2016) 7. Farah, C. S. et al. Improved surgical margin definition by narrow band imaging for resection of oral squamous cell carcinoma: A prospective gene expression profiling study. Head Neck 38, 832–839, https://doi.org/10.1002/hed.23989 (2016). , p g gi y g g q prospective gene expression profiling study. Head Neck 38, 832–839, https://doi.org/10.1002/hed.23989 (2016). prospective gene expression profiling study. Head Neck 38, 832–839, https://doi.org/10.1002/hed.23989 (2016). 8. Slootweg, P. J., Hordijk, G. J., Schade, Y., van Es, R. J. & Koole, R. Treatment failure and margin status in head and critical view on the potential value of molecular pathology. Oral Oncol 38, 500–503 (2002). p p g p pi g y p g 8. Slootweg, P. J., Hordijk, G. J., Schade, Y., van Es, R. J. & Koole, R. Treatment failure and margin status in head and neck cancer. A critical view on the potential value of molecular pathology Oral Oncol 38 500–503 (2002) g J j J J g critical view on the potential value of molecular pathology. Oral Oncol 38, 500–503 (2002). p p gy 9. Partridge, M. et al. Detection of minimal residual cancer to investigate why oral tumors recur despite seemingly adequate treatment. Clin Cancer Res 6, 2718–2725 (2000). p p gy 9. Partridge, M. et al. Detection of minimal residual cancer to investigate why oral tumors recur despite seemingly adequate treatment Clin Cancer Res 6, 2718–2725 (2000). 10. Goldenberg, D. et al. Intraoperative molecular margin analysis in head and neck cancer. Arch Otolaryngol Head Neck Surg 130, 39–44, https://doi.org/10.1001/archotol.130.1.39 (2004). 10. Goldenberg, D. et al. Intraoperative molecular margin analysis in head and neck cancer. Arch Otolaryngol Head Neck Surg 130, 39–44, https://doi.org/10.1001/archotol.130.1.39 (2004). p g 1. Jonas, S. & Izaurralde, E. Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet 16, 421–433 https://doi.org/10.1038/nrg3965 (2015). p g g ( ) 2. Calin, G. A. et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Nat Acad Sci USA 101, 2999–3004, https://doi.org/10.1073/pnas.0307323101 (2004). p g g ( ) 12. Calin, G. A. et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA 101, 2999–3004, https://doi.org/10.1073/pnas.0307323101 (2004). 1. Hussain, T. & Nguyen, Q. T. Molecular imaging for cancer diagnosis and surgery. Adv Drug Deliv Rev 66, 90–100, https://doi org/10.1016/j.addr.2013.09.007 (2014). g j 2. Eckardt, A., Barth, E. L., Kokemueller, H. & Wegener, G. Recurrent carcinoma of the head and neck: treatment strategies and survival analysis in a 20-year period. Oral Oncol 40, 427–432, https://doi.org/10.1016/j.oraloncology.2003.09.019 (2004). References 1. Hussain, T. & Nguyen, Q. T. Molecular imaging for cancer diagnosis and surgery. Adv Drug Deliv Rev 66, 90–100, https://doi. org/10.1016/j.addr.2013.09.007 (2014). 3. Braakhuis, B. J., Bloemena, E., Leemans, C. R. & Brakenhoff, R. H. Molecular analysis of surgical margins in head and neck cancer: more than a marginal issue. Oral Oncol 46, 485–491, https://doi.org/10.1016/j.oraloncology.2010.01.019 (2010). g g j gy 4. Yanamoto, S. et al. Clinicopathological risk factors for local recurrence in oral squamous cell carcinoma. Int J Oral Maxillofac Surg 41, 1195–1200, https://doi.org/10.1016/j.ijom.2012.07.011 (2012).i p g j j 5. de Carvalho, A. C. et al. Clinical significance of molecular alterations in histologically negative surgical margins of head and neck cancer patients. Oral Oncol 48, 240–248, https://doi.org/10.1016/j.oraloncology.2011.10.018 (2012). 6 Nguyen P et al High specificity of combined narrow band imaging and autofluorescence mucosal assessment of patients with head 5. de Carvalho, A. C. et al. Clinical significance of molecular alterations in histologically negative surgical margins of head and neck cancer patients. Oral Oncol 48, 240–248, https://doi.org/10.1016/j.oraloncology.2011.10.018 (2012). 6. Nguyen, P. et al. High specificity of combined narrow band imaging and autofluorescence mucosal assessment of patients with head and neck cancer. Head Neck 35, 619–625, https://doi.org/10.1002/hed.22999 (2013). p p g j gy ( ) 6. Nguyen, P. et al. High specificity of combined narrow band imaging and autofluorescence mucosal assessment of patients with head and neck cancer. Head Neck 35, 619–625, https://doi.org/10.1002/hed.22999 (2013).i 6. Nguyen, P. et al. High specificity of combined narrow band imaging and autofluoresce and neck cancer. Head Neck 35, 619–625, https://doi.org/10.1002/hed.22999 (2013).i 6. Nguyen, P. et al. High specificity of combined narrow band imaging and autofluorescence mucosal assessment of patients with head and neck cancer. Head Neck 35, 619–625, https://doi.org/10.1002/hed.22999 (2013).i p g 7. Farah, C. S. et al. Improved surgical margin definition by narrow band imaging for resection of oral squamous cell carcinoma: A 7. Farah, C. S. et al. Improved surgical margin definition by narrow band imaging for resection of oral squamous cell carcinoma: A prospective gene expression profiling study. Head Neck 38, 832–839, https://doi.org/10.1002/hed.23989 (2016). 8 Sl P J H dijk G J S h d Y E R J & K l R T f il d i i h d d k A 7. Farah, C. S. et al. www.nature.com/scientificreports/ g p y p g p Acids Res 42, e96, https://doi.org/10.1093/nar/gku354 (2014). p g g 6. Eijssen, L. M. et al. User-friendly solutions for microarray quality control and pre-processing on ArrayAnalysis.org. Nucleic Acid Res 41, W71–76, https://doi.org/10.1093/nar/gkt293 (2013).ff 46. Eijssen, L. M. et al. User-friendly solutions for microarray qu j y y Res 41, W71–76, https://doi.org/10.1093/nar/gkt293 (2013).f p g g 7. Wilson, C. L. & Miller, C. J. Simpleaffy: a BioConductor package for Affymetrix Quality Control and data analysis. Bioinformatic 21, 3683–3685, https://doi.org/10.1093/bioinformatics/bti605 (2005). 21, 3683–3685, https://doi.org/10.1093/bioinformatics/bti605 (2005). 48. Lopez-Romero, P. Pre-processing and differential expression analysis of Agilent microRNA arrays using the AgiMicroRna Bioconductor library BMC Genomics 12 64 https://doi org/10 1186/1471 2164 12 64 (2011) p g 8. Lopez-Romero, P. Pre-processing and differential expression analysis of Agilent microRNA arrays using the AgiMicroRna Bioconductor library. BMC Genomics 12, 64, https://doi.org/10.1186/1471-2164-12-64 (2011). 9. Wu, Z. & Irizarry, R. A. Preprocessing of oligonucleotide array data. Nat Biotechnol 22, 656–658; author reply658, https://doi org/10.1038/nbt0604-656b (2004).f g 0. Smyth, G. K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat App Genet Mol Biol 3, Article3, https://doi.org/10.2202/1544-6115.1027 (2004). g 1. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal o the Royal Statistical Society. Series B. Methodological 57, 289–300 (1995). he Royal Statistical Society. Series B. Methodological 57, 289–300 (1 y y g 2. Lê Cao, K., Gonzalez, I. & Dejean, S. integrOmics: an R package to unravel relationships between two omics datasets. Bioinformatics 25, 2855–2856, https://doi.org/10.1093/bioinformatics/btp515 (2009).t g 3. de Hoon, M. J., Imoto, S., Nolan, J. & Miyano, S. Open source clustering software. Bioinformatics 20, 1453–1454, https://doi org/10.1093/bioinformatics/bth078 (2004).fi g 54. Griffiths-Jones, S. miRBase: the microRNA sequence database. Methods Mol Biol 342, 129–138, https://doi.org/10.1385/1-59745- 123-1:129 (2006). 5. Chun, H. & Keles, S. Sparse partial least squares regression for simultaneous dimension reduction and variable selection. J R Stat Soc Series B Stat Methodol 72, 3–25, https://doi.org/10.1111/j.1467-9868.2009.00723.x (2010). g j 6. Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res 19, 1639–1645, https://doi org/10.1101/gr.092759.109 (2009). 7. Backes, C., Khaleeq, Q. T., Meese, E. & Keller, A. miEAA: microRNA enrichment analysis and annotation. Nucleic Acids Res 44 W110–116, https://doi.org/10.1093/nar/gkw345 (2016).i 58. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102, 15545–15550, https://doi.org/10.1073/pnas.0506580102 (2005). www.nature.com/scientificreports/ 24. Binahmed, A., Nason, R. W. & Abdoh, A. A. The clinical significance of the positive surgical margin in oral cancer. Oral Onc 43, 780–784, https://doi.org/10.1016/j.oraloncology.2006.10.001 (2007). 25. Ord, R. A. & Aisner, S. Accuracy of frozen sections in assessing margins in oral cancer resection. Journal of oral and maxillo surgery 55, 663–669 (1997).h g y ( ) 26. Ikemura, K. & Ohya, R. The accuracy and usefulness of frozen‐section diagnosis. Head & neck 12, 298–302 (1990). h 27. Spiro, R. H., Guillamondegui, O., Paulino, A. F. & Huvos, A. G. Pattern of invasion and margin assessment in patients with tongue cancer. Head & neck 21, 408–413 (1999).l g 28. Parsons, J. T., Mendenhall, W. M., Stringer, S. P., Cassisi, N. J. & Million, R. R. An analysis of factors influencing the outcome of postoperative irradiation for squamous cell carcinoma of the oral cavity. International Journal of Radiation Oncology* Biology* Physics 39, 137–148 (1997). y 9. Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366 883–892, https://doi.org/10.1056/NEJMoa1113205 (2012). p g 0. Aubry, M. et al. From the core to beyond the margin: a genomic picture of glioblastoma intratumor heterogeneity. Oncotarget 6 12094–12109, https://doi.org/10.18632/oncotarget.3297 (2015).f 12094–12109, https://doi.org/10.18632/oncotarget.3297 (2015).f g g 31. Lajer, C. B. et al. Different miRNA signatures of oral and pharyngeal squamous cell carcinomas: a prospective translational study. Br J Cancer 104, 830–840, https://doi.org/10.1038/bjc.2011.29 (2011).i f J Cancer 104, 830–840, https://doi.org/10.1038/bjc.2011.29 (2011 p g j 32. Severino, P. et al. MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA- 10b dysregulation implicated in cell proliferation. BMC Cancer 13, 533, https://doi.org/10.1186/1471-2407-13-533 (2013). y g p p p g ( ) 3. Fukumoto, I. et al. MicroRNA expression signature of oral squamous cell carcinoma: functional role of microRNA-26a/b in th y g p p p g 33. Fukumoto, I. et al. MicroRNA expression signature of oral squamous cell carcinoma: functional role of m modulation of novel cancer pathways. Br J Cancer 112, 891–900, https://doi.org/10.1038/bjc.2015.19 (2015). ukumoto, I. et al. MicroRNA expression signature of oral squamous cell carcinoma: functional role of microRNA 26a/b in the odulation of novel cancer pathways. Br J Cancer 112, 891–900, https://doi.org/10.1038/bjc.2015.19 (2015).i p g q modulation of novel cancer pathways. Br J Cancer 112, 891–900, https://doi.org/10.1038/bjc.2015.19 (2015).i y g j 34. Manikandan, M. et al. References p g p 3. Childs, G. et al. Low-level expression of microRNAs let-7d and miR-205 are prognostic markers of head and neck squamous cel carcinoma. Am J Pathol 174, 736–745, https://doi.org/10.2353/ajpath.2009.080731 (2009). g j 4. Clague, J. et al. Genetic variation in MicroRNA genes and risk of oral premalignant lesions. Mol Carcinog 49, 183–189, https://doi org/10.1002/mc.20588 (2010). g 5. Li, J. et al. MiR-21 indicates poor prognosis in tongue squamous cell carcinomas as an apoptosis inhibitor. Clin Cancer Res 15 3998–4008, https://doi.org/10.1158/1078-0432.CCR-08-3053 (2009).ii p g 6. Scapoli, L. et al. MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression. Int J Immunopatho Pharmacol 23, 1229–1234 (2010). 17. Liu, C. J. et al. miR-31 ablates expression of the HIF regulatory factor FIH to activate the HIF pathway in head and neck carcinoma. Cancer Res 70, 1635–1644, https://doi.org/10.1158/0008-5472.CAN-09-2291 (2010).i g 8. Hui, A. B. et al. Comprehensive MicroRNA profiling for head and neck squamous cell carcinomas. Clin Cancer Res 16, 1129–1139 https://doi.org/10.1158/1078-0432.CCR-09-2166 (2010). p g ( ) 9. Avissar, M., Christensen, B. C., Kelsey, K. T. & Marsit, C. J. MicroRNA expression ratio is predictive of head and neck squamous cel carcinoma. Clin Cancer Res 15, 2850–2855, https://doi.org/10.1158/1078-0432.CCR-08-3131 (2009). 20. Chang, S. S. et al. MicroRNA alterations in head and neck squamous cell carcinoma. Int J Cancer 123, 2791–2797, https://doi. org/10.1002/ijc.23831 (2008).i g j 21. Tran, N. et al. MicroRNA expression profiles in head and neck cancer cell lines. Biochem Biophys Res Commun 358, 12–17, https:// doi.org/10.1016/j.bbrc.2007.03.201 (2007). g j 2. Hsu, S. D. et al. miRTarBase update 2014: an information resource for experimentally validated miRNA-target interactions. Nucleic Acids Res 42, D78–85, https://doi.org/10.1093/nar/gkt1266 (2014).h p g g ( ) 23. Koshizuka, K. et al. The microRNA signatures: aberrantly expressed microRNAs in head and neck squamous cell carcinoma. J Hum Genet 62, 3–13, https://doi.org/10.1038/jhg.2016.105 (2017). Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 11 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Oral squamous cell carcinoma: microRNA expression profiling and integrative analyses for elucidation of tumourigenesis mechanism. Mol Cancer 15, 28, https://doi.org/10.1186/s12943-016-0512-8 (2016). 35. Cui, Q., Yu, Z., Purisima, E. O. & Wang, E. Principles of microRNA regulation of a human cellular signaling network. Mol Sys 2, 46, https://doi.org/10.1038/msb4100089 (2006). p g 6. Douglas, W. G. et al. Development of head and neck squamous cell carcinoma is associated with altered cytokine responsiveness Mol Cancer Res 2, 585–593 (2004).h , ( ) 7. Geiger, J. L., Grandis, J. R. & Bauman, J. E. The STAT3 pathway as a therapeutic target in head and neck cancer: Barriers and innovations. Oral Oncol 56, 84–92, https://doi.org/10.1016/j.oraloncology.2015.11.022 (2016).i 38. Xu, C. et al. Integrative analysis of DNA copy number and gene expression in metastatic oral squamous cell carcinoma identifies genes associated with poor survival. Mol Cancer 9, 143, https://doi.org/10.1186/1476-4598-9-143 (2010). 39. Kucia-Tran, J. A. et al. Overexpression of the oncostatin-M receptor in cervical squamous cell carcinoma is associated epithelial-mesenchymal transition and poor overall survival. Br J Cancer 115, 212–222, https://doi.org/10.1038/bjc.2016.199 (2 40 C ff l M M l Ti l i di h li ff f i M i 40. Caffarel, M. M. et al. Tissue transglutaminase mediates the pro-malignant effects of oncostatin cervical squamous cell carcinoma. J Pathol 231, 168–179, https://doi.org/10.1002/path.4222 (2013) 0. Caffarel, M. M. et al. Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma. J Pathol 231, 168–179, https://doi.org/10.1002/path.4222 (2013). 1. Junk, D. J. et al. Oncostatin M promotes cancer cell plasticity through cooperative STAT3-SMAD3 signaling. Oncogene, https://doi org/10.1038/onc.2017.33 (2017). 2. Chen, B. et al. Autocrine activity of BDNF induced by the STAT3 signaling pathway causes prolonged TrkB activation and promote human non-small-cell lung cancer proliferation. Sci Rep 6, 30404, https://doi.org/10.1038/srep30404 (2016). g p p p g p 3. Laxman, N. et al. Global miRNA expression and correlation with mRNA levels in primary human bone cells. RNA 21, 1433–1443 https://doi.org/10.1261/rna.049148.114 (2015). p https://doi.org/10.1261/rna.049148.114 (2015) p g 4. Nunez, Y. O. et al. Positively correlated miRNA-mRNA regulatory networks in mouse frontal cortex during early stages of alcoho dependence. BMC Genomics 14, 725, https://doi.org/10.1186/1471-2164-14-725 (2013).i p p g 45. Calura, E. et al. Wiring miRNAs to pathways: a topological ap 45. Calura, E. et al. Wiring miRNAs to pathways: a topological approach to integrate miRNA and mRNA expression profiles. Nu Acids Res 42, e96, https://doi.org/10.1093/nar/gku354 (2014). www.nature.com/scientificreports/ 9. Kuleshov, M. V. et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res 44, W90–97 https://doi.org/10.1093/nar/gkw377 (2016). p g g 0. Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128 https://doi.org/10.1186/1471-2105-14-128 (2013). 61. Supek, F., Bosnjak, M., Skunca, N. & Smuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6, e21800, https://doi.org/10.1371/journal.pone.0021800 (2011).t p j e21800, https://doi.org/10.1371/journal.pone.0021800 (2011).t p g j p 2. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13 2498–2504, https://doi.org/10.1101/gr.1239303 (2003). p g g 63. Kutmon, M., Lotia, S., Evelo, C. T. & Pico, A. R. WikiPathways App for Cytoscape: Making biological pathways amenable to network analysis and visualization. F1000Res 3, 152, https://doi.org/10.12688/f1000research.4254.2 (2014). Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 12 www.nature.com/scientificreports/ Acknowledgements g Financial support for this work was provided through academic consultancy funds generated and held by C.S. Farah, and a grant from the Royal Brisbane & Women’s Hospital Foundation. We thank Phan Nguyen and Martin Batstone from the Royal Brisbane & Women’s Hospital for assistance with surgical assessment of margins and follow-up data, and Maurice Stevens for assistance with funding from the Royal Brisbane & Women’s Hospital Foundation. We thank Sarah Wagner, Amy Chiang, Yvette Emmanuel, Sandra Stein and Glenn Francis formerly from the Molecular and Clinical Pathology Research Laboratory (MACH R) at Princess Alexandra Hospital for assistance with RNA extraction and microarray hybridization. We thank Dominque Gorse, Cas Simons, Kim-Anh LeCao, Rosanna Quinlivan, Roxane Legaie and Xin-Yi Chua formerly from Queensland Facility for Advanced Bioinformatics (QFAB) for assistance with bioinformatic data analysis. Author Contributions C.F. conceived and initiated this study. C.F. coordinated the experiments, the clinical sample collection and provided clinical information. A.D. performed data analysis and prepared the initial draft of the manuscript with significant input from C.F. S.F. performed bioinformatic analysis and prepared the final draft of the manuscript with significant input from C.F. and A.D. Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-19341-x Competing Interests: The authors declare that they have no competing interests. ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific REPOrTS \| (2018) 8:823 \| DOI:10.1038/s41598-018-19341-x 13
https://openalex.org/W3108408088	https://www.frontiersin.org/articles/10.3389/fphys.2020.609006/pdf	English	null	Limb-Specific Features and Asymmetry of Nerve Conduction Velocity and Nerve Trunk Size in Human	Frontiers in physiology	2,020	cc-by	6,769	ORIGINAL RESEARCH published: 03 December 2020 doi: 10.3389/fphys.2020.609006 Limb-Specific Features and Asymmetry of Nerve Conduction Velocity and Nerve Trunk Size in Human Ayaka Nobue 1,2, Yoko Kunimasa 2, Hiromu Tsuneishi 2, Kanae Sano 1, Hiroyuki Oda 2 and Masaki Ishikawa 2* Ayaka Nobue 1,2, Yoko Kunimasa 2, Hiromu Tsuneishi 2, Kanae Sano 1, Hiroyuki Oda 2 and Masaki Ishikawa 2* 1 Faculty of Health Sciences, Morinomiya University of Medical Sciences, Osaka, Japan, 2 Graduate School of Sport and Exercise Sciences, Osaka University of Health and Sport Sciences, Osaka, Japan This study aimed to simultaneously examine the differences of human nerve conduction velocity (NCV) and nerve cross-sectional area (nCSA) between the upper and lower limbs and between different regions of the upper and lower limbs. Thirty healthy subjects volunteered for the study. NCV and nCSA of the ulnar and tibial nerves were measured with the dominant and non-dominant arms and the supporting and reacting legs using supramaximal electric stimulation and peripheral nerve ultrasonography at three regions for ulnar and tibial nerves, respectively. Supramaximal electric stimulation was superficially applied to the ulnar and tibial nerves at each point. These action potentials were recorded from the digiti minimi and soleus muscles for the ulnar and tibial nerves, respectively. Our results clearly showed that the NCV, nCSA, and circumference of the ulnar and tibial nerves were higher and greater in the lower limbs than in the upper limbs. The greater the circumference, the greater the nCSA for both the upper and lower limbs. However, unlike the upper limbs, the supporting leg did not have higher NCV than the reacting leg despite its greater circumference. Therefore, nCSA can be related to the circumference but not necessarily function for NCV developments of the lower limbs. These various aspects between the upper and lower limbs suggest that NCV does not depend on the nCSA sizes or upper and lower limb circumference; the results indicate the existence of limb-specific NCV but not nCSA developments. Keywords: nerve conduction velocity (NCV), ultrasonography, peripheral nerve, electrical stimulation, rapid movement, reaction, nerve cross-sectional area Edited by: Borja Sañudo, Sevilla University, Spain Reviewed by: Rinaldo André Mezzarane, University of Brasilia, Brazil Paul McCulloch Reviewed by: Rinaldo André Mezzarane, University of Brasilia, Brazil Paul McCulloch, Midwestern University, United States Reviewed by: Rinaldo André Mezzarane, University of Brasilia, Brazil Paul McCulloch Specialty section: This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology Keywords: nerve conduction velocity (NCV), ultrasonography, peripheral nerve, electrical stimulation, rapid movement, reaction, nerve cross-sectional area Citation: Nobue A, Kunimasa Y, Tsuneishi H, Sano K, Oda H and Ishikawa M (2020) Limb-Specific Features and Asymmetry of Nerve Conduction Velocity and Nerve Trunk Size in Human. Front. Physiol. 11:609006. doi: 10.3389/fphys.2020.609006 Nobue A, Kunimasa Y, Tsuneishi H, Sano K, Oda H and Ishikawa M (2020) Limb-Specific Features and Asymmetry of Nerve Conduction Velocity and Nerve Trunk Size in Human. Front. Physiol. 11:609006. The elucidation of the neuromuscular function of the human peripheral nervous system that enables rapid and accurate limb movements can be revealed by evaluating the morphology and functional characteristics of the peripheral nerves. Previous animal studies reported that trained mice had greater nerve axon diameter than the non-trained mice (Edds, 1950; Samorajski and Rolsten, 1975) and the peripheral nerve conduction velocity (NCV) was greater with the greater peripheral nerve axon diameter (Gasser and Grundfest, 1939; Hursh, 1939). In human, The elucidation of the neuromuscular function of the human peripheral nervous system that enables rapid and accurate limb movements can be revealed by evaluating the morphology and functional characteristics of the peripheral nerves. Previous animal studies reported that trained mice had greater nerve axon diameter than the non-trained mice (Edds, 1950; Samorajski and Rolsten, 1975) and the peripheral nerve conduction velocity (NCV) was greater with the greater peripheral nerve axon diameter (Gasser and Grundfest, 1939; Hursh, 1939). In human, December 2020 \| Volume 11 \| Article 609006 Frontiers in Physiology \| www.frontiersin.org Limb-Specific Human Neuromuscular Characteristics Nobue et al. however, the analysis of the peripheral nerve size in vivo is difficult and many reports are only available on human NCV. Kim et al. (2009a) and Pawlak and Kaczmarek (2010) reported that the NCV in the trained athletes was higher in the dominant than in the non-dominant upper limbs. Hatta et al. (1995) also reported that the NCV and dominant forearm circumference were faster and greater in badminton and kendo players than those in the healthy control subjects. Consequently, they imply that the developments of the human arm circumference and its muscle size would develop the human NCV. Therefore, high resolution imaging techniques of the peripheral nerve in vivo are expected to prove the above speculation.hi to discern the lateral preference and dominancy of the lower limbs. A previous human lower limb study (Kim et al., 2009b) found no differences in the NCV of the lower limbs between the dominant and non-dominant sides. Citation: Therefore, the lower limbs could have smaller differences in the nCSA and muscle sizes between the dominant and non-dominant sides than in the upper limbs. Therefore, unlike the upper limbs, the lateral preference and dominancy of the lower limbs may not exist.h Therefore, this study aimed to simultaneously examine the nCSA size of the upper and lower limb regions with the NCV of the ulnar and tibial nerves. Our hypotheses are as follows: (1) NCV, nCSA, and limb circumference are greater in the lower limbs than in the upper limbs. (2) The nCSA at any upper and lower limb region depends upon their circumferences. However, the lower limb shows no significant correlation between the size of the lower limb nCSA and its NCV as is the case with the previous upper limb study (Nobue and Ishikawa, 2015). Functionally, the proximal parts of the limbs have higher and thinner NCV and nCSA and vice versa than the distal parts. (3) Unlike the upper limbs, the NCV and nCSA of the lower limbs do not show any lateral preference despite the varying circumferences of both legs. The diameter size of the peripheral nerve fiber is very small (proximately 10–30 μmm). So far, the resolution of current in vivo human imaging technology cannot identify the cross-sectional area (CSA) of the nerve fibers. Therefore, the factors enhancing human NCV are not fully understood in the nerve fiber level. However, recent high-resolution imaging techniques of the peripheral nerve ultrasonography allow direct measurements of the cross-sectional area of the nerve trunk (nCSA), which is a bundle of various nerve fibers. Although there is no evidence for a correlation between nCSA and either axon diameter or NCV, this peripheral nerve ultrasonography combined with NCV calculation evoked by electrical stimulation makes it possible to further evaluate in vivo human peripheral nerve morphology and function. MATERIALS AND METHODS p p p gy Unlike the upper limbs, there have only been a few reports of NCV for the lower limbs, especially for a comparison of NCV for both the upper and lower limbs (Kim et al., 2009b). From the literature, the NCV of the upper limbs would be higher than that of the lower limbs (Mayer, 1963). However, the peripheral nerve ultrasonography showed that the lower limbs had greater nCSA than the upper limbs (Bedewi et al., 2017, 2018). These NCV and nCSA reports are not in line with the principle that the greater the peripheral nerve axon diameter, the higher the NCV (Edds, 1950; Samorajski and Rolsten, 1975). This conflict needs to be thoroughly examined during the simultaneous comparison between both human upper and lower limbs and at different regions because nCSA was not uniformly developed from the distal to proximal parts (Nobue and Ishikawa, 2015). The regional specificity of the nCSA developments and functions in both the upper and lower limbs also need to be fully discussed. In addition, the postural lower limb muscles had greater innervation ratio calculated by the number of muscle fibers dominated by axons than in the upper limb fine regulator muscles (Feinstein et al., 1955). Therefore, the branch unit of efferent nerve fibers in the greater innervation ratio muscle could be greater nerve axon size, and therefore, the lower limbs could have greater nCSA than the upper limbs. In this case, the simultaneous NCV measurements for both the upper and lower limbs together with nCSA measurements can solve the above-mentioned discrepancies and demonstrate the existence of the limb-specific NCV profiles. Th fi f h l l f d d Subjectsh j Thirty participants who have no history of any neurological, peripheral neuropathy, or other disorders of the upper and lower limbs, as well as no bilateral differences of the forearm and shank length, volunteered for this study [25 male and 5 female; age 19.8 ± 1.6 (18–25) years; body mass 65.5 ± 15.6 kg; height 172.0 ± 6.7 cm]. All subjects were competitive and active athletes who regularly attended local competitions for more than 6 years [tennis, baseball, track and field (sprint, javelin throw, high jump, hurdle, and decathlon), rugby, or soccer]. The dominant hand was confirmed by the Edinburgh Handedness Inventory (Oldfield, 1971), and the dominant (reacting) and non-dominant (supporting) legs were confirmed by the Waterloo Footedness Questionnaire (Elias et al., 1998). Informed consent was obtained before the experiment, which was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of the Osaka University of Health and Sport Sciences (authorization number 19-8). Frontiers in Physiology \| www.frontiersin.org Motor Nerve Conduction Velocityh Motor Nerve Conduction Velocity The motor response from each muscle was collected using the Signal software (Signal version 7.01, Cambridge Electronic Design Limited, United Kingdom) at a sampling rate of 10 kHz (Power 1,401, Cambridge Electronics Design Limited, United Kingdom). The compound muscle action potentials (CMAPs) were evoked by the electrical stimulation (DS7A, Digitimer Ltd., United Kingdom; 0.2 ms duration constant current square wave pulses) of the ulnar and tibial nerves starting from minimal and progressing to supramaximal stimuli intensity. As shown in Figure 1A, the CMAPs are evoked from the abductor digiti minimi muscle after electrical stimulation of ulnar nerve at the same nCSA measured points. A B C FIGURE 1 \| The measurement setup for the ulnar and tibial nerves and the representative EMG responses with the compound muscle action potentials of the measured points of the cross-sectional area (CSA) and positions of nerve electrical stimulation of the ulnar and tibial nerves. (A) The measured positions of the circumstance, nerve cross-sectional area (nCSA), and electrical stimulation for the ulnar nerve: 100 mm proximal to the medial epicondyle of the humerus (UNprox), 30 mm distal to the medial epicondyle of the humerus (UNmid), and 30 mm proximal to the ulnar head (UNdis). (B) The measured positions of the circumstance, nCSA and electrical stimulation for the tibial nerve: 100 mm proximal to the popliteal fossa (TNprox), at the popliteal fossa (TNmid), and 50 mm proximal to the soleus muscle (TNdis). (C) The onset of the M wave was measured by the compound muscle action potential recorded from each stimulation point. A B C A B C FIGURE 1 \| The measurement setup for the ulnar and tibial nerves and the representative EMG responses with the compound muscle action potentials of the measured points of the cross-sectional area (CSA) and positions of nerve electrical stimulation of the ulnar and tibial nerves. (A) The measured positions of the circumstance, nerve cross-sectional area (nCSA), and electrical stimulation for the ulnar nerve: 100 mm proximal to the medial epicondyle of the humerus (UNprox), 30 mm distal to the medial epicondyle of the humerus (UNmid), and 30 mm proximal to the ulnar head (UNdis). (B) The measured positions of the circumstance, nCSA and electrical stimulation for the tibial nerve: 100 mm proximal to the popliteal fossa (TNprox), at the popliteal fossa (TNmid), and 50 mm proximal to the soleus muscle (TNdis). Upper and Lower Limb Circumferences pp The circumferences of the upper and lower limbs were measured around the maximal girth of the forearm and calf and at the measured nCSA points of UNprox, UNmid, UNdis, TNprox, TNmid, and TNdis, respectively (Figures 1A,B). The CSA for each arm and leg region was calculated from each circumference: Protocols Firstly, the upper and lower limb circumferences were measured using a measuring tape. The nCSA of the ulnar and tibial nerves of the participants were measured by ultrasonography [Noblus, Hitachi Aloka Medical Ltd., a high-frequency (18 MHz) linear array ultrasound transducer; image resolution: 0.08 mm] in the sitting position with the forearm flexed at 120° and in the abdominal position, respectively. After the nCSA measurements, NCVs of the ulnar and tibial nerves were measured using the standard techniques of supramaximal i The specificities of the lateral preference and dominancy of the upper limbs have been well examined but not in the lower limbs. McGrath et al. (2015) suggested the difficulty December 2020 \| Volume 11 \| Article 609006 2 Limb-Specific Human Neuromuscular Characteristics Nobue et al. lower leg nCSAs were averaged by nCSA at TNprox and TNmid and at TNmid and TNdis, respectively. percutaneous stimulation with a constant current stimulator (DS7A, Digitimer Ltd., United Kingdom) and surface electrode recording (P-EMG plus, Oisaka Electronic Equipment, Japan) on each limb of each subject. Nerve Cross-Sectional Areah The ulnar and tibial nerves were scanned at three regions in the upper and lower limbs, respectively (Figures 1A,B). In the upper limb, the first region was at 100 mm proximal point to the medial epicondyle of the humerus (UNprox), the second region was at 30 mm distal point to the medial epicondyle of the humerus (UNmid), and the third region was at 30 mm proximal point to the ulnar head (UNdis). In the tibial nerves, the first region was at 100 mm proximal point to the popliteal fossa (TNprox), the second region was at the popliteal fossa point (TNmid), and the third region was at 50 mm proximal point to the soleus muscle belly (TNdis). As mentioned above, the nCSA size, which is a bundle of nerve fibers was measured by the ultrasonographic images at each region of the ulnar and tibial nerves, respectively (Nobue and Ishikawa, 2015). From these ultrasonographic images, the boundary of the nerve circumference was traced, and the upper and lower limb nCSAs were separately analyzed at each point (UNprox, UNmid, and UNdis; TNprox, TNmid, and TNdis, respectively) by ImageJ software (ver 1.45 s, National Institutes of Health, Unites States). The mean upper and lower limb nCSAs were calculated by the measured three points at each limb, respectively. For the comparison between the different regions, the upper arm and forearm nCSAs were averaged by nCSA at UNprox and UNmid and at UNmid and UNdis, respectively. Moreover, the thigh and CSA at each measured point = ( ) 1 4 2 p each circumference The upper arm and forearm circumferences were averaged by circumferences at UNprox and UNmid, and at UNmid and UNdis, respectively. Furthermore, the thigh and lower leg circumferences were averaged by circumferences at TNprox and TNmid, and at TNmid and TNdis, respectively. Measured Parameters Nerve Cross-Sectional Area RESULTS For comparison between upper and lower limbs (averaged variables between lateral parts), Table 1 shows the maximum circumference, NCV, and nCSA, respectively. The lower limb had significantly greater maximum circumferences, NCV, and nCSA than the upper limb, respectively (p < 0.05). h Upper arm NCV of the ulnar nerve: the distance from UNprox to UNmid, the latency from UNprox to UNmid. h Upper arm NCV of the ulnar nerve: the distance from UNprox to UNmid, the latency from UNprox to UNmid. For the lateral comparison at upper limbs (averaged variables between upper arm and forearm), the maximum forearm circumferences, upper limb NCV, and upper limb nCSA were significantly greater in the dominant than in the non-dominant arms (264 ± 29 vs. 256 ± 31 mm, 56.7 ± 6.2 vs. 54.5 ± 4.0 m s−1, and 6.9 ± 1.6 vs. 6.2 ± 1.2 mm, respectively: p < 0.05). For the lateral comparison at lower limbs (averaged variables between thigh and lower leg), the maximum lower leg circumference was greater in the supporting than in the reacting legs (376 ± 19 vs. 373 ± 18 mm, p < 0.05). The tibial nerve nCSA did not show any significant differences between the supporting and reacting legs (23.1 ± 4.7 vs. 23.6 ± 5.0 mm2, respectively). However, the mean lower limb NCV was significantly lower in the supporting than in the reacting legs (55.7 ± 11.3 vs. 62.5 ± 10.7 m s−1, p < 0.05). Forearm NCV of the ulnar nerve: the distance from UNmid to UNdis, the latency from UNmid to UNdis. Upper limb NCV of the ulnar nerve: the average value of these above two NCVs. Thigh NCV of the tibial nerve: the distance from TNprox to TNmid, the latency from TNprox to TNmid. Thigh NCV of the tibial nerve: the distance from TNprox to TNmid, the latency from TNprox to TNmid. Lower leg NCV of the tibial nerve: the distance from TNmid to TNdis, the latency from TNmid to TNdis. Lower leg NCV of the tibial nerve: the distance from TNmid to TNdis, the latency from TNmid to TNdis. Lower limb NCV of the tibial nerve: the average value of these above two NCVs. Skin and core body temperatures (around the soleus and abductor digiti minimi muscles) of each subject were monitored (CORE, greenTEG AG, Switzerland) at each trial to avoid the influence of temperature on NCV. RESULTS During measurements, we confirmed that the skin and core body temperatures stayed constant at each subject. More detail comparisons were performed for examining region and lateral specificities. In the circumference of the upper limb, the rmANOVA with lateral dominance and region as factors showed no interaction between all variables and revealed main effects of lateral dominance [F(1,29) = 19.54, p < 0.001] and region [F(1,29) = 521.42, p < 0.001], respectively (Figure 2A). In the ulnar NCV, the rmANOVA with lateral dominance and region as factors showed no interaction between Motor Nerve Conduction Velocityh (C) The onset of the M wave was measured by the compound muscle action potential recorded from each stimulation point. December 2020 \| Volume 11 \| Article 609006 Frontiers in Physiology \| www.frontiersin.org 3 Limb-Specific Human Neuromuscular Characteristics Nobue et al. The active electrode was attached to the belly of the abductor digiti minimi muscle (the muscle innervated by the ulnar nerve) and a ground electrode was attached to the ulnar head. Examination was performed with the subjects sitting and the forearm flexed at 120°. Similarly, as shown in Figure 1B, the CMAPs are evoked from the soleus muscle after electrical stimulation of tibial nerve at the same nCSA measured points. The active electrode was attached to the belly of the soleus muscle (the muscle innervated by the tibial nerve) and a ground electrode was attached to the malleolus lateralis. Examination was performed with the subjects lying prone and the ankle in a neutral position. The stimulation regions for both upper and lower limbs were marked with an aqueous marker, and the distances between stimulation regions were measured using a measuring tape. The latency of the stimulus artifact at each point was detected as the onset of the M-wave (Figure 1C). NCV was calculated by dividing the distance between each stimulating point by the differences between the latency responses (Kimura, 2013). These values were used as follows: significance of the main effects of each parameter and interaction between lateral comparison as well as regions for the upper and lower limbs, respectively. When no transform was found that made the variable normally distributed, nonparametric Wilcoxon signed rank tests were used to test for differences between groups and the significance levels were Bonferroni corrected. The correlations between each parameter were evaluated using the Pearson’s correlation coefficients after the distribution of the variables was passed for normality. The confidence level was set at p < 0.05 to determine statistical significances for all data. SPSS 25.0 software was used for statistical analyses. Frontiers in Physiology \| www.frontiersin.org Statistical Analyses In the ulnar nCSA, the rmANOVA with lateral dominance and region as factors showed no interaction between all variables and revealed effects of lateral dominance [F(1,29) = 10.42, p = 0.003] and region [F(1,29) = 29.19, p < 0.001], respectively (Figure 2C). In the tibial nCSA, the rmANOVA showed no interaction between all variables and revealed with lateral preference and region as factors showed no interaction between all variables and revealed main effects of region [F(1,29) = 213.99, p < 0.001] but not lateral preference [F(1,29) = 3.526, p = 0.078], respectively (Figure 2F). Figure 3 shows a semi-log plot of the nCSA at each region vs. the CSA calculated from its circumference at each region of the upper and lower limbs. According to the Pearson’s correlation, the data of all limbs clearly follow a straight line, which indicates that all nCSA data of both limbs maintain a relatively constant value to its circumference (Figure 3; r = 0.90, p < 0.001), although the tibial nerve had a much greater nCSA than the ulnar nerve. p y In the circumference of the lower limb, the rmANOVA with lateral preference and region as factors showed no interaction between all variables and revealed main effects of region [F(1,29) = 295.83, p < 0.001] but not lateral preference [F(1,29) = 0.34, p = 0.564], respectively (Figure 2D). In the tibial NCV, the rmANOVA with lateral preference and region as factors showed no interaction between all variables and revealed effects of lateral preference [F(1,29) = 10.26, p = 0.003] and region [F(1,29) = 56.71, p < 0.001], respectively (Figure 2E). A further examination of the relationships between the ulnar NCV and nCSA at each region for the upper limbs showed A B C D E F FIGURE 2 \| Limb circumference, nerve conduction velocity (NCV), and nCSA nerve cross-sectional area for the upper and lower limbs. (A) The upper arm and forearm arm circumferences for the dominant and non-dominant arms are shown, respectively. (B) The ulnar NCV of the upper arm and forearm are shown for the dominant and non-dominant arms, respectively. (C) The ulnar nCSA of upper arm and forearm are shown for the dominant and non-dominant arms, respectively. (D) The thigh and lower leg circumferences for the supporting and reacting legs are shown, respectively. Statistical Analyses Results were presented as means ± standard deviations. For comparison between the upper and lower limbs, the mean maximal circumferences, NCVs and nCSA for each subject were averaged for the right and left arms and for the right and left legs, respectively. Prior to all statistical analyses for this comparison, the distribution of the variables was passed for normality. Thus, statistical analyses were performed using the paired t-test between the upper and lower limbs. For the lateral comparison, the variables were compared between dominant and non-dominant arms as well as between the supporting and reacting legs, respectively. For comparison between regions for each upper and lower limb, the variables were compared between forearm and upper arm and between thigh and lower leg, respectively. To consider the statistical test of interaction between these two comparisons, Mauchly’s test of sphericity was performed on the data, and a two-way repeated-measures ANOVA (rmANOVA) was used to test for TABLE 1 \| Comparison of the measured parameters for the upper and lower limbs. Upper limb Ulnar nerve Lower limb Tibial nerve Maximum circumferences (mm) 260 ± 30 374 ± 18* Motor nerve conduction velocity (m s−1) 55.6 ± 4.3 59.1 ± 9.0* Nerve cross-sectional area (mm2) 6.6 ± 1.2 23.3 ± 4.1* Values are expressed as means ± SD. The ulnar and tibial nCSAs were averaged by the three measured points for both the dominant and nondominant arms and for both the supporting and reacting legs, respectively. Shows significant differences between the upper and lower limbs (p < 0.05). TABLE 1 \| Comparison of the measured parameters for the upper and lower limbs Frontiers in Physiology \| www.frontiersin.org December 2020 \| Volume 11 \| Article 609006 4 Limb-Specific Human Neuromuscular Characteristics Nobue et al. all variables and revealed main effects of region [F(1,29) = 18.90, p < 0.001] but not lateral dominance [F(1,29) = 2.891, p = 0.100], respectively (Figure 2B). In the ulnar nCSA, the rmANOVA with lateral dominance and region as factors showed no interaction between all variables and revealed effects of lateral dominance [F(1,29) = 10.42, p = 0.003] and region [F(1,29) = 29.19, p < 0.001], respectively (Figure 2C). all variables and revealed main effects of region [F(1,29) = 18.90, p < 0.001] but not lateral dominance [F(1,29) = 2.891, p = 0.100], respectively (Figure 2B). Statistical Analyses (E) The tibial NCV of the thigh and lower leg are shown for the supporting and reacting legs, respectively. (F) The tibial nCSA of the thigh and lower leg are shown for the supporting and reacting legs, respectively. , †, and ns indicate repeated two-way ANOVA analysis showing the main effect of lateral dominance (preference), region and interaction. , , and †† are significantly higher values as compared with the others ( p < 0.01, *p < 0.001, ††p < 0.001, ns, not significant), respectively. B C A B C A A B B C A E D F E D FIGURE 2 \| Limb circumference, nerve conduction velocity (NCV), and nCSA nerve cross-sectional area for the upper and lower limbs. (A) The upper arm and forearm arm circumferences for the dominant and non-dominant arms are shown, respectively. (B) The ulnar NCV of the upper arm and forearm are shown for the dominant and non-dominant arms, respectively. (C) The ulnar nCSA of upper arm and forearm are shown for the dominant and non-dominant arms, respectively. (D) The thigh and lower leg circumferences for the supporting and reacting legs are shown, respectively. (E) The tibial NCV of the thigh and lower leg are shown for the supporting and reacting legs, respectively. (F) The tibial nCSA of the thigh and lower leg are shown for the supporting and reacting legs, respectively. , †, and ns indicate repeated two-way ANOVA analysis showing the main effect of lateral dominance (preference), region and interaction. , , and †† are significantly higher values as compared with the others ( p < 0.01, **p < 0.001, ††p < 0.001, ns, not significant), respectively. December 2020 \| Volume 11 \| Article 609006 Frontiers in Physiology \| www.frontiersin.org 5 Limb-Specific Human Neuromuscular Characteristics Nobue et al. FIGURE 3 \| Relationship between the nerve and CSA for the upper and lower limbs. Semi-log plots of nCSA and the estimated CSA of each circumference are plotted for both the upper and lower limbs. no significant correlation for both dominant and non-dominant arms together, and for the dominant and non-dominant arms, respectively (Figures 4A,B). Similarly, at the lower limbs, no significant correlation was found between the tibial nerve NCV and nCSA (Figures 4C,D). In the upper limbs, positive correlations were found between the forearm nCSAs and the forearm circumferences (Figure 5B; r = 0.41, p < 0.01). Statistical Analyses In the lower limbs, a weak positive correlation was found between the lower leg nCSAs and their circumferences (r = 0.28, p < 0.05; Figure 5D). DISCUSSION Our results clearly showed that the lower limbs had higher and greater NCV, nCSA, and circumference than the upper limbs. However, NCV did not show any relationships with nCSA and circumference for both the upper and lower limbs. Unlike the upper limbs, the reacting leg had higher NCV than the supporting leg, despite the supporting leg having FIGURE 3 \| Relationship between the nerve and CSA for the upper and lower limbs. Semi-log plots of nCSA and the estimated CSA of each circumference are plotted for both the upper and lower limbs. A B C D FIGURE 4 \| Relationships between motor NCV and nCSA for the upper and lower limbs, respectively. Relationships between motor NCV and nCSA of the upper arm (A) and forearm (B) for the dominant (●) and non-dominant arms (○) as well as of the thigh (C) and lower leg (D) for the supporting (●) and reacting legs (○). A B A B D C D C FIGURE 4 \| Relationships between motor NCV and nCSA for the upper and lower limbs, respectively. Relationships between motor NCV and nCSA of the upper arm (A) and forearm (B) for the dominant (●) and non-dominant arms (○) as well as of the thigh (C) and lower leg (D) for the supporting (●) and reacting legs (○). December 2020 \| Volume 11 \| Article 609006 Frontiers in Physiology \| www.frontiersin.org 6 Limb-Specific Human Neuromuscular Characteristics Nobue et al. A B C D FIGURE 5 \| Relationships between nCSA and circumferences for the upper and lower limbs, respectively. Relationships between nCSA of the upper arm (A) and forearm (B) for the dominant (●) and non-dominant arms (○) as well as of the thigh (C) and lower leg (D) for the supporting (●) and reacting legs (○). A B B B C D C D FIGURE 5 \| Relationships between nCSA and circumferences for the upper and lower limbs, respectively. Relationships between nCSA of the upper arm (A) and forearm (B) for the dominant (●) and non-dominant arms (○) as well as of the thigh (C) and lower leg (D) for the supporting (●) and reacting legs (○). testing place of the leg muscles and nerves, which were much more distal in the previous study (abductor hallucis muscle and tibial nerve) than in the present study (soleus muscle and tibial nerve). DISCUSSION Additionally, the NCV measured in the proximal part of the upper limb can be higher than that in the distal part (Trojaborg and Sindrup, 1969). Another possibility of the conflicting result is the influence of the tested subjects. The present study had active athletes as participants; however, those in the previous study were not (mentioned as just normal subjects). The NCV of the active athletes may be developed compared with the normal healthy subjects. When taken together, our results clearly showed that the lower limbs had greater and higher nCSA and NCV than the upper limbs. However, all nCSA data of each region in both the upper and lower limbs maintain a relatively constant value to its circumference and NCV did not show any relationships with nCSA for both the upper and lower limbs. Therefore, these aspects of NCV and nCSA between the upper and lower limbs indicate the existence of limb-specific NCV characteristics and non-limb- specific nCSA developments. greater circumference than the reacting leg. Therefore, the absolute nCSA size can be related to its circumference but not necessarily function to the NCV developments for the limbs. These varying aspects between the upper and lower limbs indicate the existence of limb-specific NCV developments but not nCSA developments. Frontiers in Physiology \| www.frontiersin.org CONCLUSION Direct measurements of the human NCV and nCSA clearly showed the morphological and functional differences between the upper and lower limbs and between regions of both limbs. The different aspects between the upper and lower limbs and between regions suggest that NCV does not depend on either the nCSA sizes or circumference of both upper and lower limbs and indicate the existence of limb-specific NCV developments but not nCSA developments. DATA AVAILABILITY STATEMENT Trojaborg and Sindrup (1969) reported that NCV measured in the proximal part of upper limb was higher than that in the distal part. In this case, the nerve axon diameter may be possible to be greater in the proximal part than in the distal part due to the less nerve branching in the proximal part. However, there have not been any reports about the comparison of the nerve axon diameter at different regions. In the nCSA level of the present study, the nCSA in the proximal parts were greater than that in the distal parts for both the upper and lower limbs, depending on their circumferences (Figures 2, 5). However, the distal parts of both limbs had higher NCV than the proximal parts. These results were not in line with those of the upper limbs in the previous in vivo study (Trojaborg and Sindrup, 1969). Further examination of the region specific NCV and nerve size in both the upper and lower limbs are needed to solve this inconsistency. Taken together, the present study suggests that the nCSA at any regions in both the upper and lower limbs can be depended on their circumferences. In addition, our results imply the region-specificity in both the upper and lower limbs, where the distal parts of limbs can have higher NCV as well as thinner nCSA and vice versa than the proximal parts. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. ETHICS STATEMENT The studies involving human participants were reviewed and approved by the Human Ethics Committee of Osaka University of Health and Sport Sciences. The patients/participants provided their written informed consent to participate in this study. Lateral Preferences of Neuromuscular Features in the Upper and Lower Limbs the size of the nerve trunk (nCSA), which is a bundle of nerve fibers, using peripheral nerve ultrasonography. In this size level, nCSA could contain a variety of fiber types, not only the efferent fibers but also afferent fibers. Therefore, further considerations in the diameter level of the myelinated nerve fibers and the distinction of the efferent fibers from different fiber types of mixed peripheral nerve should be given in future studies with more detail high-resolution ultrasonography. In this study, the subject was selected with no bilateral differences of the forearm and shank length and was recruited to minimize the effects of the different distances between the nodes of Ranvier in the myelinated axons. Further validation needs to expand the various subject groups. pp As shown by the previous study (Nobue and Ishikawa, 2015), the present study confirmed that the dominant arms had greater circumference and nCSA than the non-dominant arms. Furthermore, the dominant arm had higher NCV than the non-dominant arms. Therefore, the dominant upper limb can have greater and higher nCSA and NCV than the non-dominant upper limb, respectively. In the lower limbs, however, the supporting leg had lower NCV than the reacting leg. In addition, the nCSA cannot be necessarily high in the big supporting leg compared with the small reacting leg. In the reaction movements of the lower limbs, not only the reacting but also the opposite supporting legs work as the inherent functions of supporting the body weight prior to performing movements, which requires effective coordination between both legs (Promsri et al., 2020). Meanwhile, the reaction movements of the upper limbs could be focused by testing one arm but not another arm. Thereby, unlike the upper limbs, both lower limbs can have different functions to the reaction movements. Thus, the limb-specific function to quick response movements may lead to different results of lateral preferences of neuromuscular features for both the upper and lower limbs, respectively. Limb Specificity of Neuromuscular Features As surmised by the reports of Bedewi et al. (2017, 2018), our results clearly confirmed that the lower limbs had an approximately 3.5 times greater human nCSA than the upper limbs, despite the lower limbs having approximately 1.4 times greater maximum circumference than the upper limbs. Additionally, the lower limbs NCV (59.1 ± 9.0 m s−1) was greater than that in the upper limbs (55.6 ± 4.3 m s−1). This result does not necessarily coincide with those of the previous study, which showed that the lower limbs had lower NCV (45.5 ± 3.8 m s−1) than the upper limbs (58.9 ± 2.2 m s−1; Mayer, 1963). This conflicting result could be related to the December 2020 \| Volume 11 \| Article 609006 Frontiers in Physiology \| www.frontiersin.org 7 Limb-Specific Human Neuromuscular Characteristics Nobue et al. AUTHOR CONTRIBUTIONS AN, YK, KS, HO, and MI contributed to the study concept and design. AN, KS, HO, and MI contributed to methodological developments. AN, YK, and HT carried out the experiment, the data analysis, and interpretation. AN and MI co-wrote the paper. All authors contributed to the article and approved the submitted version. REFERENCES Mayer, R. F. (1963). Nerve conduction studies in man. Neurology 13, 1021–1030. doi: 10.1212/wnl.13.12.1021 Bedewi, M. A., Abodonya, A., Kotb, M., Kamal, S., Mahmoud, G., Aldossari, K., et al. (2018). Estimation of ultrasound reference values for the lower limb peripheral nerves in adults: a cross-sectional study. Medicine 97:e0179. doi: 10.1097/MD.0000000000010179 McGrath, T. M., Waddington, G., Scarvell, J. M., Ball, N. B., Creer, R., Woods, K., et al. (2015). The effect of limb dominance on lower limb functional performance--a systematic review. J. Sports Sci. 34, 289–302. doi: 10.1080/ 02640414.2015.1050601 Nobue, A., and Ishikawa, M. (2015). Relationship between the fascia size of ulnar nerve and the nerve conduction velocity in humans. Jpn. J. Phys. Fitness Sports Med. 64, 427–434. doi: 10.7600/JSPFSM.64.427 Bedewi, M. A., Abodonya, A., Kotb, M., Mahmoud, G., Kamal, S., Alqabbani, A., et al. (2017). Estimation of ultrasound reference values for the upper limb peripheral nerves in adults: a cross-sectional study. Medicine 96:e9306. doi: 10.1097/MD.0000000000009306 Oldfield, R. C. (1971). The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia 9, 97–113. doi: 10.1016/0028-3932(71)90067-4f Edds, M. V. Jr. (1950). Hypertrophy of nerve fibers to functionally overloaded muscles. J. Comp. Neurol. 93, 259–275. doi: 10.1002/cne.900930207 Pawlak, M., and Kaczmarek, D. (2010). Field hockey players have different values of ulnar and tibial motor nerve conduction velocity than soccer and tennis players. Arch. Ital. Biol. 148, 365–376. doi: 10.4449/aib.v148i4.1208f Elias, L. J., Bryden, M. P., and Bulman-Fleming, M. B. (1998). Footedness is a better predictor than is handedness of emotional lateralization. Neuropsychologia 36, 37–43. doi: 10.1016/s0028-3932(97)00107-3 Promsri, A., Haid, T., Werner, I., and Federolf, P. (2020). Leg dominance effects on postural control when performing challenging balance exercises. Brain Sci. 10:128. doi: 10.3390/brainsci10030128 Feinstein, B., Lindegard, B., Nyman, E., and Wohlfart, G. (1955). Morphologic studies of motor units in normal human muscles. Acta Anat. 23, 127–142. doi: 10.1159/000140989 Samorajski, T., and Rolsten, C. (1975). Nerve fiber hypertrophy in posterior tibial nerves of mice in response to voluntary running activity during aging. J. Comp. Neurol. 159, 553–558. doi: 10.1002/cne.901590407 Gasser, H. S., and Grundfest, H. (1939). Axon diameters in relation to the spike dimensions and the conduction velocity in mammalian a fibers. Am. J. Phys. 127, 393–414. doi: 10.1152/ajplegacy.1939.127.2.393 Trojaborg, W., and Sindrup, E. W. (1969). Motor and sensory conduction in different segments of the radial nerve in normal subjects. J. Neurol. Neurosurg. Psychiatry 32, 354–359. FUNDING The diameter size of the peripheral nerve fiber is proximately 10–30 μmm. So far, the resolution of current in vivo human imaging technology cannot identify an axon diameter of the nerve fibers. Therefore, in the present study, we have measured This work was supported by Japan Society for Promotion of Science (KAKENHI) grant nos. 20K19499, 26702026, 15KK0261, 17H02156, and 18K10874. December 2020 \| Volume 11 \| Article 609006 Frontiers in Physiology \| www.frontiersin.org 8 Limb-Specific Human Neuromuscular Characteristics Nobue et al. Frontiers in Physiology \| www.frontiersin.org December 2020 \| Volume 11 \| Article 609006 REFERENCES doi: 10.1136/jnnp.32.4.354 Hatta, A., Nishihira, Y., Takemiya, T., Shimoda, M., and Changming, L. (1995). Ulnar nerve conduction velocity in athletes: comparison between kendo, badminton, soft tennis players and nonathletes. Adv. Exerc. Sports Physiol. 2, 177–184.i Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. t Hursh, J. B. (1939). Conduction velocity and diameter of nerve fibers. Am. J. Phys. 127, 131–139. doi: 10.1152/ajplegacy.1939.127.1.131 Kim, S., Nishihira, Y., and Hatta, A. (2009b). Changes in the peripheral motor nerve conduction velocity and its distribution in the lower limbs with long- term exercise. Adv. Exerc. Sports Physiol. 15, 95–100. Copyright © 2020 Nobue, Kunimasa, Tsuneishi, Sano, Oda and Ishikawa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Kim, S., Nishihira, Y., Higashiura, T., Hatta, A., Hayashi, Y., Hayashi, K., et al. (2009a). The effects of long-term exercise training on the motor nerve conduction velocity (MCV) in long-distance wheelchair athletes. Jpn. Society Exerc. Sports Physiol. 16, 17–24. p y Kimura, J. (2013). Electrodiagnosis in diseases of nerve and muscle: Principles and practice. 4th Edn. Oxford: Oxford University Press. December 2020 \| Volume 11 \| Article 609006 Frontiers in Physiology \| www.frontiersin.org 9
https://openalex.org/W3025947767	https://www.scielo.br/j/aabc/a/X9mCGG5JR3dqWTXPc5wjvxD/?format=pdf&lang=en	English	null	Identification of caffeic acid and rutin by UHPLC MS/MS and antioxidant activity of Commelina erecta Lineu. in cell culture	Anais da Academia Brasileira de Ciências	2,020	cc-by	5,645	Identification of caffeic acid and rutin by UHPLC MS/MS and antioxidant activity of Commelina erecta Lineu. in cell culture FELIPE A.M. OTSUKA, RODRIGO B. SANTOS, LARISSA F. CHAVES, ROSANGELA S. SANTOS, ADRIANO B. CHAVES FILHO, SAYURI MIYAMOTO & HUMBERTO R. MATOS Abstract: The Commelina erecta L. (C. erecta) also known as erva-de-santa-luzia is reported by local population to have medical properties against some pathological conditions. In this study, two extracts of C. erecta leaves (aqueous and ethanolic) were phytochemically analysed and evaluated for their in-vitro antioxidant activities by DPPH, TBARS, NO assays and cell viability assays. The ultra-high performance liquid chromatography followed by tandem mass spectrometry analysis showed the presence of rutin and caffeic acid in aqueous and ethanolic extract. The total polyphenols in aqueous and ethanolic extracts found were 142.7 ± 3.0 and 123.1 ± 5.8 μg/mL of GAE, respectively. The ethanolic extract (5 mg/mL) inhibits TBARS by 33.8%, and the aqueous extract (5 mg/mL) exhibited scavenger property against nitric oxide derivatives to an extent of 77.8%. In cell culture, both extracts improved cell survivability under H2O2 induced oxidative stress. Thus, C. erecta extract is a good candidate to become a phytotherapic medicine. Key words: Antioxidant activity, caffeic acid, Commelina erecta L., polyphenols. An Acad Bras Cienc (2020) 92(1): e20190491 DOI 10.1590/0001-3765202020190491 Anais da Academia Brasileira de Ciências \| Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc \| www.fb.com/aabcjournal An Acad Bras Cienc (2020) 92(1): e20190491 DOI 10.1590/0001-3765202020190491 Anais da Academia Brasileira de Ciências \| Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc \| www.fb.com/aabcjournal An Acad Bras Cienc (2020) 92(1): e20190491 DOI 10.1590/0001-3765202020190491 Anais da Academia Brasileira de Ciências \| Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc \| www.fb.com/aabcjournal An Acad Bras Cienc (2020) 92(1) Plant material Folin-Ciocalteau method (Singleton & Rossi 1965) of phenol assay was carried out in triplicate, read in spectrophotometer at 765 nm and results were expressed in μg/mL of gallic acid equivalent (GAE). The plants were harvested in Sergipe, city of São Cristovão, Brazil, location coordinates 10°58’35.7”S 37°15’25.8”W during September 2012. They were classified by Federal University of Sergipe, herbarium and assigned the voucher number ASE 2319. The leaves were separated and washed, then air dried at ambient temperature for 48 hours, totally to obtain 122,22 g of dried leaves to prepare aqueous and ethanolic extract. i. Aqueous extract Dried leaves (20 g) were used for extraction in a similar way to tea preparation. Briefly, infusion process started shaking dried leaves in 500 mL of warm water (70 °C) until the system reach ambient temperature (25 °C), in the meanwhile the mixture was stirred. The extract was filtered with qualitative filter paper and lyophilized in a TE-211 (Tecnal, Brazil), obtaining 1.78 g (8.9% of yielding) of extract powder. Extract was protected from light until used for analysis. Antioxidant activity of 2,2-diphenyl-1- picrylhydrazyl (DPPH) assay The assay was done following Brand-Williams method with modifications (Brand-Williams et al. 1995). Briefly, was measured through spectrophotometer the absorption of the methanolic radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution at 515 nm. The assay was conducted dissolving the crude extract in five different concentrations (1 – 5 mg/mL), then 50 µL of each extract solution was mixed with 2 mL of DPPH 90 µM solution. The assay was done in quadruplicate and each sample was read with a gap of 5 minutes for a total of 15 minutes starting from 0 minutes. Butylated hydroxytoluene (BHT) solution (1 mg/mL) was used as positive control and the results were expressed as inhibition percent (IP) of DPPH radical. The IP was calculated as following: IP = Acontrol - (Asample - Ablank)/Acontrol * 100. Where Acontrol is the absorbance of the DPPH only measured at 20 minutes, Asample is the absorbance of the DPPH + sample solution measured at 20 minutes, and Ablank is the absorbance of the solvent used. INTRODUCTION of redox balance between the production of reactive oxygen or nitrogen species and antioxidant defences cause metabolic deregulation and signalling dysfunction (Behl & Moosmann 2002) and hence diseases. Substances able to neutralize reactive species are called antioxidants, these substances can prevent the damage to living organisms caused by reactive species (Saravanan et al. 2014). The phenolic compounds for instance are known to have antioxidant and anticancer property (Scalbert et al. 2005). However, there is a great lack of chemical information about antioxidant compounds in C. erecta. The family of plants called Commelinaceae belongs to equisetopsida class, it is native of tropical America and well distributed in tropics with about 155 genus and 500 species (Mirbel 1804). Commelina erecta L. (C. erecta) in Brazil, is popularly called erva-de-santa-luzia, and has an ethnopharmacological relevance as its leaves extract finds applications against eyes infections, skin rashes, hepatic disorders, as diuretic and antirheumatic (Alonso Paz et al. 1995, Lombardo 1983). The oxidative stress is implicated in mediating several diseases, for instance, diabetes, neurodegenerative diseases (López- Alarcón et al. 2014, Shi & Pan 2012). The loss Medicinal herbs are used worldwide as part of traditional medicine to improve health and An Acad Bras Cienc (2020) 92(1) An Acad Bras Cienc (2020) 92(1) ANTIOXIDANT ACTIVITY OF Commelina erecta LINEU. FELIPE A.M. OTSUKA et al. decrease disease risk factors and therefore C. erecta leaf extracts were chosen. The goals of this study were to screen the C. erecta leaf extracts for antioxidant activity using in vitro assays, and to establish the active principle by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/ MS). extract was filtered with qualitative filter paper and roto-evaporated in a MA-120 (Marconi, Brazil), obtaining 1 g (yielding 5%) of extract powder that was protected from light. Both extracts were resuspended in their respective extraction solutions in five different concentrations (1 – 5 mg/mL) and crude extract was divided for antioxidant screening, cell viability analysis and UHPLC analysis. When necessary, extract powder was stored at -20 °C. Thiobarbituric acid reactive substances (TBARS) assay The assay was performed according to the method of Hartwig et al. (1993) with modifications to quantify lipid peroxidation. The supernatant was then read on spectrophotometer at 532 nm. The assay was conducted in triplicate and the concentration of thiobarbituric acid reactive substances (TBARS) was calculated with the molar extinction coefficient (ε = 157000 L mol−1 cm−1). ii. Ethanolic extract Dried leaves (20 g) were used for ethanolic extraction. Briefly, the leaves were shaken with 600 mL of ethanol (90%) at ambient temperature (25 °C) for 2 hours with constant mixing. The An Acad Bras Cienc (2020) 92(1) e20190491 2 \| 10 ANTIOXIDANT ACTIVITY OF Commelina erecta LINEU. FELIPE A.M. OTSUKA et al. serum (10%), streptomycin (100 µg/ml) and penicillin (100 units/mL). Cell viability by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay The cellular viability under oxidative stress by hydrogen peroxide was based on work done by Denizot & Lang (1986). Groups were divided as: Control (treated with phosphate buffer), H2O2 (Treated only with hydrogen peroxide 100 μM), Caffeic acid (treated with caffeic acid 50 μM under H2O2 treatment), and three groups of extracts called “Aq/Et Com” (aqueous or ethanolic commelina extracts: 0.25, 0.5, and 1 mg/mL) treated with H2O2. The cells L-929 were cultivated in a 96 wells culture plate with 2 × 104 cells/well, for 24 hours. Then, the tested cells were washed and incubated with the extract’s solutions done in DMEM for 2 hours and the control cells were washed and treated with phosphate buffer solution. After that, the cells were washed and incubated with 100 μM of H2O2 for 10 minutes. The cells were washed and 188 μL of DMEM plus 112 μL of 3-(4,5-301 dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide (MTT) (5 mg/mL) were added and incubated for 2 hours. The medium was removed and the precipitated (formazan) was solubilized in 150 μL of sodium dodecyl sulfate (SDS) 50% and 150 μL of dimethylformamide (pH 4.8). The assay was done in quadruplicate and read at 595 nm in spectrophotometer (LIBRA S60, Biochrom). Scavenging of nitric oxide The assay was done following Green et al. (1982) method to quantify nitric oxide derivatives (NOx, x = 2 or 3) with modifications. Briefly, a solution of sodium nitroprusside (SNP) 5 mM in phosphate buffer 0.1 M (pH = 7.0) was incubated with the extract solutions to a proportion of 10 µL of extract to 4 mL of SNP. Then, 1 mL of the Griess reagent (sulfanilamide 1% in phosphoric acid 5% and N-1-naphthyl-ethylenediamine 0.1%) was added in the mixture and the absorbance was read at 546 nm for each 30 minutes for a total of 150 minutes at 25 ºC. The concentration of the extract solutions ranged from 0.5 – 5 mg/ mL, results were expressed as IP of NOx after 150 minutes. Assay was done in quadruplicate. Cell cultures Murine fibroblasts from the NCT clone 929 strain (L929 cells), originated from adipose connective tissue was used to test the aqueous extract and human lung`s fibroblasts IMR-90 (ATCC® CCL-186™) to test the ethanolic extract to study the protective effect of the extracts against the oxidative stress mediated by H2O2. The cells were incubated at 37 °C under a humid atmosphere with 5% of CO2 and cultivated in a modified media Dulbecco with Eagle medium (DMEM), glucose (1000 mg/L), L-glutamine (1000 mg/L), sodium bicarbonate (3700 mg/L), fetal bovine Statistical analyses and the samples were centrifuged at 2500 g for 10 min. The supernatant was filtered with a 0.22 µm Durapore® (PVDF) and 5 µL were injected in the UHPLC/ ESI-TOFMS system. Student’s t-test and ANOVA followed by Bonferroni post-test were used to evaluate the significant difference among the means of the different concentrations and the controls, analysis was performed using GraphPad Prism version 5.0 for Windows. The results were expressed as mean ± standard deviation. Linear correlation coefficient (Pearson product- moment) was calculated to measure linearity between two variables. Different statistical significance was defined as p<0.05. The chemical characterization was performed by ESI-TOFMS (Triple TOF 6600, Sciex, Concord, US) interfaced with an ultra-high performance liquid chromatography (UHPLC Nexera, Shimadzu, Kyoto, Japan). The samples were loaded into a Phenomenex Kinetex® (C18 column, 2.6 µm, 2.1 mm i.d. x 100 mm) with a flow rate of 0.3 mL min-1 and the oven temperature maintained at 25 °C. For reverse- phase LC, mobile phase A consisted of water, while mobile phase B composed of acetonitrile. Mobile phases A and B contained formic acid (at a final concentration of 0.05%) for experiment performed in negative ionization mode. The linear gradient during analysis was as follows: from 10 to 36.7% B over the first 5 min., from 36.7 to 90% B during 5-9 min, hold at 90% B from 9-14 min., decreased from 90 to 10% B during 14-15 min., and hold at 10% B from 15-20 min. UHPLC-MS/MS analysis Aqueous extract powder was resuspended using 100 mg of extract per mL of water and pre-treated with a clean-up method described by Vinson et al. (2001) with modifications. Water soluble phenols were determined using 0.1 mL of extract solution in a 0.5 mL mixture containing 50% methanol/water. After an extraction time of 3 hours under 90 °C, 1 mL of methanol was added An Acad Bras Cienc (2020) 92(1) e20190491 3 \| 10 ANTIOXIDANT ACTIVITY OF Commelina erecta LINEU. FELIPE A.M. OTSUKA et al. RESULTS AND DISCUSSION Many studies involving polyphenols have shown their antioxidant potential, therefore these compounds can modulate positively against some deleterious processes such as hepatical cirrhosis and cancer (Morry et al. 2017). Table I represents the screening of antioxidant role of both extracts (aqueous and ethanolic). The concentration of polyphenols expressed as gallic acid equivalents (GAE) of the aqueous and ethanolic extract for the tested concentrations (1 – 5 mg/mL) increased from 29.7 ± 0.6 to 142.7 ± 3.0 µg/mL linearly (r = 0.998) and 45.4 ± 0.7 to 123.1 ± 5.8 µg/mL (r = 0.998) of GAE respectively. Phenolic compounds, which include a broad class of known antioxidants such as flavonoids and phenolic acids, perform their action through reduction of several oxidizing agents. Thus, the total phenolic content of an extract is an estimative of its reducing power against free radicals, which mechanism of action relies on the donation of the hydrogen from the phenol group, and self-stabilization through the aromaticity (Yehye et al. 2015). The MS was operated in negative ionization mode, and the scan range set at a mass-to- charge ratio of 100-1000 Da. Data acquisition using Analyst® 1.7.1 was performed with 100 ms acquisition time for MS1 scan and 25 ms acquisition time to obtain the MS2 of rutin and caffeic acid. An ion spray voltage of -4.5 kV and the cone voltage at -80 V were set to analysis. Additional parameters included curtain gas set at 25 psi, nebulizer and heater gases at 50 psi and interface heater of 450 °C. The post-acquisition MRM-like data was used for quantification of rutin [M-H]- (m/z 609.1 → 300.0276) and caffeic acid [M-H] - (m/z 179.0 → 135.0452). The collision energies used for each compound were -45 eV for rutin and -20 eV for caffeic acid. The MS/MS data was analyzed with PeakView®. Regarding the DPPH scavenging, both extracts were able to reduce DPPH formation in a concentration dependent manner, but An Acad Bras Cienc (2020) 92(1) e20190491 4 \| 10 ANTIOXIDANT ACTIVITY OF Commelina erecta LINEU. FELIPE A.M. OTSUKA et al. Table I. Antioxidant activity screening of the aqueous and ethanolic extract from C. erecta leaves. RESULTS AND DISCUSSION Sample Concentration Total polyphenols (μg/mL of GAE) DPPH Inhibition Percent (%) TBARS (μM) Aqueous Ethanolic Aqueous Ethanolic BHT Aqueous Ethanolic BHT 1 mg/mL 2 mg/mL 3 mg/mL 4 mg/mL 5 mg/mL 29.7 ± 0.6 63.1 ± 2.6 91.3 ± 14.7 110.8 ± 2.0 142.7 ± 3.0 45.4 ± 0.7 53.0 ± 6.8 77.8 ± 7.2 119.7 ± 7.4 123.1 ± 5.8 0 0.7 ± 0.2 1.3 ± 0.4 2.4 ± 0.8 5.4 ± 0.7 0 0.6 ± 0.3 1.2 ± 0.5 2.0 ± 0.5 4.6 ± 0.8 43.0 - - - - 5.7 ± 0.1 5.9 ± 0.1 5.9 ± 0.1 5.9 ± 0.2 5.8 ± 0.1 3.5 ± 0.2 3.5 ± 0.2 3.4 ± 0.3 3.3 ± 0.2 3.0 ± 0.1 3.4 ± 0.1* - - - - Linear coefficient (r) 0.993 0.932 0.935 0.933 - - - - Data are mean measurements ± standard deviation, n = 4. Butylated hydroxytoluene (BHT) was used as a reference antioxidant. In TBARS assay * means statistical difference between aqueous extract and BHT using t-student, p<0.05. Table I. Antioxidant activity screening of the aqueous and ethanolic extract from C. erecta leaves. Data are mean measurements ± standard deviation, n = 4. Butylated hydroxytoluene (BHT) was used as a reference antioxidant. In TBARS assay * means statistical difference between aqueous extract and BHT using t-student, p<0.05. each of the tested concentrations possess the same potential to inhibit TBARS as the BHT control group. On the other hand, the aqueous extract did not change the TBARS levels in this assay. Aqueous extracts of plants may do not provide reliable TBARS measurements because the egg yolk is a lipid based matrix, so the phenolic compounds extracted by water are not lipophilic enough to protect against the lipid peroxidation of the egg yolk. As the ethanolic extraction contains lipophilic polyphenols that works better in lipid environment, such as lipid membranes, was seen a decrease in TBARS concentration. The advantage of lipophilic extracts in this assay has been discussed as the “polar paradox”, which happen in trials to measure the oxidative damage of an emulsion system composed by oil : water (like the TBARS assay) plus polar antioxidants from aqueous extract that will be less effective than non-polar antioxidants from ethanolic extracts to prevent oxidative damage (Koleva et al. 2002). An Acad Bras Cienc (2020) 92(1) e20190491 5 \| 10 RESULTS AND DISCUSSION were less efficient (IP = ~5% for the higher concentration) compared to the control BHT (IP = ~43%), a well know antioxidant (Yehye et al. 2015). Evaluation of antioxidant activity by measuring DDPH absorbance is widely used, mainly on plant extracts to predict the respective free radical scavenger potential (Conforti et al. 2002). Chemical substances may perform antioxidant activity by different ways, such as single electron transfer (Nimse & Pal 2015), metal ion-chelation activity (De Souza & De Giovani 2004) and scavenging of peroxyl radicals (Sies & Stahl 1995). The low efficiency observed in DPPH scavenging assay may came from the inability of such compounds current in the extract perform their optimal antioxidant activity, which depend on the chemical structure and solution properties (pH, solvent dieletric constant) (Cerón-Carrasco et al. 2010; Medina et al. 2013). Finally, mimicking the lipid peroxidation throughout the MDA production by fatty acids oxidation within membrane cells (Janero 1990), the response against TBARS with the extracts were tested using the egg yolk as lipid matrix. The ethanolic extract did not show statistical difference from BHT group, so we presume that The results of the NO (nitric oxide) scavenger activity of extracts through inhibition of NOx (nitric oxide derivatives) are shown in Table II. According to the data, both extracts possessed high inhibition potential against An Acad Bras Cienc (2020) 92(1) e20190491 5 \| 10 ANTIOXIDANT ACTIVITY OF Commelina erecta LINEU. FELIPE A.M. OTSUKA et al. NOX formation and increased the inhibition as extract concentration rises. The maximum IP reached for ethanolic extract (5 mg/mL) was 85.6% ± 1.9 and the aqueous extract (5 mg/ mL) reached 77.8% ± 2.5 after 150 minutes. We could not find any report of C. erecta extracts on the evaluation of NO inhibition. Since NO is an important modulator of inflammatory process and participates in cytotoxicity pathways mediated by bacteria (Cerqueira & Yoshida 2002), the efficient NO scavenging by the tested extracts is suggestive of their anti-inflammatory and antioxidant activity potentials. hydrogen peroxide is a model where reactive oxygen species (ROS) are formed in excess, decreasing cell viability, because H2O2 is a ROS and it reacts with biomolecules such as lipid membranes, proteins and DNA, resulting on cell growth impairment by stopping the replication process of the DNA and activating apoptosis pathways (Redza-Dutordoir & Averill-Bates 2016, Soria-Valles et al. 2015). Figure 2 shows the UHPLC-MS/MS results. RESULTS AND DISCUSSION Comparing peaks retention times (RT) with standards it was possible to identify caffeic acid (a) and rutin (b). Caffeic acid showed higher signal intensity in ethanolic extract. More detailed characterization of caffeic acid and rutin availability in Commelina erecta extract is shown in MS/MS analysis of the extracts. Fragmentation of the peak corresponding to caffeic acid (m/z 179.0) displayed a fragment corresponding to the loss of CO2 at m/z 135.0452. For rutin (m/z 609.1), the main fragment at m/z 300.0287 was derived from the fragmentation of the glycosidic bond. The chromatogram profile (Fig. 2C) showed 2 clear peaks one at RT=2.9 min and the other at RT=4.1 min in the channel corresponding to caffeic acid transition (m/z = To evaluate the antioxidant activity in cell culture the MTT method was used with L-929 and IMR-90 cells under oxidative stress induced by hydrogen peroxide (Denizot & Lang 1986), that indirectly measures the cell viability through the conversion of the yellow MTT to an insoluble formazan salt by mitochondria. As shown in the Figure 1, both the extracts protected against the oxidative stress induced by hydrogen peroxide (H2O2) by increasing the survivability of cells compared with the non-treated cells, presumably by antioxidant mechanisms like free radical scavenger. Cell damage induced by Table II. Inhibitory effect of the ethanolic and aqueous extract from C. erecta leaves on nitric oxide scavenger assay. Aqueous extract Ethanolic extract Extract concentration (mg/mL) IP NOX concentration (µM) IP NOX concentration (µM) 0.5 51.6 ± 5.3 8.7 ± 1.9* 62.8 ± 6.6 6.4 ± 1.7 1 55.9 ± 7.6 7.9 ± 1.5* 74.3 ± 6.9 4.5 ± 1.2 2.5 64.2 ± 3.8 6.4 ± 0.7* 83.4 ± 3.3 2.9 ± 0.6** 5 77.8 ± 2.5 4.0 ± 0.5* 85.6 ± 1.9 2.5 ± 0.5** Water - 17.9 ± 0.7 - - Ethanol - - 52.3 ± 10.7 8.3 ± 2.5 Data of concentration are presented as mean ± standard deviation n = 4 after 150 minutes of reaction. * means statistically different between Water control group vs Aqueous extract group using t-Student p<0.05 and ** means statistically different between Ethanol control group vs Ethanolic extract group using t-Student p<0.05. itory effect of the ethanolic and aqueous extract from C. erecta leaves on nitric oxide scavenger Table II. Inhibitory effect of the ethanolic and aqueous extract from C. erecta leaves on nitric ox assay. Data of concentration are presented as mean ± standard deviation n = 4 after 150 minutes of reaction. * means statistically different between Water control group vs Aqueous extract group using t-Student p<0.05 and ** means statistically different between Ethanol control group vs Ethanolic extract group using t-Student p<0.05. RESULTS AND DISCUSSION Data of concentration are presented as mean ± standard deviation n = 4 after 150 minutes of reaction. * means statistically different between Water control group vs Aqueous extract group using t-Student p<0.05 and ** means statistically different between Ethanol control group vs Ethanolic extract group using t-Student p<0.05. Data of concentration are presented as mean ± standard deviation n = 4 after 150 minutes of reaction. * means statistically different between Water control group vs Aqueous extract group using t-Student p<0.05 and ** means statistically different between Ethanol control group vs Ethanolic extract group using t-Student p<0.05. An Acad Bras Cienc (2020) 92(1) e20190491 6 \| 10 ANTIOXIDANT ACTIVITY OF Commelina erecta LINEU. FELIPE A.M. OTSUKA et al. Figure 1. Cell viability by MTT assay in L-929 cells in presence of the aqueous extract of C. erecta leaves (a) and ethanolic extract treatment in IMR-90 cells (b). Control group = no treatment (just phosphate buffer), H2O2 group = treated with H2O2 100 µM, Caffeic acid group = treated with caffeic acid (50 µM) plus H2O2, Aq and Et Com groups = treated with aqueous and ethanol extract (0.25, 0.5 and 1 mg/mL) plus H2O2. * Statistically different between control group p<0.05 and ** statistically difference between H2O2 group p<0.05. For multiple comparisons ANOVA followed by Bonferroni’s test was applied. Figure 1. Cell viability by MTT assay in L-929 cells in presence of the aqueous extract of C. erecta leaves (a) and ethanolic extract treatment in IMR-90 cells (b). Control group = no treatment (just phosphate buffer), H2O2 group = treated with H2O2 100 µM, Caffeic acid group = treated with caffeic acid (50 µM) plus H2O2, Aq and Et Com groups = treated with aqueous and ethanol extract (0.25, 0.5 and 1 mg/mL) plus H2O2. * Statistically different between control group p<0.05 and ** statistically difference between H2O2 group p<0.05. For multiple comparisons ANOVA followed by Bonferroni’s test was applied. 179.0 → 135.04) suggesting the presence of an unknown molecule similar to caffeic acid (m/z = 135.04) but more lipophilic. Additionally, for the channel monitoring rutin (m/z = 609.1→ 300.0276), the chromatogram profile showed a single peak at RT=3.8 min. Interestingly rutin intensity was greater in aqueous compared to ethanolic extract. by either reactive oxygen and nitrogen species (Lee et al. 2016). RESULTS AND DISCUSSION Caffeic acid has been reported with antinistrosative effects in Alzheimer disease and other inflammatory models (Mehrotra et al. 2011; Wang et al. 2016). The extracts tested were remarkable by effectively increasing cell viability under damage by ROS and combat NO toxicity because of the presence of polyphenols. The ability of the C. erecta extracts as an antioxidant and as an antinitrosative agent is attributed to the presence of caffeic acid (Kono et al. 1995; Pinto et al. 2015). It is assumed that C. erecta extracts possess antioxidant activity primarily against NOx, the oxidized state from the free radical nitric oxide (NO) (Green et al. 1982), because the presence of caffeic acid, based on results of Table II and Figure 2. The suggested mechanism by how caffeic acid inhibits NO is not totally understood, but was shown that nitration reaction can happen at the ring and in the propanoate double bond of caffeic acid (D’ischia 2005). Recent studies have demonstrated important role of antioxidants protecting cells against oxidative stress induced An Acad Bras Cienc (2020) 92(1) e20190491 7 \| 10 CONCLUSIONS As many plants are accepted by population for medical uses, a study about their potential chemicals and their role as antioxidants is crucial for rational use. The study demonstrates An Acad Bras Cienc (2020) 92(1) e20190491 7 \| 10 ANTIOXIDANT ACTIVITY OF Commelina erecta LINEU. FELIPE A.M. OTSUKA et al. Figure 2. Mass spectra and chromatogram profile of Caffeic acid in the channel m/z 179.0 → 135.0452 (a) and Rutin in the channel m/z 609.1 → 300.0276 (b) identified in C. erecta extracts (c). Liquid chromatography run conditions were: column Phenomenex Kinetex® (C18 column, 2.6 µm, 2.1 mm i.d. x 100 mm) with a flow rate of 0.3 mL min-1 and the oven temperature maintained at 25 °C. The collision energies used for each compound were -45 eV for rutin and -20 eV for caffeic acid. Figure 2. Mass spectra and chromatogram profile of Caffeic acid in the channel m/z 179.0 → 135.0452 (a) and Rutin in the channel m/z 609.1 → 300.0276 (b) identified in C. erecta extracts (c). Liquid chromatography run conditions were: column Phenomenex Kinetex® (C18 column, 2.6 µm, 2.1 mm i.d. x 100 mm) with a flow rate of 0.3 mL min-1 and the oven temperature maintained at 25 °C. The collision energies used for each compound were -45 eV for rutin and -20 eV for caffeic acid. that both extracts showed antioxidant activity against oxidative stress and NOx screening assays. Further the treatment of hydrogen peroxide challenged cell cultures with C. erecta extracts raised the cell viability. The chemical characterization of the C. erecta extracts showed the presence of caffeic acid even in different run conditions and other substances not yet characterized. Thus, this plant has antioxidant potential to minimize risk associated with diseases that are mediated by oxidative stress. REFERENCES activity: a comparative study on three testing methods. Phytochem Anal 13: 8-17. ALONSO PAZ E, CERDEIRAS MP, FERNANDEZ J, FERREIRA F, MOYNA P, SOUBES M, VÁZQUEZ A, VERO S & ZUNINO L. 1995. Screening of Uruguayan medicinal plants for antimicrobial activity. J Ethnopharmacol 45: 67-70. ALONSO PAZ E, CERDEIRAS MP, FERNANDEZ J, FERREIRA F, MOYNA P, SOUBES M, VÁZQUEZ A, VERO S & ZUNINO L. 1995. Screening of Uruguayan medicinal plants for antimicrobial activity. J Ethnopharmacol 45: 67-70. KONO Y, SHIBATA H, KODAMA Y & SAWA Y. 1995. The suppression of the N-nitrosating reaction by chlorogenic acid. Biochem J 312: 947-953. LEE CT, YU LE & WANG JY. 2016. Nitroxide antioxidant as a potential strategy to attenuate the oxidative/nitrosative stress induced by hydrogen peroxide plus nitric oxide in cultured neurons. Nitric Oxide 54: 38-50. BEHL C & MOOSMANN B. 2002. Oxidative nerve cell death in Alzheimer’s disease and stroke: Antioxidants as neuroprotective compounds. Biol Chem 383: 521-536. BRAND-WILLIAMS W, CUVELIER ME & BERSET C. 1995. Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci Technol 28: 25-30. LOMBARDO A. 1983. Flora Montevidensis. [s.l.] Intendencia Municipal de Montevideo. LÓPEZ-ALARCÓN C, ARENAS A, LISSI E & SILVA E. 2014. The role of protein-derived free radicals as intermediaries of oxidative processes. Biomol Concepts 5: 119-130. CERÓN-CARRASCO JP, BASTIDA A, REQUENA A, ZÚÑIGA J & MIGUEL B. 2010. A theoretical study of the reaction of β-carotene with the nitrogen dioxide radical in solution. J Phys Chem B 114: 4366-4372. MEDINA ME, IUGA C & ALVAREZ-IDABOY JR. 2013. Antioxidant activity of propyl gallate in aqueous and lipid media: A theoretical study. Phys Chem Chem Phys 15: 13137-13146. CERQUEIRA NF & YOSHIDA WB. 2002. Óxido nítrico: revisão. Act Cir Bras 17: 417-423. MEHROTRA A, SHANBHAG R, RAO-CHAMALLAMUDI M, SINGH VP & MUDGAL J. 2011. Ameliorative effect of caffeic acid against inflammatory pain in rodents. Eur J Pharmacol 666: 80-86. CONFORTI F, STATTI GA, TUNDI R, MENICHINI F & HOUGHTON P. 2002. Antioxidant activity of methanolic extract of Hypericum triquetrifolium Turra aerial part. Fitoterapia 73: 479-483. MIRBEL CFBD. 1804. Histoire naturelle, générale et particulière, des plantes. F. Dufart, v. 8, Paris, p. 177. DENIZOT F & LANG R. 1986. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89: 271-277. MORRY J, NGAMCHERDTRAKUL W & YANTASEE W. 2017. Acknowledgments We thank Osvaldo Andrade Santos of the Universidade Federal de Sergipe and Perinto Calafange, for kindly provided the Commelina erecta samples for analysis, and Lydia F. Yamaguchi and Prof. Masuo J. Kato from the Institute of Chemistry, University of Sao Paulo for providing caffeic acid and rutin standards. The authors are grateful for the technical and financial support from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Instituto Nacional de Ciência e Tecnologia de Processos Redox em Biomedicina (INCT REDOXOMA) - REDOXOMA). An Acad Bras Cienc (2020) 92(1) e20190491 8 \| 10 ANTIOXIDANT ACTIVITY OF Commelina erecta LINEU. FELIPE A.M. OTSUKA et al. Correspondence to: Humberto Reis Matos E-mail: hrmatos10@hotmail.com Correspondence to: Humberto Reis Matos E-mail: hrmatos10@hotmail.com REFERENCES Marechal Rondon, s/n, Rosa Elze, 49100-000 São Cristovão, SE, Brazil 2University of São Paulo, Department of Biochemistry, Institute of Chemistry, Av. Prof. Lineu Prestes, 748, Butantã, 05508-900 São Paulo, SP, Brazil SHI YC & PAN TM. 2012. Red mold, diabetes, and oxidative stress: A review. Appl Microbiol Biot 94: 47-55. SIES H & STAHL W. 1995. Vitamins E and C, beta-carotene, and other carotenoids as antioxidants. Am J Clin Nutr 62: 1315S-1321S. SINGLETON VL & ROSSI JA. 1965. Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am J Enol Vit 16: 144-158. SORIA-VALLES C, OSORIO FG, GUTIÉRREZ-FERNÁNDEZ A, DE LOS ANGELES A, BUENO C, MENÉNDEZ P, MARTÍN-SUBERO JI, DALEY GQ, FREIJE JM & LÓPEZ-OTÍN C. 2015. NF-κB activation impairs somatic cell reprogramming in ageing. Nat Cell Biol 17: 1004-1013. VINSON JA, SU X, ZUBIK L & BOSE P. 2001. Phenol antioxidant quantity and quality in foods: fruits. J Agric Food Chem 49: 5315-5321. WANG Y, WANG Y, LI J, HUA L, HAN B, ZHANG Y, YANG X, ZENG Z, BAI H, YIN H & LOU J. 2016. Effects of caffeic acid on learning deficits in a model of Alzheimer’s disease. Int J Mol Med 38: 869-875. 2University of São Paulo, Department of Biochemistry, Institute of Chemistry, Av. Prof. Lineu Prestes, 748, Butantã, 05508-900 São Paulo, SP, Brazil YEHYE WA, RAHMAN NA, ARIFFIN A, ABD-HAMID SB, ALHADI AA, KADIR FA & YAEGHOOBI M. 2015. Understanding the chemistry behind the antioxidant activities of butylated hydroxytoluene (BHT): A review. Eur J Med Chem 101: 295-312. An Acad Bras Cienc (2020) 92(1) e20190491 10 \| 10 REFERENCES Oxidative stress in cancer and fibrosis: Opportunity for therapeutic intervention with antioxidant compounds, enzymes, and nanoparticles. Redox Biol 11: 240-253. DE SOUZA RFV & DE GIOVANI WF. 2004. Antioxidant properties of complexes of flavonoids with metal ions. Redox Rep 9: 97-104. NIMSE SB & PAL D. 2015. Free radicals, natural antioxidants, and their reaction mechanisms. RSC Adv 5: 27986-28006. D’ISCHIA M. 2005. Nitrosation and nitration of bioactive molecules: Toward the basis of disease and its prevention. CR CHIM 8: 797-806. PINTO IFD, SILVA RP, CHAVES-FILHO AB, DANTAS LS, BISPO VS, MATOS IA, OTSUKA FAM, SANTOS AC & MATOS HR. 2015. Study of antiglycation, hypoglycemic, and nephroprotective activities of the green dwarf variety coconut water ( Cocos nucifera L.) in alloxan-induced diabetic rats. J Med Food 18: 802-809. GREEN LC, WAGNER DA, GLOGOWSKI J, SKIPPER PL, WISHNOK JS & TANNENBAUM SR. 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 126: 131-138. HARTWIG A, KLYSZCZ-NASKO H, SCHLEPEGRELL R & BEYERSMANN D. 1993. Cellular damage by ferric nitrilotriacetate and ferric citrate in V79 cells: interrelationship between lipid peroxidation, DNA strand breaks and sister chromatid exchanges. Carcinogenesis 14: 107-112. REDZA-DUTORDOIR M & AVERILL-BATES DA. 2016. Activation of apoptosis signalling pathways by reactive oxygen species. BBA - Mol Cell Res 1863: 2977-2992. SARAVANAN S, ARUNACHALAM K & PARIMELAZHAGAN T. 2014. Antioxidant, analgesic, anti-inflammatory and antipyretic effects of polyphenols from Passiflora subpeltata leaves - A promising species of Passiflora. Ind Crop Prod 54: 272-280. JANERO DR. 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 9: 515-540. JANERO DR. 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 9: 515-540. SCALBERT A, JOHNSON IT & SALTMARSH M. 2005. Polyphenols: antioxidants and beyond. Am J Clin Nutr 81: 215S-217S. KOLEVA II, VAN-BEEK TA, LINSSEN JP, DE GROOT A & EVSTATIEVA LN. 2002. Screening of plant extracts for antioxidant An Acad Bras Cienc (2020) 92(1) e20190491 9 \| 10 FELIPE A.M. OTSUKA et al. ANTIOXIDANT ACTIVITY OF Commelina erecta LINEU. FELIPE A.M. OTSUKA1 https://orcid.org/0000-0003-1868-751X RODRIGO B. SANTOS1 https://orcid.org/0000-0003-1677-9584 LARISSA F. CHAVES1 https://orcid.org/0000-0002-0484-0600 ROSANGELA S. SANTOS1 https://orcid.org/0000-0003-3546-2873 ADRIANO B. CHAVES FILHO2 https://orcid.org/0000-0002-2321-6504 SAYURI MIYAMOTO2 https://orcid.org/0000-0002-5714-8984 HUMBERTO R. MATOS1 https://orcid.org/0000-0003-3898-9515 1Federal University of Sergipe, Department of Physiology, Av. Author contributions How to cite OTSUKA FAM, SANTOS RB, CHAVES LF, SANTOS RS, CHAVES FILHO AB, MIYAMOTO S & MATOS HR. 2020. Identification of caffeic acid and rutin by UHPLC MS/MS and antioxidant activity of Commelina erecta Lineu. in cell culture. An Acad Bras Cienc 92: e20190491. DOI 10.1590/0001- 3765202020190491. Otsuka FAM, Filho ABC and Matos HR designed the research. Filho ABC, Santos RB, Santos RS, Chaves LF and Miyamoto S characterized the phytochemical species. Otsuka FAM and Filho ABC did the antioxidant assays. Otsuka FAM, Santos RS and Matos HR wrote and contributed in reviewing the manuscript. Manuscript received on April 26, 2019; accepted for publication on June 11, 2019 An Acad Bras Cienc (2020) 92(1) e20190491 10 \| 10 An Acad Bras Cienc (2020) 92(1) e20190491 10 \| 10
https://openalex.org/W4384115369	https://zenodo.org/records/8142723/files/TAI...%D0%98%D0%A1%D0%A1%D0%98%D2%9A%D0%9B%D0%98%D0%9A%20%D0%A2%D0%90%D0%AA%D0%9C%D0%98%D0%9D%D0%9E%D0%A2%D0%98%20%D0%A2%D0%98%D0%97%D0%98%D0%9C%D0%9B%D0%90%D0%A0%D0%98%D0%94%D0%90%20%D0%AF%D0%A1%D0%A1%D0%98%20%D2%9A%D0%A3%D0%81%D0%A8%20%20%20%D0%9A%D0%9E%D0%9B%D0%9B%D0%95%D0%9A%D0%A2%D0%9E%D0%A0%D0%9B%D0%90%D0%A0%D0%98%D0%9D%D0%98%D0%9D%D0%93%20%D0%98%D0%A8%D0%9B%D0%90%D0%A8%20%D0%9F%D0%90%D0%A0%D0%90%D0%9C%D0%95%D0%A2%D0%A0%D0%9B%D0%90%D0%A0%D0%98.pdf	Russian	null	ИССИҚЛИК ТАЪМИНОТИ ТИЗИМЛАРИДА ЯССИ ҚУЁШ КОЛЛЕКТОРЛАРИНИНГ ИШЛАШ ПАРАМЕТРЛАРИ	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,014	TECHNIQUE AND IT. JOURNAL 2023 TECHNIQUE AND IT. JOURNAL 2023 TECHNIQUE AND IT. JOURNAL 2023 А.Даулетбаева, Р.Айтбаев Аннотация: Мазкур мақолада иссиқлик таъминоти тизимларида ясси қуёш коллекторларининг ишлаш параметрлари бугинги кундаги давлат даражасидаги долзарб масалалардан бири ҳисобланади. Айниқса, бу муаммоларни ҳал этишда уларни оптималлаштириш алоҳида аҳамият касб этиши айтилади Калит сўзлар: иссиқлик таъминоти, ясси қуёш коллекторлари, қуёш энергиясидан, сунъий насосли циркуляцияли қуёшли иссиқлик таъминоти, қайта тикланувчи энергия, Иссиқлик таъминоти тизимларида Жаҳонда қуёш энергиясидан амалий фойдаланишнинг энг устувор йўналишларидан бири – қуёшли иссиқлик таъминоти тизимларидир. Ривожланган мамлакатларда, жумладан АҚШ, Ҳитой, Ҳиндистон, Япония, Германия, Швеция ва Россия каби давлатларда, қуёш энергиясидан фойдаланиш соҳасидаги илмий тадқиқотларда юқори иқтисодий самарадорликка эга, арзон ва содда қуёш коллекторлари ва тизимларини яратиш, уларнинг нархи ва улардан фойдаланиш вақтида сарф харажатларини пасайтириш масалалари етакчи ўрин эгалламоқда. Бу борада иссиқлик таъминоти тизимларида ясси қуёш коллекторларининг ишлаш параметрларини оптималлаштириш орқали уларнинг самарадорлигини ошириш муҳим аҳамият касб этади. Жаҳоннинг етакчи илмий марказларида қуёшли иссиқлик таъминоти тизимларини такомиллаштиришга, хусусан ясси қуёш коллекторларининг ишлаш параметрларини оптималлаштириш орқали уларнинг самарадорли- TECHNIQUE AND IT. JOURNAL 2023 TECHNIQUE AND IT. JOURNAL 2023 гини оширишни янги усулларни яратишга қаратилган илмий тадқиқотлар олиб борилмоқда. Ушбу йўналишда бир ва икки контурли, табиий термосифон ва сунъий насосли циркуляцияли қуёшли иссиқлик таъминоти тизимларининг қуёш контурида иссиқлик ташувчисини кичик сарф билан ишлаши ва талаб этилган хароратгача бир марта қиздириш ҳисобига энг мақбул режими таъминлаш орқали қуёш коллекторларининг самарадорли- гини ошириш жараёнларини моделлаштириш бўйича тадқиқотлар устивор ҳисобланмоқда. Шу билан бирга, қуёшли иссиқлик таъминоти тизимларида қуёш радиациясини стационар бўлмаган келиб тушиш шароитда табиий термосифон циркуляция ҳаракатининг моделини ва қуёш коллекторларининг иссиқлик ишлаб чиқариш унумдорлигини аниқлаш усулини такомиллаш- тириш долзарб вазифалардан бири деб ҳисобланмоқда. Республикамизда қайта тикланувчи энергия манбаларидан кенг фойда- ланиш ва самарадорлигини оширишга ёрдам берадиган янги технология- ларни яратиш бўйича тадқиқотлар ўтказиш ва уларни амалда қўллаш бўйича кенг кўламли чора-тадбирлар амалга оширилмоқда. 2022-2026 йилларга мўлжалланган Янги Ўзбекистоннинг тараққиёт стратегиясида, жумладан «Иқтисодиётни электр энергияси билан узлуксиз таъминлаш ҳамда “Яшил иқтисодиёт” технологияларини барча соҳаларга фаол жорий этиш, иқтисоди- ётнинг энергия самарадорлигини 20 фоизга ошириш»1 бўйича вазифалари белгиланган. Ушбу вазифаларни амалга оширишда, хусусан, қайта тик- ланувчи энергия манбалари асосида янги ишлаб чиқариш қувватларини қу- риш ва мавжудларини модернизация қилиш, жумладан иссиқлик таъминоти тизимларида ясси қуёш коллекторларининг ишлаш параметрларини оптимал- лаштириш орқали уларнинг самарадорлигини ошириш муҳим ҳисобланади. Иссиқлик таъминоти тизимларида ясси қуёш коллекторларини ишлаш Иссиқлик таъминоти тизимларида ясси қуёш коллекторларини ишлаш 2 TECHNIQUE AND IT. JOURNAL 2023 TECHNIQUE AND IT. TECHNIQUE AND IT. JOURNAL 2023 А.Даулетбаева, Р.Айтбаев JOURNAL 2023 параметрларини оптималлаштириш орқали уларнинг самарадорлигини оширишга йўналтирилган тадқиқотлар жахоннинг етакчи олий таълим муассасалари ва илмий марказларида, хусусан, University of Wisconsin– Madison (АҚШ), Shanghai jiao Tong University (Хитой халқ Республикаси), Indian Institute of Technology Dehli (Ҳиндистон), Indian Institute of Technology Roorkee (Ҳиндистон), National Institute of Technology Hamirpur (Ҳиндистон), Tamkang Unversitiy (Тайвань), Unversitiy of Tanta (Миср), Universiti Kebangsaan Malaysia (Малайзия), Alternate Hydro Energy Centre (Ҳиндистон), Xi’an jiaotong University (Хитой халқ Республикаси), , King Mongkut’s University of Technology Thonbure (Тайланд), University of Tokyo (Япония), “МЭИ” Миллий тадкикот университетида (Россия Федерацияси), Россия Фанлар Академияси Сибирь бўлимининг иссиқлик физикаси илмий текшириш институтида, Ўзбекистон Фанлар Академиясининг «Физика- Қуёш» ИИЧБсида (Ўзбекистон Республи- каси), Тошкент архитектура- қурилиш институтида (Ўзбекистон Республика- си) ва Фарғона политехника институтида (Ўзбекистон Республикаси) кенг микёсларда олиб борилмоқда. Дунё амалиётида қуёшли иситиш тизимларини лойиҳалаш ва гелио қурилмаларининг такомиллаштирилган конструкцияларини ҳисоблаш ва уларда амалга ошириладиган гидродинамик хамда иссиклик жараёнларини жадаллаштириш усуллари ишлаб чикилган. Қуёшли cув қиздиргичларнинг самарадорлигини ошириш хусусиятларини ўрганиш бир қатор олимлар, хусусан Дж.А. Даффи, У.А. Бекман, С.А. Кляйн, Г.О. Лёф, Х.С. Хоттел, Дж. Твайделл, А.Уэйр ва бошқаларнинг ишларида кўриб чиқилган. Бу ишларда қуёшли сув иситгичи қурилмаларининг конструктив ва режим параметрла- рини оптималлаштириш ва самарадорлигига боғлиқлиги ўрганилган. С.И. Смирнов, О.С. Попель, С.Е. Фрид, В.И. Виссарионов, В.А. Бутузов, М.Д. Дунё амалиётида қуёшли иситиш тизимларини лойиҳалаш ва гелио қурилмаларининг такомиллаштирилган конструкцияларини ҳисоблаш ва уларда амалга ошириладиган гидродинамик хамда иссиклик жараёнларини жадаллаштириш усуллари ишлаб чикилган. Қуёшли cув қиздиргичларнинг самарадорлигини ошириш хусусиятларини ўрганиш бир қатор олимлар, хусусан Дж.А. Даффи, У.А. Бекман, С.А. Кляйн, Г.О. Лёф, Х.С. Хоттел, Дж. Твайделл, А.Уэйр ва бошқаларнинг ишларида кўриб чиқилган. Бу ишларда қуёшли сув иситгичи қурилмаларининг конструктив ва режим параметрла- рини оптималлаштириш ва самарадорлигига боғлиқлиги ўрганилган. С.И. Смирнов, О.С. Попель, С.Е. Фрид, В.И. Виссарионов, В.А. Бутузов, М.Д. 3 3 TECHNIQUE AND IT. JOURNAL 2023 TECHNIQUE AND IT. JOURNAL 2023 Рабинович, А.Р. Ферт, Н.В. Харченко, Б.М. Хрусталев, В.В. Кувшинов, Р.А. Захидов, Р.Р. Авезов, Ю.К. Рашидов, А.И.Исманжанов, Ш.И. Клычев, Г.Н. Узаков, Н.Р. Авезова ва бошқаларнинг ишларида ўрганилган. Шу билан бирга айтиб ўтиш керакки, қуёш коллекторларидан самарали фойдаланиш улар ўрнатилган иссиқлик таъминоти тизимларининг тузилиши, уларда оқиб ўтадиган иссиқлик ва гидродинамик жараёнлар ва ишлаш параметрларига боғлик бўлиб, хали тўлиқ ўрганилмаган ва оптималлаштирилмаган. Фойдаланилган адабиётлар: 1. Рашидов Ю.К., Исмоилов М.М., Ясси қуёш коллекторларини ишлаш параметрларини оптиммаллаштири. Фарғона политехника институти Иқтидорли талабалар, магистрантлар, докторантлар ва мустақил изланувчилар Online илмий-амалий анжуман 2020 йил 16-17 ноябрь 1. Рашидов Ю.К., Исмоилов М.М., Ясси қуёш коллекторларини ишлаш параметрларини оптиммаллаштири. TECHNIQUE AND IT. JOURNAL 2023 TECHNIQUE AND IT. JOURNAL 2023 А.Даулетбаева, Р.Айтбаев Фарғона политехника институти Иқтидорли талабалар, магистрантлар, докторантлар ва мустақил изланувчилар Online илмий-амалий анжуман 2020 йил 16-17 ноябрь 2. Рашидов Ю.К., Исмоилов М.М. Ясси қуёш коллекторларини ўзини ўзи дренаж қилишнинг ишончли усули. Фарғона политехника институти “Замонавий бино-иншоотларини ва уларнинг конструкцияларини лойихалаш, барпо этиш, реконструкция ва модернизация қилишнинг долзарб муаммолари” мавзусида Республика Online илмий-амалий анжуман. 2021 йил 21-22 апрел 2. Рашидов Ю.К., Исмоилов М.М. Ясси қуёш коллекторларини ўзини ўзи дренаж қилишнинг ишончли усули. Фарғона политехника институти “Замонавий бино-иншоотларини ва уларнинг конструкцияларини лойихалаш, барпо этиш, реконструкция ва модернизация қилишнинг долзарб муаммолари” мавзусида Республика Online илмий-амалий анжуман. 2021 йил 21-22 апрел 3. Рашидов Ю.К., Орзиматов Ж.Т., Рашидов К.Ю., Исмоилов М.М. Разроботка и расчёт энергоэкономичных самодренируемых гелиоустановк с активным элементов Республика илмий-техника анжумани илмий мақолалар тўплами “Мухандислик коммуникация тизимларида янги технологиялар” мавзусидаги 1-2 май 2018-йил Тошкент. 4. Мадалиев Э.Ў., Исмоилов М.М., “Разработка и исследование солнечно- воздушных коллекторов Профессор-ўқитувчилар илмий-амалий анжумани Фарғона Политехника Институти.2018 Йил 7-8 Ноябрь кунлари. 4 TECHNIQUE AND IT. JOURNAL 2023 TECHNIQUE AND IT. JOURNAL 2023 5. Рашидов Ю.К., Исмоилов М.М., К вопросу повышения эффективности плоских солнечных коллекторов в системах теплоснабжения путём оптимизации их режимных параметров. «Муҳандислик коммуникацияларини лойиҳалаш, қуриш ва фойдаланишда инновацион технологиялар» Мавзусидаги республика Илмий ва илмий-техник анжумани Фарғона политехника институти 2019 йил 29-30 март Фарғона 5
https://openalex.org/W4284880772	https://www.jour.fnisc.ru/index.php/vlast/article/download/9057/8838	Russian	null	Механизмы формирования новых элит на Северном Кавказе в условиях социально-политических трансформаций (позднеимперский – раннесоветский периоды): историографический обзор	Vlastʹ	2,022	cc-by	2,925	13 0 13 0 2 0 2 2 ’ 0 3 ВЛ А С Т Ь ДАРЧИЕВА Светлана Валерьевна – кандидат исторических наук, доцент; старший науч- ный сотрудник Северо-Осетинского института гуманитарных и социальных исследований им. В.И. Абаева Владикавказского научного центра РАН (362035, Россия, Республика Северная Осетия, г. Владикавказ, ул. Галковского, 200; svetik-dar70@mail.ru) ДАРЧИЕВА Светлана Валерьевна – кандидат исторических наук, доцент; старший науч- ный сотрудник Северо-Осетинского института гуманитарных и социальных исследований им. В.И. Абаева Владикавказского научного центра РАН (362035, Россия, Республика Северная Осетия, г. Владикавказ, ул. Галковского, 200; svetik-dar70@mail.ru) МЕХАНИЗМЫ ФОРМИРОВАНИЯ НОВЫХ ЭЛИТ НА СЕВЕРНОМ КАВКАЗЕ В УСЛОВИЯХ СОЦИАЛЬНО-ПОЛИТИЧЕСКИХ ТРАНСФОРМАЦИЙ (ПОЗДНЕИМПЕРСКИЙ – РАННЕСОВЕТСКИЙ ПЕРИОДЫ): ИСТОРИОГРАФИЧЕСКИЙ ОБЗОР Аннотация. Всестороннее изучение влияния элитарных групп на политическое и социально-экономиче- ское развитие страны в различные исторические эпохи является весьма актуальной исследовательской проблемой. В современный период масштабных социально-экономических и политических преоб- разований, на данном этапе научно-технического развития российского общества возрастает роль элиты как особого социального слоя, который, с одной стороны, инициирует происходящие в обществе изменения, а с другой – принимает активное участие в их реализации, а также осмысливает пути и пер- спективы дальнейшего исторического развития России. В статье рассматриваются основные проблемы элитообразования в России (на примере Северного Кавказа) в первой трети XX в. в свете современной российской историографии. Ключевые слова: Государственная дума, Временное правительство, историография, политические и партийные элиты, национальная интеллигенция, Северный Кавказ П рогрессивное развитие общества в большой степени зависит от той избран- ной группы выдающихся по профессиональным и моральным каче- ствам людей, которых принято обозначать одним емким термином – элита. Английский историк, социолог, философ и культуролог А. Тойнби высказал мысль, что, когда общество идет за элитой, оно развивается, когда элита идет за обществом, оно деградирует. Тойнби исходил из идеального представления об избранной группе людей, а именно из того, что элита – это выдающиеся по профессиональным качествам, высокоморальные люди, которые имеют право вести за собой остальное общество. Однако жизнь, конечно, сложнее сложив- шихся дефиниций. П В изучении элиты в большей степени преуспели политологи, в меньшей – историки, социологи. Выделилось целое направление политической науки – элитология. В трудах Г. Моски, В. Парето, Р. Михельса и др. были обосно- ваны теории функционирования и циркуляции элит [Моска 1994; Pareto 1993; Михельс 1990]. Основатели теории элиты (элитизма) задались вопросом, кто имеет и может взять на себя миссию управлять массами, а также обосновали теории функционирования и циркуляции элит. Политические элиты возникли в России не вчера. В советский период основ- ные положения теории элит рассматривали лишь некоторые исследователи. Например, А.А. Галкин разрабатывал эту тему на примере «внешних» элит, изучая элитные группы зарубежных стран [Галкин 2004]. К критике элитных теорий в советский период обращались такие исследователи, как Г.К. Ашин, 131 2022’03 ВЛАСТЬ П.С. Гуревич, Ю.Н. Давыдов и др. [Ашин 2010; Гуревич 2001; Давыдов, Роднянская 1980]. В настоящее время наблюдается повышение научного и общественного инте- реса к элитологии как науке об элитном слое в системе социально-политической стратификации населения страны, региона и т.п. МЕХАНИЗМЫ ФОРМИРОВАНИЯ НОВЫХ ЭЛИТ НА СЕВЕРНОМ КАВКАЗЕ В УСЛОВИЯХ СОЦИАЛЬНО-ПОЛИТИЧЕСКИХ ТРАНСФОРМАЦИЙ (ПОЗДНЕИМПЕРСКИЙ – РАННЕСОВЕТСКИЙ ПЕРИОДЫ): ИСТОРИОГРАФИЧЕСКИЙ ОБЗОР Существующая исто- риография представлена самыми разнообразными авторами, такими как 13 2 2 0 2 2 ’ 0 3 2 0 2 2 ’ 0 3 2 0 2 2 ’ 0 3 ВЛ А С Т Ь С.В. Дарчиева, А.В. Дарчиев, В.С. Томеллери, Д.Н. Прасолов, Ф.Х. Хадонова, Д.Н. Шипов и многими другими [Дарчиева, Дарчиев 2018; Томеллери 2020; Прасолов 2021; Хадонова 2020; Шипов 2007]. р ; ; ] В период между Февральской и Октябрьской революциями 1917 г. осущест- влялся переход от традиционной общественно-политической организации общества и традиционного государства к новой форме политического и соци- ального устройства России, возникал новый вектор социально-политического развития российского социума. В это время представители северокавказских народов (горская знать, казачество, буржуазия, интеллигенция и представи- тели исламского духовенства) создали исполнительные комитеты на местах, а затем государственное образование – Союз объединенных горцев Северного Кавказа. Таким образом, они не только расширили свое участие в управлении регионом, но и попытались взять власть в свои руки. В период между двумя революциями наметились изменения в представлении горской элиты о меха- низмах выдвижения во власть: местные политические деятели Февральской революции, которые позже сформировали Горское правительство, выступали за всеобщие выборы и пропорциональность этнического представительства в органах власти. р Историография, анализирующая различные аспекты политической борьбы на Северном Кавказе в период между Февральской и Октябрьской революци- ями в России в 1917 г. и в период Гражданской войны, достаточно обширна: это работы М.Х. Алисхановой, А.Г. Кажарова, А.К. Кулумбековой, Т.Н. Литвиновой, В.Б. Лобанова, Т.Х. Матиева, А.Т. Царикаева и др. [Алисханова, Ибрагимов 2021; Кажаров 2018; Кулумбекова 2021; Литвинова 2017; Лобанов 2013; 2021; Матиев 2020; Царикаев 2017]. В этих работах предпринят комплексный анализ деятельности различных политических сил в годы революций и Гражданской войны на территории Северного Кавказа. Авторы ввели огромный массив источников из центральных и региональных архивохранилищ, проанализиро- вали советскую, эмигрантскую и современную литературу. Сделана попытка после советского жестко регламентированного и одностороннего изложения восполнить пробелы в изучении Гражданской войны на Северном Кавказе. В то же время работы, изданные в советское время, продолжают иметь огромное значение, поскольку в них содержится богатый фактический и статистический материал, так и не переизданный в постсоветский период. П Г й й 1921 б р , р р После окончания Гражданской войны в 1921 г. большевики поставили вопрос о формировании новой власти как в центре, так и на национальных окраинах страны. Разрушение институтов позднеимперского периода и отход от преж- них государственных принципов создали совершенно новую политическую ситуацию, в которой большевики начали формировать политическую систему и рекрутировать новую властную элиту. МЕХАНИЗМЫ ФОРМИРОВАНИЯ НОВЫХ ЭЛИТ НА СЕВЕРНОМ КАВКАЗЕ В УСЛОВИЯХ СОЦИАЛЬНО-ПОЛИТИЧЕСКИХ ТРАНСФОРМАЦИЙ (ПОЗДНЕИМПЕРСКИЙ – РАННЕСОВЕТСКИЙ ПЕРИОДЫ): ИСТОРИОГРАФИЧЕСКИЙ ОБЗОР Это связано не только с важно- стью повышения качества управленческой деятельности в политической системе общества как таковой, но и с тем, что при различных политических режимах судьбы миллионов людей (большинства) во многом зависят от решений, при- нимаемых правящим (элитарным) меньшинством. Среди отечественных поли- тологов, философов, социологов, занимающихся исследованием элит, следует назвать Л.П. Буеву, О.В. Гаман-Голутвину, А.А. Королева, А.К. Магомедова и др. [Буева 2010; Гаман-Голутвина 2000; Королев 2010; Магомедов 1994]. Среди широкого круга разноплановой литературы, анализирующей процесс общественных трансформаций в России начала XX в., необходимо выделить коллективную монографию под редакцией В.В. Трепавлова «Этнические элиты в национальной политике России», в которой авторы рассмотрели вопрос инкорпорации этнических элит в элиту общегосударственную [Этнические элиты… 2017]. В начале XX в. Государственная дума стала важнейшим фактором разви- тия политической культуры народов России и способствовала формирова- нию новых национальных элит. Северный Кавказ не стал исключением. Под лозунгом конституционного диалога с властью местная политическая элита стремилась выработать собственную линию поведения. Обозначились этно- конфессиональные и региональные интересы, и оформились программные требования решения национального вопроса. Национально-региональные фракции Государственной думы координировали деятельность политических сил в регионах, притягивали к себе сторонников проведения преобразова- ний политической и культурной жизни нерусских народов империи. За время работы Думы (1906–1917 гг.) депутатский корпус подвергся эволюции, при- ведшей к тому, что только меньшая часть состава Думы продолжала работать в рамках думского парламентаризма. После Февральской революции 1917 г. создается Временный комитет Государственной думы, комиссары которого бесперебойно обеспечивали работу государственного аппарата, способство- вали проведению демократических реформ в управлении страной и активно участвовали в политической жизни своих регионов. Северокавказские депу- таты Государственной думы различных созывов (К.Л. Бардиж, И.И. Гайдаров, М.М. Далгат, М.А. Караулов, Ф.А. Щербина, Т.Э. Эльдарханов) участвовали как во Временном правительстве, так и в национальных и региональных прави- тельствах. Они составили весомое кадровое ядро национальных политических элит, закрепившихся в новых независимых государствах, которые появились после распада Российской империи. Широкий спектр вопросов, связанных с зарождением и деятельностью российского парламента, взаимоотношениями Думы с правительством и верховной властью, а также причины краха монархии находятся в центре внимания исследователей М.М. Айбатова, А.А. Андреева, С.В. Дарчиевой, А.А. Чемакина и др. [Айбатов 2020; Андреев 2020; Дарчиева 2015; Чемакин 2018]. Пристальный интерес у отечественных и зарубежных ученых вызывает де- ятельность людей, живших и творивших в переломное время. В связи с этим публикуются воспоминания известных государственных, политических и общественных деятелей, печатаются их исторические портреты. Одной из центральных становится тема роли личности в истории. МЕХАНИЗМЫ ФОРМИРОВАНИЯ НОВЫХ ЭЛИТ НА СЕВЕРНОМ КАВКАЗЕ В УСЛОВИЯХ СОЦИАЛЬНО-ПОЛИТИЧЕСКИХ ТРАНСФОРМАЦИЙ (ПОЗДНЕИМПЕРСКИЙ – РАННЕСОВЕТСКИЙ ПЕРИОДЫ): ИСТОРИОГРАФИЧЕСКИЙ ОБЗОР Под рекрутированием и механизмами рекрутирования элиты понимаются, по определению О. Гаман-Голутвиной, «принципы выдвижения в ее состав новобранцев, неизбежно разнящиеся в зависимости от общественного строя и исторической эпохи (такими принци- пами, попеременно либо синхронно, были кровное родство, наследование, владение собственностью, профессиональная компетентность, партийная принадлежность, личная преданность, старшинство или выслуга лет, протек- ционизм и т.д.)» [Гаман-Голутвина 2000: 100]. В 1920-х гг. выдвижению управ- ленцев от «станка» и «сохи» в партийно-государственные органы придавалось первостепенное значение. Для укрепления советской власти в национальных окраинах начинается поли- тика коренизации, которая выражалась в назначении на руководящие долж- ности представителей национальных меньшинств, в создании национально- 133 2022’03 2022’03 ВЛАСТЬ территориальных автономий, ведении делопроизводства и поощрении СМИ на национальных языках. Коренизация подразумевала и «формирование эле- ментов управленческого аппарата кадрами, знающими язык, быт и психологию местного населения» [Сталин 1952: 116-117]. Спецификой этой политики было то, что на фоне подготовки и привлечения национальных кадров осуществля- лось массовое командирование на окраины руководителей из центра. Одной из важнейших задач советского национально-государственного стро- ительства на Северном Кавказе в 1920–1930-е гг. было создание эффективно работающего партийно-советского аппарата. Залог этой эффективности пар- тийное руководство видело в обеспечении принципа классового подхода при формировании его кадрового состава и недопущении социально чуждых эле- ментов (кулачество, мусульманское духовенство, либеральная интеллигенция и т.д.). В документальной публикации «ЦК РКП(б)-ВКП(б) и национальный вопрос» представлены архивные документы, охватывающие 1933–1945 гг. Этот период характеризовался переходом сталинского руководства от национально дифференцированной политики в отношении этнических обществ к курсу на унификацию и нивелирование этнонационального многообразия1. В доку- ментах сборника отражены основные направления развития советской наци- ональной политики в 1930-е гг.: формирование пятиуровневого статуса наци- ональных образований, ликвидация национально-территориальных округов, постепенный отказ от коренизации аппарата [Царикаев 2017: 190]. р р [ р ] Проблематика, связанная с изучением раннесоветской политической элиты, не имела до недавнего времени в отечественной политической науке не только своей исследовательской традиции, но и развернутой историографии. В моно- графии М.С. Восленского «Номенклатура» на значительном историческом массиве анализируется специфика политического господства правящей элиты в номенклатурной системе [Восленский 1991]. Однако следует отметить, что в последнее время изучение советской политической элиты, истории ее ста- новления и развития, механизмов генезиса и трансформаций привлекает все большее внимание отечественных исследователей. В этой связи отме- тим работу Т.Ю. Красовицкой «Национальные элиты как социокультурный феномен советской государственности (октябрь 1917–1923 г.). Автор акцен- тирует внимание на понимании феномена «элиты качества», также анализи- рует социокультурный феномен национальных элит, механизм формирования элиты и превращения ее по своему должностному статусу в «элиту положения» [Красовицкая 2007: 5-6]. 1 ЦК РКП(б)-ВКП(б) и национальный вопрос: в 2 кн. Кн. 2. 1933–1945 (сост.: Л.С. Гатагова, Л.П. Кошелева, Л.А. Роговая, Дж. Кадио). М.: РОССПЭН. 2009. 1095 с. МЕХАНИЗМЫ ФОРМИРОВАНИЯ НОВЫХ ЭЛИТ НА СЕВЕРНОМ КАВКАЗЕ В УСЛОВИЯХ СОЦИАЛЬНО-ПОЛИТИЧЕСКИХ ТРАНСФОРМАЦИЙ (ПОЗДНЕИМПЕРСКИЙ – РАННЕСОВЕТСКИЙ ПЕРИОДЫ): ИСТОРИОГРАФИЧЕСКИЙ ОБЗОР Историографический обзор позволяет сделать вывод, что в исторической литературе накоплен весомый фактический и аналитический материал, каса- ющийся отдельных аспектов рассматриваемой нами проблемы. Список литературы Айб М М 2020 Список литературы Айбатов М.М. 2020. Политико-правовая деятельность депутатов Северного Кавказа в Государственной Думе Российской империи в начале XX в. – Юридический вестник Дагестанского государственного университета. Т. 36. № 4. С. 14-18. Алисханова М.Х., Ибрагимов М.М. 2021. К вопросу о революционном дви- жении в Чечне в 1905–1907 гг. – Современная научная мысль. № 6. С. 80-85. Андреев А.А. 2020. Организация выборов среди туземного и инородческого населения в первую Государственную Думу. – Научный диалог. № 3. С. 298-316 Андреев А.А. 2020. Организация выборов среди туземного и инородческого населения в первую Государственную Думу. – Научный диалог. № 3. С. 298-316. 13 4 2 0 2 2 ’ 0 3 2 0 2 2 ’ 0 3 ВЛ А С Т Ь Ашин Г.К. 2010. Элитология: история, теория, современность. М.: Изд-во МГИМО(У). 598 с. ( ) Буева Л.П. 2010. Крах управленческих стратегий. – Элита России в прошлом и настоящем: социально-психологические и исторические аспекты: сборник науч- ных статей. М.: Изд-во Московского гуманитарного ун-та. 496 с. Восленский М.С. 1991. Номенклатура. Господствующий класс Советского Союза. М.: МП «Октябрь», «Советская Россия». 624 с. Г А А 2004 Р й М О Галкин А.А. 2004. Размышления о политике и политической науке. М.: Оверлей. 278 с. Гаман-Голутвина О.В. 2000. Определение основных понятий элитологии. – Полис. Политические исследования. № 3. C. 97-103. Гуревич П.С. 2001. Философия человека. Ч. 2. М.: Изд-во ИФ РАН. 209 c. Давыдов Ю.Н., Роднянская И.Б. 1980. Социология контркультуры: критиче- ский анализ. М.: Наука. 264 с. Дарчиева С.В. 2015. Государственная Дума Российской империи и вопросы эко- номического, политического и культурного развития Северного Кавказа (1906– 1917 гг.): монография. Владикавказ: ИПЦ СОИГСИ ВНЦ РАН и РСО-А. 260 с. Дарчиева С.В., Дарчиев А.В. 2018. Либеральная интеллигенция в революции 1917 г.: И.В. Баев о кризисе власти в России. – Былые годы. Российский истори- ческий журнал. № 50(4). С. 1744-1751. ур ( ) Кажаров А.Г. 2018. Формирование Горской АССР и проблемы националь- ного самоопределения Кабарды и Балкарии (1920–1921 гг.). – Гуманитарные и юридические исследования. № 1. С. 48-55. Королев А.А. 2010. Элиты России через призму исторической психологии. – Элита России в прошлом и настоящем: социально-психологические и истори- ческие аспекты: сборник научных статей. М.: Изд-во Московского гуманитар- ного ун-та. Красовицкая Т.Ю. 2007. Национальные элиты как социокультурный феномен советской государственности (октябрь 1917–1923 г.): документы и материалы. М.: Изд-во ИРИ РАН. 446 с. Кулумбекова А.К. 2021. Взаимоотношения казачества и коренных народов Терской области в 1900–1921 гг.: исторический аспект. Владикавказ: Изд-во Цопанова А.Ю. 234 с. Литвинова Т.Н. Список литературы Айб М М 2020 2017. Политическая институционализация и борьба элит на Северном Кавказе в период революций 1917 года и Гражданской войны. – Журнал социологии и социальной антропологии. № 20(4). С. 154-168. Лобанов В.Б. 2013. История антибольшевистского движения на Северном Кавказе, 1917–1920 гг.: на материалах Терека и Дагестана. СПб: Полторак. 424 с. Лобанов В.Б. 2021. Административно-территориальное деление Терека и Дагестана в период революции и Гражданской войны, 1917–1920 гг. – Кавказалогия: электронный журнал. № 1. С. 67-80. Доступ: https://kbsu.ru/ wp-content/uploads/2021/04/lobanov_kavkazologija_2021_1.pdf (проверено 30.04.2022). Магомедов А.К. 1994. Политические элиты российской провинции. – Мировая экономика и международные отношения. № 4. С. 72-79. Магомедов А.К. 1994. Политические элиты российской провинции. – Мировая экономика и международные отношения. № 4. С. 72-79. Матиев Т.Х. 2020. Горское национальное движение в революциях и Гражданской войне на Северном Кавказе (1917–1921 гг.). Назрань: ИНГНИИ им. Ч.Э. Ахриева. 402 с. Матиев Т.Х. 2020. Горское национальное движение в революциях и Гражданской войне на Северном Кавказе (1917–1921 гг.). Назрань: ИНГНИИ им. Ч.Э. Ахриева. 402 с. Матиев Т.Х. 2020. Горское национальное движение в революциях и Гражданской войне на Северном Кавказе (1917–1921 гг.). Назрань: ИНГНИИ им. Ч.Э. Ахриева. 402 с. Михельс Р. 1990. Социология политической партии в условиях демократии. – Диалог. № 3. С. 55-60. Михельс Р. 1990. Социология политической партии в условиях демократии. – Диалог. № 3. С. 55-60. Моска Г. 1994. Правящий класс. – Социс. Социологические исследования. № 10 С. 187-198. 135 2022’03 ВЛАСТЬ Прасолов Д.Н. 2021. «Вся Кабарда встретит этот акт с чувством великого нравственного удовлетворения…» Статья Г. Баева «О присоединении Малой Кабарды к Большой». – Кавказология: электронный журнал. № 4. С. 86-107. Доступ: https://doi.org/10.31143/2542-212X-2021-4-86-107 (проверено 30.04.2022). ) Сталин И.В. 1952. Сочинения. 1921–1923. М.: Госполитиздат. Т. 5. 446 с. Сталин И.В. 1952. Сочинения. 1921–1923. М.: Госполитиздат. Т. 5. 446 с. Томеллери В.С. 2020. Из истории осетиноведения: переписка Георгия (Гаппо) Васильевича Баева и Георгия Ахвледиани. – Вестник Волгоградского государ- ственного университета. Сер. 2. Языкознание. Т. 19. № 6. С. 92-122. Хадонова Ф.Х. 2020. О Гаппо Баеве (материалы к биографии). – Вестник Северо-Осетинского государственного университета им. К.Л. Хетагурова. № 1. С 83 93 Томеллери В.С. 2020. Из истории осетиноведения: переписка Георгия (Гаппо) Васильевича Баева и Георгия Ахвледиани. – Вестник Волгоградского государ- ственного университета. Сер. 2. Языкознание. Т. 19. № 6. С. 92-122. Хадонова Ф.Х. 2020. О Гаппо Баеве (материалы к биографии). – Вестник Северо-Осетинского государственного университета им. К.Л. Хетагурова. № 1. С. 83-93. Царикаев А.Т. 2017. Проблемы национально-государственного строитель- ства на Северном Кавказе в 1920–1930-е гг. в новейших документальных публикациях. Список литературы Айб М М 2020 Исторические, философские, политические и юридические науки, культурология. Вопросы теории и практики. – Грамота. № 12(86): в 5 ч. Ч. 2. С. 188-191. Чемакин А.А. 2018. Истоки русской национал-демократии: 1896–1914 годы. СПб: Владимир Даль. 651 с. Шипов Д.Н. 2007. Воспоминания и думы о пережитом. М.: РОСПЭН. 679 с. Этнические элиты в национальной политике России (отв. ред. В.В. Трепавлов). 2017. М.: Изд-во ИРИ РАН; Центр гуманитарных инициатив. 477 с. Pareto V. 1933. Le traite de sociologie generale. Paris: Payot. 864 p. DARCHIEVA Svetlana Valer'evna, Cand.Sci. (Hist.), Associate Professor; Senior Researcher at I. Abaev North Ossetian Institute of Humanities and Social Studies, Vladikavkaz Scientific Center of the Russian Academy of Sciences (200 Galkovskogo St, Vladikavkaz, Republic of North Ossetia–Alania, Russia, 362035; svetik-dar70@mail.ru) DARCHIEVA Svetlana Valer'evna, Cand.Sci. (Hist.), Associate Professor; Senior Researcher at I. Abaev North Ossetian Institute of Humanities and Social Studies, Vladikavkaz Scientific Center of the Russian Academy of Sciences (200 Galkovskogo St, Vladikavkaz, Republic of North Ossetia–Alania, Russia, 362035; svetik-dar70@mail.ru) MECHANISMS FOR THE FORMATION OF NEW ELITES IN THE NORTH CAUCASUS UNDER CONDITIONS OF SOCIO-POLITICAL TRANSFORMATIONS (LATE IMPERIAL – EARLY SOVIET PERIODS): HISTORIOGRAPHICAL REVIEW Abstract. The progressive development of society largely depends on the select group of people – the elite. A comprehensive study of the influence of elite groups on the political and socio-economic development of the country in different historical eras is a very relevant research problem. In the modern period of large-scale socio-economic and political transformations and scientific and technological development of Russian society the role of the elite as a special social stratum is increasing. On the one hand, it initiates the changes taking place in society, and on the other hand, takes an active part in their implementation, and comprehends the ways and prospects for the further historical development of Russia. The article deals with the main problems of elite formation in the North Caucasus in the first third of the 20th century in the light of modern Russian historiography. Keywords: State Duma, Provisional Government, historiography, political and party elites, national intelligentsia, North Caucasus
https://openalex.org/W2589580339	https://www.nature.com/articles/ncomms14319.pdf	English	null	Unraveling the processes shaping mammalian gut microbiomes over evolutionary time	Nature communications	2,017	cc-by	15,170	1 Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 2 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 3 Laboratoire d Ecologie Alpine, CNRS, University of Grenoble Alpes, FR-38041, Grenoble Cedex 9, France. 4 Organismic and Evolutionary Biology, Harvard University, 26 Oxford St, Cambridge, Massachusetts 02138, USA. 5 The Broad Institute of MIT and Harvard, 415 Main Street Cambridge, Massachusetts 02142, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to E.A. (email: ejalm@mit.edu). ARTICLE Received 26 Jul 2016 \| Accepted 8 Dec 2016 \| Published 23 Feb 2017 Received 26 Jul 2016 \| Accepted 8 Dec 2016 \| Published 23 Feb 2017 DOI: 10.1038/ncomms14319 Results h l Phylogenetic decomposition of community dissimilarities. Gut microbiome content in operational taxonomic units (OTUs) can vary greatly between two communities (that is, hosts). Usually, these compositional dissimilarities (b-diversity26) are described with a single measure, either using taxonomic metrics, such as the Sørensen metric27, or using phylogenetic metrics, such as UniFrac28. However, relying on a single number to describe community dissimilarities may be too simplistic if different factors shape compositions in OTUs at different bacterial phylogenetic scales25. Yet, there is no robust framework in the literature that integrates compositional b-diversity along a phylogenetic timescale. p y g We developed a new method (BDTT; for b-diversity through time) to account for this potential temporal scale disparity between factors, here to separate the inﬂuence of host diet and phylogeny on gut microbiota compositions along the timescale of bacterial evolution (Supplementary Fig. 2). BDTT computes compositional turnover (using Sørensen or Bray–Curtis metrics, see Methods) between communities at different time period along the bacterial phylogenetic timescale, producing a proﬁle of b- diversities29. From the leaves to the root, the bacterial tree (here, reconstructed de novo from all 16S rRNA reads) is continuously sliced, either by time or evolutionary distance. For each time period, the tree of bacteria is cut, yielding clades that can serve as OTUs in downstream analysis. Microbiome composition is then determined for each mammalian species in terms of these new OTUs, and pairwise compositional dissimilarities are computed. Importantly, the BDTT proﬁle provides a phylogenetic decomposition of the broadly used UniFrac metric28 (Supplementary Fig. 2 and Supplementary Note 1), Finally, at each time period, b-diversities are correlated to host diet and phylogenetic distances, and the comparison of the amount of b- y Here we characterized the variation in mammalian gut microbiome compositions in light of host diet and phylogeny. We are deﬁning diet with a coarse granularity, using nine large dietary categories from the EltonTraits database23. A percentage for each species is assigned to each of the nine categories (see ‘Host phylogeny and dietary data’ in the Methods and Supplementary Table 1). Hence, this study focuses on the impact that large dietary shifts had on microbiomes at the scale of mammalian evolution. Furthermore, we employ ‘host phylogeny’ as a composite term that encompasses all traits that change roughly clock-like along the phylogeny of hosts and that might inﬂuence microbiome compositions, such as genetic or immunological factors24. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 T he evolution of binary symbiotic relationships between animal hosts and individual microbial symbionts is well documented1–3. However, little is known about the mechanisms that shape community structure during the long- term symbiosis of hosts and their gut microbiomes. If sister host lineages share similar bacteria, gut communities may recapitulate host phylogeny. This pattern, referred to as ‘phylosymbiosis’4–6, does not imply a process and vertical inheritance of symbionts. We deﬁne ‘vertical inheritance’ as the restricted transmission of bacterial lineages within- rather than between-host lineages, as in dispersal exclusively to conspeciﬁcs; this could include but would not require transmission from mother to offspring. Phylosymbiosis may arise from the neutral vertical inheritance of symbionts (for example, co-speciation associated with allopatric speciations of hosts, see Supplementary Fig. 1), and/or from selective vertical inheritance, for instance, with intimate co-evolution between host and microbes7–9. But it can also arise independently of vertical inheritance, if closely related hosts with similar genetic or behavioral traits select similar bacteria from the environment10 (Supplementary Fig. 1). The signal of phylosymbiosis can erode over time or even disappear, when a selective trait such as diet is decoupled from host phylogeny, promoting the horizontal acquisition of bacterial symbionts from the environment or from distantly related hosts11,12. T microbiome compositions independently of the signiﬁcant dietary shifts that occurred during mammalian evolution, and if vertical inheritance is generating this correlation with host phylogeny, these associations should be stronger in recent regions of the bacterial tree (as co-speciation events are not possible prior to the evolution of mammals). However, if vertical inheritance is not involved in generating this diet-independent correlation with host phylogeny, associations with host phylogeny should be seen at timescales of bacterial evolution that are decoupled from host evolution. For example, a given mammalian clade might select non-vertically inherited bacteria within a bacterial lineage that arose prior to the emergence of mammals. Thus a phylogenetically informed approach incorporating compositional disparities along the bacterial phylogenetic timescale may allow us to disentangle the individual contributions of host phylogeny and major dietary shifts and to better understand how and to what extent the different types of bacterial inheritance (vertical and horizontal) have driven gut community evolution. Using such an approach, we show that diet drives the horizontal acquisition of bacterial lineages that belong to ancient bacterial clades and that host phylogeny predicts the presence of more recent bacterial lineages. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 We show that gut microbiomes have recorded the information of major dietary shifts that occurred during the evolution of mammals, allowing us to predict ancient diets from the reconstruction of ancient microbiomes. Associations between microbiome compositions and host phylogeny are universal in mammals and stronger among recently diverged mammals. Finally, our results suggest that co-speciation between bacterial lineages and their mammalian hosts partly drives these patterns of phylosymbiosis. y In mammals, both at the intrahost and interhost species levels, whether host evolutionary history (host genetics and phylogeny, respectively) or host diet exerts a stronger inﬂuence in shaping the gut microbiome is controversial. Several studies11,13–16 have claimed that diet is the main driver of gut microbiome composition. Some authors have claimed that host genetics has a minor contribution relative to environmental factors in humans and chimpanzees15,17,18, while others have found evidence for a stronger impact of host genetics19,20. At the interhost level, it has recently been shown that two gut bacterial families possess lineages that harbour patterns of co-speciation in four hominid hosts7, highlighting the inﬂuence of host phylogeny at short evolutionary scales. However, previous investigations have disagreed on whether or not11,21 a signal for phylosymbiosis exists at larger evolutionary scales, across all mammalian gut microbiomes4,11,21,22. Because of all these uncertainties, and because we anticipate that correlation with host phylogeny would be generated by a mix of vertical and horizontal inheritance, while the correlation with diet would be primarily driven by horizontal inheritance, it is unknown whether mammalian gut microbiomes primarily evolve through vertical or horizontal inheritance over evolutionary time. NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications Unraveling the processes shaping mammalian gut microbiomes over evolutionary time Mathieu Groussin1,2,, Florent Mazel3,, Jon G. Sanders4, Chris S. Smillie1,2,5, Se´bastien Lavergne3, Wilfried Thuiller3 & Eric J. Alm1,2,5 Whether mammal–microbiome interactions are persistent and speciﬁc over evolutionary time is controversial. Here we show that host phylogeny and major dietary shifts have affected the distribution of different gut bacterial lineages and did so on vastly different bacterial phylogenetic resolutions. Diet mostly inﬂuences the acquisition of ancient and large microbial lineages. Conversely, correlation with host phylogeny is mostly seen among more recently diverged bacterial lineages, consistent with processes operating at similar timescales to host evolution. Considering microbiomes at appropriate phylogenetic scales allows us to model their evolution along the mammalian tree and to infer ancient diets from the predicted microbiomes of mammalian ancestors. Phylogenetic analyses support co-speciation as having a signiﬁcant role in the evolution of mammalian gut microbiome compositions. Highly co-speciating bacterial genera are also associated with immune diseases in humans, laying a path for future studies that probe these co-speciating bacteria for signs of co-evolution. 1 Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 2 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 3 Laboratoire d Ecologie Alpine, CNRS, University of Grenoble Alpes, FR-38041, Grenoble Cedex 9, France. 4 Organismic and Evolutionary Biology, Harvard University, 26 Oxford St, Cambridge, Massachusetts 02138, USA. 5 The Broad Institute of MIT and Harvard, 415 Main Street Cambridge, Massachusetts 02142, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to E.A. (email: ejalm@mit.edu). 1 NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications ARTICLE ARTICLE We also measured to what extent the covariation between host phylogeny and diet drives gut community b-diversities. We observed that, at all bacterial phylogenetic scales, the covariation between host phylogeny and diet only weakly explains bacterial community dissimilarity (R2 ranging from about 0.01 to about 0.04, see Supplementary Fig. 9c and ‘The covariance between host phylogenetic and dietary distances poorly explains community dissimilarities’ in Supplementary Note 5). Altogether, these results suggest that, even though broad shifts in diet can be locally correlated with host phylogeny34,35, the impacts of these factors on changes in the microbiome can be primarily observed in different microbial lineages. We applied BDTT to a data set of 33 mammalian gut microbiomes11, composed of 44,444 dereplicated and chimera- free amplicons of the 16S rRNA gene (V2 region), from which we reconstructed the time-calibrated bacterial phylogenetic tree. Host phylogenetic distances between our 33 mammals were deduced from a time-calibrated ultrametric phylogenetic tree of mammals30, which was recently updated31,32. Diet distances were deduced from EltonTraits23, a database that compiles dietary attributes for all mammalian species. Phylogeny and diet shape microbiomes at different scales. Consistent with our hypothesis, BDTT was able to disentangle the effects that the major dietary shifts had on gut microbiome compositions from those of host phylogeny. This was possible because host phylogeny shapes gut microbiome compositions mainly near the leaves of the bacterial tree (Mantel test; R2 ¼ 0.38 at 100 Myr ago, P valueo0.001; R2 ¼ 0.03 at 1,000 Myr ago, P value40.05), while host diet mostly determines the distribution of more ancient bacterial lineages among hosts (Mantel test; R2 ¼ 0.08 at 100 Myr ago, P valueo0.001; R2 ¼ 0.22 at 1,000 Myr ago, P valueo0.001) (Fig. 1a,b). g ) ( g ) We performed multiple control experiments to evaluate the robustness of this signal of phylogenetic scale disparity between host phylogeny and diet (Supplementary Figs 4–7 and Supplementary Note 3 and 4). We measured the inﬂuence of the intrahost species variability of microbiome compositions, the inﬂuence of topological uncertainties in the tree of bacteria and the inﬂuence of differences in statistical power at different depth of the bacterial tree. We also used branch lengths in the bacterial tree expressed in average substitution/site as a proxy of time in the BDTT approach. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 diversity explained by each factor across bacterial timescales may reveal the phylogenetic levels at which their individual inﬂuence is greatest. We run BDTT on simulated data sets (see ‘Validation of BDTT on simulated data’ in the Methods) and we show that it is able to disentangle the effect of different factors shaping community assembly at different phylogenetic scales (Supplementary Fig. 3 and Supplementary Note 2). As mammals diverged recently in comparison to bacteria, the effect of host phylogeny on recent bacterial lineages is consistent with, although not direct proof of, co-diversiﬁcation between hosts and their gut microbiomes. The effect of diet on more ancient bacterial lineages is consistent with the evolutionary conservation of functional traits involved in the digestion of dietary compounds in ancient bacterial taxa25,33. Note that BDTT allows us to statistically disentangle the temporally separated portions of the contributions of host phylogeny and diet (when deﬁned with a coarse granularity), not the totality of the processes themselves. Note that, for BDTT to be informative, it is not necessary that bacterial time estimates (in millions of years ago (Myr ago)) be strictly accurate, but rather that the relative order of branching is conserved from relatively ancient to relatively modern divergences. Branch lengths expressed in the expected number of substitution per site were also used to approximate time and to cluster sequences into OTUs at different slices in our BDTT approach, yielding to identical patterns (Supplementary Figs 2 and 4). To further resolve the roles of host diet and phylogeny, we asked whether the more recent bacterial lineages that correlate with host phylogeny are nested within ancient diet-related bacterial lineages or whether they are independent from them (Supplementary Fig. 9a,b). After removing the bacterial lineages correlated with broad categories of diet (Fig. 1a), we found that the correlations between host phylogeny and bacterial composition within recent time slices decreased but remained very strong (Fig. 1c), close to their previous level (Mantel test; R2 ¼ 0.34 versus 0.38 at 100 Myr ago). This means that although some bacteria related to host phylogeny at recent timescales are nested within higher clades also related to broad categories of host diet, a large part of the bacteria associated with host phylogeny are different from the bacteria associated with diet (see also ‘Bacterial lineages correlating with host phylogeny and diet lowly overlap’ in Supplementary Note 5). NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications Results h l We hypothesized that major changes in diet and host phylogeny may have driven vertical and horizontal inheritance of bacterial lineages at different bacterial phylogenetic scales6,25. For example, when mammalian lineages shifted their diet towards herbivory, they might have horizontally acquired herbivorous-speciﬁc bacterial lineages, and those bacterial lineages could even predate the divergence of the mammals. Furthermore, if host phylogeny shapes gut NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 (a,b) Bacterial lineages that diverge recently in evolutionary history show high levels of correlation with host phylogenetic distances (blue) while correlation with host dietary distances (orange) is greatest for more ancient lineages. (a) Lines show correlations between the pairwise Sørensen compositional dissimilarities and pairwise host phylogenetic or dietary distances (dashed lines: 95% null envelope). (b) Individual bacterial lineages correlated with diet or phylogeny (circles). Pie charts represent the percentage of lineages that correlate signiﬁcantly with each factor at different times. The phylogenetic scale is common to plots a,b. (c) When diet-associated lineages (in b) are removed, correlation with host phylogeny (dark blue) still holds (light blue), but correlation with host diet does not (orange versus brown). (d) Each bacterial lineage having a signiﬁcant correlation with diet (b) was called herbivorous- or carnivorous-speciﬁc if it is only found in herbivores or carnivores, respectively. Herbivory is associated with bacterial lineages that arise earlier in bacterial evolution than those associated with carnivory. Figure 1 \| Phylogenetic-scale disparities in mammalian gut microbiomes. (a,b) Bacterial lineages that diverge recently in evolutionary history show high levels of correlation with host phylogenetic distances (blue) while correlation with host dietary distances (orange) is greatest for more ancient lineages. (a) Lines show correlations between the pairwise Sørensen compositional dissimilarities and pairwise host phylogenetic or dietary distances (dashed lines: 95% null envelope). (b) Individual bacterial lineages correlated with diet or phylogeny (circles). Pie charts represent the percentage of lineages that correlate signiﬁcantly with each factor at different times. The phylogenetic scale is common to plots a,b. (c) When diet-associated lineages (in b) are removed, correlation with host phylogeny (dark blue) still holds (light blue), but correlation with host diet does not (orange versus brown). (d) Each bacterial lineage having a signiﬁcant correlation with diet (b) was called herbivorous- or carnivorous-speciﬁc if it is only found in herbivores or carnivores, respectively. Herbivory is associated with bacterial lineages that arise earlier in bacterial evolution than those associated with carnivory. As we could predict host diet, we hypothesized that we might be able to infer the diet of ancestral mammals by reconstructing their microbiome. We further hypothesized this should only be possible if we use an appropriate phylogenetic cutoff (as before, B300 Myr ago or B94% OTUs) to describe community structure. In particular, reconstruction of widely used 97% OTUs should not be able to reconstruct ancestral dietary patterns. ARTICLE We controlled for the inﬂuence of the unequal sampling size across samples by performing rarefaction of the OTU tables, and we controlled for the differences in granularity between the host phylogenetic and dietary distance matrices. All of these experimental controls support that our analysis uncovers a genuine and robust biological signal of scale disparity between the two factors. Finally, we described the process of mammalian diet evolution and then used this process to simulate traits along the phylogeny of hosts. We used these simulated traits to run BDTT and compared the simulated correlation proﬁles with the correlation proﬁle obtained when using observed diets. We show that the peak of correlation between microbiome compositions and diet at ancient timescales is not solely an echo of host phylogeny and conclude that the contribution of diet on microbiome compositions is partially decoupled from the host phylogenetic history (see Supplementary Fig. 8 and ‘The contribution of diet on microbiome compositions is partially decoupled from the host phylogenetic history’ in Supplementary Note 3). Herbivory and carnivory act at distinct timescales. We also discovered a timescale disparity between herbivore- and carnivore-associated bacterial lineages. Herbivory is associated with bacterial lineages that arise deeper in the bacterial phylogeny (4200 Myr ago) than those associated with carnivory, which are conﬁned in a limited range of phylogenetic scales (between 150 and 300 Myr ago, see Fig. 1d). This suggests that herbivory and carnivory are associated with bacterial lineages that emerged at different evolutionary timescales and that traits allowing bacterial lineages to thrive within a mammalian herbivorous gut appeared early in bacterial evolution, while those permitting specialization in carnivore guts arrived much later, which is consistent with the late appearance of carnivorous animals36. Omnivores do not harbour omnivore-speciﬁc bacteria. The fact that some bacterial lineages may have functionally adapted to thrive in gut environmental conditions that are diet-speciﬁc and not host-speciﬁc raises the question: do generalist hosts harbour distinct microbes that are also ‘generalists’ and can digest both plant and animal material or do they include a mixture of herbivorous and carnivorous bacteria? Upon closer inspection, we found specialist bacterial lineages associated with both herbivores and carnivores (Fig. 2a), suggesting functional adaptations to these two types of diet-related gut environments. However, we did not ﬁnd specialist bacteria associated with omnivores, MMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications 3 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Past Lineage correlated with: Both Approximate time (Ma) 2,000 1,000 500 100 Present Fusobacteria Firmicutes Fibrobacteres Carnivorous specific Herbivorous specific Bacteroidetes Tenericutes Spirochaetes Proteobacteria Verrucomicrobia Actinobacteria Host phylogeny (HP) Number of carni.-and herbi.-specific lineages Ratio of carni.-versus herbi.-specific lineages 20 10 0 600 500 400 300 200 100 Approximate time (Ma) HP without diet-associated lineages Host diet (HD) HD without diet-associated lineages 0.4 0.3 0.2 0.1 0 100 80 60 40 20 0 Correlation with community dissimilarity (R2) 0.4 0.3 0.2 0.1 Correlation with community dissimilarity (R2) Host diet Host phylogeny Host diet Host phylogeny a b c d Figure 1 \| Phylogenetic-scale disparities in mammalian gut microbiomes. (a,b) Bacterial lineages that diverge recently in evolutionary history show high levels of correlation with host phylogenetic distances (blue) while correlation with host dietary distances (orange) is greatest for more ancient lineages. (a) Lines show correlations between the pairwise Sørensen compositional dissimilarities and pairwise host phylogenetic or dietary distances (dashed lines: 95% null envelope). (b) Individual bacterial lineages correlated with diet or phylogeny (circles). Pie charts represent the percentage of lineages that correlate signiﬁcantly with each factor at different times. The phylogenetic scale is common to plots a,b. (c) When diet-associated lineages (in b) are removed, correlation with host phylogeny (dark blue) still holds (light blue), but correlation with host diet does not (orange versus brown). (d) Each bacterial lineage having a signiﬁcant correlation with diet (b) was called herbivorous- or carnivorous-speciﬁc if it is only found in herbivores or carnivores, respectively. Herbivory is associated with bacterial lineages that arise earlier in bacterial evolution than those associated with carnivory. NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Past Lineage correlated with: Both Approximate time (Ma) 2,000 1,000 500 100 Present Fusobacteria Firmicutes Fibrobacteres Bacteroidetes Tenericutes Spirochaetes Proteobacteria Verrucomicrobia Actinobacteria 0.4 0.3 0.2 0.1 Correlation with community dissimilarity (R2) Host diet Host phylogeny Host diet Host phylogeny a b a b Lineage correlated with: 0.4 0.3 0.2 0.1 Correlation with community dissimilarity Verrucomicrobia Actinobacteria Host phylogeny (HP) Number of carni.-and herbi.-specific linea Ratio of carni.-versus herbi.-specific lineag 20 10 600 500 400 300 200 100 Approximate time (Ma) HP without diet-associated lineages Host diet (HD) HD without diet-associated lineages 0.4 0.3 0.2 0.1 0 100 80 60 40 20 Correlation with community dissimilarity (R2) community dissimilarity (R ) c d Carnivorous specific Herbivorous specific Proteobacteria Number of carni.-and herbi.-specific lineages Ratio of carni.-versus herbi.-specific lineages 20 10 0 Approximate time (Ma) 100 80 60 40 20 0 d Actinobacteria Host phylogeny (HP) 600 500 400 300 200 100 Approximate time (Ma) HP without diet-associated lineages Host diet (HD) HD without diet-associated lineages 0.4 0.3 0.2 0.1 0 Correlation with community dissimilarity (R2) co u ty d ss a ty ( ) c d d d Host phylogeny (HP) Approximate time (Ma) Correlation with community dissimilarity (R2) community dissimilarity (R2) Figure 1 \| Phylogenetic-scale disparities in mammalian gut microbiomes. (a,b) Bacterial lineages that diverge recently in evolutionary history show high levels of correlation with host phylogenetic distances (blue) while correlation with host dietary distances (orange) is greatest for more ancient lineages. (a) Lines show correlations between the pairwise Sørensen compositional dissimilarities and pairwise host phylogenetic or dietary distances (dashed lines: 95% null envelope). (b) Individual bacterial lineages correlated with diet or phylogeny (circles). Pie charts represent the percentage of lineages that correlate signiﬁcantly with each factor at different times. The phylogenetic scale is common to plots a,b. (c) When diet-associated lineages (in b) are removed, correlation with host phylogeny (dark blue) still holds (light blue), but correlation with host diet does not (orange versus brown). (d) Each bacterial lineage having a signiﬁcant correlation with diet (b) was called herbivorous- or carnivorous-speciﬁc if it is only found in herbivores or carnivores, respectively. Herbivory is associated with bacterial lineages that arise earlier in bacterial evolution than those associated with carnivory. Figure 1 \| Phylogenetic-scale disparities in mammalian gut microbiomes. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Pig Carnivorous Omnivorous Herbivorous OTUs Mammals OTU table Axis 1 Axis 2 Axis 3 PCA diet ~ Axis 1 + Axis 2 + Axis 3 Multinomial logistic regression Predicted diet probability for each diet category Projection on PCA 3 Coordinates (x1 x2 x3) ML reconstructed ancient community x1 x2 x3 0 1 0 ... 1 Prediction b a Spec. Bear Bears Bush Dog Hyena Armadillo Human Chimpanzee Black Lemur Colobus Gorilla Capybara 2 1 0 −1 −2 OTUs Mammals OTU table Axis 1 Axis 2 Axis 3 PCA diet ~ Axis 1 + Axis 2 + Axis 3 Multinomial logistic regression Predicted diet probability for each diet category Projection on PCA 3 Coordinates (x1 x2 x3) ML reconstructed ancient community x1 x2 x3 0 1 0 ... 1 Prediction b b Kangaroo Hyrax Afr. Eleph Callimicos Baboon Orang-outan Squirrel Rabbit Lion Polar Bear Horse Zebra Black Rhin Springbok Gazelle Big Horn Urial Giraffe Okapi R T Lemur Saki V.W. Pig −3 −2 −1 0 1 2 −3 Carnivorous ↔ Omnivorous ↔ Herbivorous (77.6%) Omnivorous ↔ Carnivorous (15.2%) Diet-specific bacteria Actinobacteria Firmicutes Fusobacteria Proteobacteria Black Bear V.W. Pig Carnivorous Omnivorous Herbivorous a Spec. Bear Bears Bush Dog Hyena Armadillo Human Chimpanzee Black Lemur Colobus Gorilla Capybara 2 1 0 −1 −2 b a Carnivorous ↔ Omnivorous ↔ Herbivorous (77.6%) Carnivorous Omnivorous Herbivorous Capybara Squirrel Rabbit Baboon Colobus Gorilla Human Chimpanzee Orang-outan Saki Callimicos Black Lemur R-T Lemur Urial Bighorn Sheep Gazelle Springbok Giraffe Okapi V. W. Pig Black Rhinoceros Zebra Horse Lion Hyena Black Bear Polar Bear Spec. Bear Bush Dog Armadillo Hyrax Afr. Elephant Kangaroo c (Ma) 150 (Ma) 150 100 50 0 50 100 0 Figure 2 \| Gut microbiomes predict extant and ancestral mammalian diets. (a) Diet-correlated bacterial lineages (squares coloured by phylum) and mammals (black dots) separated by dietary distances are jointly projected in the same ordination space using the OMI59 mapping procedure (see Methods). Predicted niche breadth59 of a bacterial lineage is depicted with an ellipse. The time period selected to deﬁne bacterial lineages is B300 Myr ago. Bacterial niche breadths show that many bacterial groups are herbivore or carnivore speciﬁc. However, no bacterial lineage has a niche breadth encompassing omnivores only (omnivores are in brown). (b) Workﬂow for microbiome-based prediction of ancient diet. (c) Inference of ancient mammalian diets. Left: trait-based reconstruction using 1,534 mammals34. Right: Microbiota-based reconstruction with the 33 mammals under study. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 To test our hypotheses, we reconstructed ancestral microbiome compositions for each mammalian ancestor using a maximum likelihood (ML)-based approach37, that accounts for the vertical and horizontal inheritance of OTUs along the host phylogeny. We used a model that integrates over the numerous events of gain and loss of bacterial lineages that occurred during the millions of years of host evolution, with varying rates across host lineages. We then used our microbiome-based model to predict ancestral diets from these ancestral microbiomes. We compared our microbiome-based predictions to those obtained with a classic macro-evolutionary, trait-based model that considers diet as a suggesting that omnivores contain a combination of herbivorous and carnivorous bacterial groups. Figure 2a shows these results using a joint projection of the diet-associated bacterial lineages with hosts that are separated by diet on the same ordination space (see also Supplementary Note 6). Gut microbiomes can predict ancient mammalian diets. We next investigated whether the presence of these diet-speciﬁc bacteria is sufﬁcient to predict diet in extant hosts. To test this, we built a multinomial regression model (see Methods and Fig. 2b) to predict diet in mammalian hosts from their microbiome composition. We evaluated the accuracy of our model using cross-validation experiments, and we show that our microbiome-based method is accurate and performs well at estimating host diet (Supplementary Fig. 10a–c and ‘Accuracy of our microbiome-based method of diet prediction’ in Supplementary Note 7). NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Kangaroo Hyrax Afr. Elephant Callimicos Baboon Orang-outan Squirrel Rabbit Lion Polar Bear Horse Zebra Black Rhino. Springbok Gazelle Big Horn Urial Giraffe Okapi R T Lemur Saki V.W. Pig −3 −2 −1 0 1 2 −3 Carnivorous ↔ Omnivorous ↔ Herbivorous (77.6%) Omnivorous ↔ Carnivorous (15.2%) Diet-specific bacteria Actinobacteria Firmicutes Fusobacteria Proteobacteria Black Bear V.W. Pig Carnivorous Omnivorous Herbivorous OTUs Mammals OTU table Axis 1 Axis 2 Axis 3 PCA diet ~ Axis 1 + Axis 2 + Axis 3 Multinomial logistic regression Predicted diet probability for each diet category Projection on PCA 3 Coordinates (x1 x2 x3) ML reconstructed ancient community x1 x2 x3 0 1 0 ... 1 Prediction Carnivorous Omnivorous Herbivorous Capybara Squirrel Rabbit Baboon Colobus Gorilla Human Chimpanzee Orang-outan Saki Callimicos Black Lemur R-T Lemur Urial Bighorn Sheep Gazelle Springbok Giraffe Okapi V. W. Pig Black Rhinoceros Zebra Horse Lion Hyena Black Bear Polar Bear Spec. Bear Bush Dog Armadillo Hyrax Afr. Elephant Kangaroo b a c Spec. Bear (Ma) 150 (Ma) 150 100 50 0 Bears Bush Dog Hyena Armadillo Human Chimpanzee Black Lemur Colobus Gorilla Capybara 2 1 0 −1 −2 50 100 0 Figure 2 \| Gut microbiomes predict extant and ancestral mammalian diets. (a) Diet-correlated bacterial lineages (squares coloured by phylum) and mammals (black dots) separated by dietary distances are jointly projected in the same ordination space using the OMI59 mapping procedure (see Methods). Predicted niche breadth59 of a bacterial lineage is depicted with an ellipse. The time period selected to deﬁne bacterial lineages is B300 My ago. Bacterial niche breadths show that many bacterial groups are herbivore or carnivore speciﬁc. However, no bacterial lineage has a niche breadth encompassing omnivores only (omnivores are in brown). (b) Workﬂow for microbiome-based prediction of ancient diet. (c) Inference of ancient li di t L ft t it b d t ti i 1 534 l 34 Ri ht Mi bi t b d t ti ith th 33 l d t d Kangaroo Hyrax Afr. Elephant Callimicos Baboon Orang-outan Squirrel Rabbit Lion Polar Bear Horse Zebra Black Rhino. Springbok Gazelle Big Horn Urial Giraffe Okapi R T Lemur Saki V.W. Pig −3 −2 −1 0 1 2 −3 Carnivorous ↔ Omnivorous ↔ Herbivorous (77.6%) Omnivorous ↔ Carnivorous (15.2%) Diet-specific bacteria Actinobacteria Firmicutes Fusobacteria Proteobacteria Black Bear V.W. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 clades across several branches of the mammalian phylogeny (Supplementary Fig. 11 and Supplementary Note 8). Most of the divergent predictions, as shown in Fig. 2c, are located in ancient, poorly sampled areas of the mammalian tree (carnivorous Chiroptera, Afrotheria, Xenarthra and Marsupialia are absent in the present data set, see ‘Microbiome-based and trait-based inference of ancestral diets in mammals’ in Supplementary Note 7). the signal for phylosymbiosis might be uniform or variable across mammalian clades, and it might be weak or strong. In the following, we address these questions with communities deﬁned at a recent bacterial phylogenetic scale (B120 Myr ago, OTUs with B97% similarity, see Methods), where correlation with host phylogeny is high. p y g y g After 4100 millions of years of mammalian evolution, many gains and losses of bacterial lineages occurred, possibly randomizing the genuine signal of compositional change that mirrors the mammalian phylogeny. To measure phylosymbiosis, we modelled the dynamics of gain and loss of bacterial lineages using the same phylogenetic model37 that we previously used in the section ‘Gut microbiomes can predict ancient mammalian diets’. We quantiﬁed phylosymbiosis with a hierarchical approach along the tree of mammals, by reconstructing ancestral communities for each mammalian ancestor (that is, for each node of the host phylogeny). We reasoned that if the size of a reconstructed community for an ancestor of a given mammalian clade is higher than randomly expected under a null model in which host–bacteria associations are shufﬂed, then mammals belonging to this clade harbour a phylosymbiotic signal, because their gut microbiota share more bacterial lineages between each other than randomly expected. We computed the magnitude of the phylosymbiosis signal along the mammalian host tree, for each ancestor, by computing a standard effect size (SES) per branch/node: We further compared the precision of the predictions obtained with the microbiome- and trait-based methods on a similar taxonomic sampling (33 species). We found that the entropy of the ancestral dietary probability distributions, which measures the uncertainty in these distributions, is signiﬁcantly lower with microbiome data (Wilcoxon test, W ¼ 232, P value ¼ 0.0002), indicating a higher precision of the microbiome-based method (Supplementary Fig. 10d–f and ‘Microbiome-based and trait- based inference of ancestral diets in mammals’ in Supplementary Note 7). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Finally, bacterial phylogenetic groups summarized at 120 Myr ago (or B97% 16S similarity) or 600 Myr ago (B91% similarity) were much less accurate at predicting diet (see ‘Reconstruction of ancient diets from microbiomes deﬁned at alternative bacterial phylogenetic scales’ in Supplementary Note 7). These results underscore the importance of identifying the correct phylogenetic resolution with which to deﬁne communities, in order to study patterns of host–microbiome evolution. Overall, our results show that gut microbiomes record the information of past dietary adaptations with the horizontal acquisition of diet-speciﬁc bacteria and that signals of these adaptations still remain in the microbiomes of extant mammals. SESphylosymbiosis ¼ SESphylosymbiosis ¼ Size ancestral OTU contentObs mean size ancestral OTU contentNull ð Þ s:d: size ancestral OTU contentNull ð Þ ð1Þ The phylosymbiosis signal is strong in mammals. Phylo- symbiosis is a pattern describing higher compositional similarity (that is, low b-diversity) between bacterial communities colonizing closely related hosts compared with distantly related hosts4–6. The correlation between microbiome composition and host phylogeny at shallow bacterial phylogenetic scales observed in Fig. 1 does not universally describe the variation and magnitude of phylosymbiosis across the host mammalian tree: For a given ancestor, the higher the SES is, the stronger the signal for phylosymbiosis is. Although previous parsimony and distance-based methods provided conﬂicting results on the existence of a phylosymbiosis signal4,11, we observe that most of the mammalian clades, both young and ancient (up to 80 Myr ago), harbour a signiﬁcant (permutation test, P valueo0.05) and strong phylosymbiosis signal (Fig. 3a). The magnitude of For a given ancestor, the higher the SES is, the stronger the signal for phylosymbiosis is. Although previous parsimony and distance-based methods provided conﬂicting results on the existence of a phylosymbiosis signal4,11, we observe that most of the mammalian clades, both young and ancient (up to 80 Myr ago), harbour a signiﬁcant (permutation test, P valueo0.05) and strong phylosymbiosis signal (Fig. 3a). The magnitude of Phylosymbiosis signal 50 Kangaroo Afr. Elephant Hyrax Armadillo Bush Dog Spec. Bear Polar Bear Black Bear Hyena Lion Horse Zebra Black Rhinoceros V. W. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 At each ancestor, the probability vector for each diet category is transformed into a linear variable bound between 0 (carnivorous) and 1 (herbivorous). Between two ancestors, diet is assumed to evolve linearly. Animal images courtesy of Julien Renaud. Carnivorous Omnivorous Herbivorous Capybara Squirrel Rabbit Baboon Colobus Gorilla Human Chimpanzee Orang-outan Saki Callimicos Black Lemur R-T Lemur Urial Bighorn Sheep Gazelle Springbok Giraffe Okapi V. W. Pig Black Rhinoceros Zebra Horse Lion Hyena Black Bear Polar Bear Spec. Bear Bush Dog Armadillo Hyrax Afr. Elephant Kangaroo c (Ma) 150 (Ma) 150 100 50 0 50 100 0 c Figure 2 \| Gut microbiomes predict extant and ancestral mammalian diets. (a) Diet-correlated bacterial lineages (squares coloured by phylum) and mammals (black dots) separated by dietary distances are jointly projected in the same ordination space using the OMI59 mapping procedure (see Methods). Predicted niche breadth59 of a bacterial lineage is depicted with an ellipse. The time period selected to deﬁne bacterial lineages is B300 Myr ago. Bacterial niche breadths show that many bacterial groups are herbivore or carnivore speciﬁc. However, no bacterial lineage has a niche breadth encompassing omnivores only (omnivores are in brown). (b) Workﬂow for microbiome-based prediction of ancient diet. (c) Inference of ancient mammalian diets. Left: trait-based reconstruction using 1,534 mammals34. Right: Microbiota-based reconstruction with the 33 mammals under study. At each ancestor, the probability vector for each diet category is transformed into a linear variable bound between 0 (carnivorous) and 1 (herbivorous). Between two ancestors, diet is assumed to evolve linearly. Animal images courtesy of Julien Renaud. with the fossil record38. For these reasons, we considered this latter reconstruction as a reference, to which microbiota-based reconstructions were compared. Interestingly, we found that microbiome-based inferences agreed at 70% of ancestral nodes to these reference trait-based predictions (Fig. 2c). We observed that transitions towards herbivory are associated with multiple and convergent horizontal gains of herbivorous-speciﬁc bacterial discrete variable with three different states (Carnivorous, Omnivorous and Herbivorous). We performed a trait-based diet reconstruction with (i) the present mammalian data set (33 mammals) and (ii) a much larger taxonomic sampling (1,534 species of mammals34). This large set of species more exhaustively samples mammalian diversity and previously provided trait-based diet reconstructions that are in agreement NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications 5 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Indeed, when running reconciliations considering mammalian herbivores only, we observed that the frequency of all OTUs having more co-speciation than host swap was similar to the frequency observed with all mammals (Z-test, 70% versus 67%, P value40.05). However, it does not rule out the possibility that ﬁne-grained differences in diet that are locally correlated with host phylogeny and, which are not captured by our diet distance metric, have promoted co-speciation or, alternatively, have created spurious co-speciation signals (see Supplementary Discussion). Co-speciation between hosts and their gut bacterial symbionts. Phylosymbiosis does not necessarily imply vertical inheritance, it only reﬂects congruency between gut bacterial compositions and host phylogeny. We next asked whether this signal could have been generated through vertical inheritance, via co-speciations between symbionts and mammals. Besides co-speciation, at least two other possible mechanisms can generate the same signals of correlation with host phylogeny and of phylosymbiosis10 (Fig. 3a and Supplementary Fig. 1): horizontal inheritance through host swaps within related hosts, and environmental ﬁltering by closely related hosts that select similar symbionts from the environment. Of these various mechanisms, co-speciation is more likely to result in congruent splits between the symbiont and host phylogenetic trees. We used a probabilistic model of host tree/symbiont tree reconciliation implemented in ALE39–41 to measure the magnitude of topological congruence between symbiont and host trees and the number of bacterial lineages experiencing more co-speciation than host-swap events. Altogether, while most bacterial lineages do show evidence of horizontal inheritance, our results also suggest that vertical inheritance is an evolutionary path followed by numerous mammalian gut symbionts and extend the recent results on patterns of co-speciation between gut bacterial lineages and Hominids7 to larger evolutionary scales (see Supplementary Note 9). Co-speciating bacteria are associated with disease in humans. We next investigated the characteristics of bacterial lineages that harbour high co-speciation levels. Interestingly, at the scale of mammals, we found that many widely studied genera showed low levels of vertical inheritance with hosts (Fig. 4d, Supplementary Fig. 13 and Supplementary Note 9). These ﬁndings are consistent with the notion that these metabolically important and cosmo- politan organisms provide similar and probably diet-related functions across diverse hosts and thus are more likely to be transferred across hosts or acquired from the environment with little host selectivity. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 We found that, within the 67% of bacterial clades showing more co-speciation than host swap, only 16% show no signiﬁcant rate differences (Fig. 4a). phylosymbiosis (SES, equation 1) decays with evolutionary time (Fig. 3b), consistent with multiple turnovers over millions of years of host evolution and/or with the long-term evolution of host traits, progressively selecting for different bacteria. Contrary to some previous conclusions11, this turnover of gut microbes during mammalian evolution is not too rapid to erase the phylosymbiosis signal. At the shallow phylogenetic scale at which we measured phylosymbiosis, the correlation between community dissimilarities and host dietary distances is weak but signiﬁcant (Fig. 1a). We measured to what extent the phylosymbiosis signal is affected by dietary shifts. At each focal node in the mammalian phylogeny, we estimated the magnitude of dietary shift after the divergence of the two descendant lineages. We used our trait-based ancestral diet reconstructions using 1,534 mammalian species and measured the average diet distance with the two descendant nodes. As phylosymbiosis is negatively and linearly correlated to node ages (Fig. 3), we compared for each focal mammalian node the residuals of this regression to the magnitude of dietary shift at this node. If ancestral dietary shifts have an impact on phylosymbiosis, we expect to observe a negative correlation between phylosymbiosis residuals and the magnitudes of dietary shift. We show that the effect of dietary shift poorly explains the decrease in phylosym- biosis compared with what would be expected by age: the regression has a negative but weak slope (R ¼ 0.15, Supplementary Fig. 12). We conclude that dietary transitions along mammalian evolution had minor effect on the part of the microbiota that correlates to host phylogeny. Among the OTUs that have a distribution across hosts that correlates with host phylogeny, 89% have more co-speciation than host swap (Fig. 4a,c), supporting that co-speciation is likely to be a major driver of the correlation with host phylogeny. Moreover, 31% of these bacteria show no sign of host swap and have fully congruent phylogenies with the host phylogeny. It is possible, however, that iterative bacterial specializations on related host linages (or host-swap speciation) locally yield concordant nodes in symbionts/host topologies, inﬂating the amount of detected co-speciations42 (see Supplementary Discussion). We did not observe that strong changes in diet are driving the co-speciation signals that we measured. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Pig Okapi Giraffe Springbok Gazelle Bighorn Sheep Urial R-T Lemur Black Lemur Callimicos Saki Orang-outan Chimpanzee Human Gorilla Colobus Rabbit Squirrel Capybara Baboon 10 (Standard effect size) 0 (Ma) 150 100 50 0 50 40 30 20 10 0 20 40 60 Age (Ma) 80 100 Phylosymbiosis signal (standard effect size) a b Figure 3 \| Strong phylosymbiosis signal in mammals. (a) Long-term persistence of host–microbiota associations in mammalian evolution. Mammalian phylogeny with internal nodes coloured according to the degree of possession of clade-speciﬁc compositions of bacteria (clade-speciﬁc phylosymbiosis signal) (see Methods). Black dots denote mammalian clades that do not harbour a signiﬁcant phylosymbiotic signal (Permutation test, 999 permutations, P value40.05). The weak phylosymbiosis signal in Rodents or Afrotheria is likely due to a poor sampling within these two groups. (b) Decay of the phylosymbiosis signal with time (x axis: age of the corresponding nodes). 6 NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10 1038/ncomms14319 \| www nature com/naturecommunications Phylosymbiosis signal 50 Kangaroo Afr. Elephant Hyrax Armadillo Bush Dog Spec. Bear Polar Bear Black Bear Hyena Lion Horse Zebra Black Rhinoceros V. W. Pig Okapi Giraffe Springbok Gazelle Bighorn Sheep Urial R-T Lemur Black Lemur Callimicos Saki Orang-outan Chimpanzee Human Gorilla Colobus Rabbit Squirrel Capybara Baboon 10 (Standard effect size) 0 (Ma) 150 100 50 0 a b 50 40 30 20 10 0 20 40 60 Age (Ma) 80 100 Phylosymbiosis signal (standard effect size) b a Phylosymbiosis signal Figure 3 \| Strong phylosymbiosis signal in mammals. (a) Long-term persistence of host–microbiota associations in mammalian evolution. Mammalian phylogeny with internal nodes coloured according to the degree of possession of clade-speciﬁc compositions of bacteria (clade-speciﬁc phylosymbiosis signal) (see Methods). Black dots denote mammalian clades that do not harbour a signiﬁcant phylosymbiotic signal (Permutation test, 999 permutations, P value40.05). The weak phylosymbiosis signal in Rodents or Afrotheria is likely due to a poor sampling within these two groups. (b) Decay of the phylosymbiosis signal with time (x axis: age of the corresponding nodes). 3 \| Strong phylosymbiosis signal in mammals. (a) Long-t NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications 6 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 an overestimation of the number of co-speciation events. To control for these effects for each individual OTU, we evaluated whether the observed rate difference between co- speciation and host swap is signiﬁcantly higher than expected under a null model (see Methods). NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Observed bacterial lineages After controlling for overfitting Null expectation 0.0 Frequency 1.0 Co-spe > H-swap Non-specific Co-spe ≤ H-swap a Gazelle B. Rhinoceros Okapi Zebra Giraffe Urial Sheep Springbok Horse V. Warty Pig Bighorn Sheep Clost_1 Clost_2 Clost_3 Clost_4 Clost_6 Clost_5 Clost_7 Clost_8 Clost_9 Clost_10 0 50 Ma Hosts Clostriales spp. b d All OTUs OTUs correlating with host phylogeny c 1.0 0.0 0.5 Co-speciation rate * Observed bacterial lineages After controlling for overfitting Null expectation OTUs correlate OTUs do not correlate With host phylogeny 0.0 Average co-speciation rate per genus Mitsuokella Catonella-like Fabibacter-like Caldicoprobacter-like Geosporobacter-like Sporobacter-like Alkalitalea-like Rikenella-like Alloprevotella Sarcina Streptococcus Roseburia Clostridium XI Clostridium sensu stricto Bacteroides Peptococcus-like Cellulosilyticum Gemmiger-like 0.01 e Subdoligranulum 0.5 1.0 0.8 0.6 0.4 0.2 0.00 0.05 Relative abundance 0.15 0.10 Non-IBD CD UC IBD * * Figure 4 \| Large-scale vertical inheritance of mammalian gut symbionts. (a) Frequencies of bacterial lineages harbouring either more co-speciation (Co-spe.) or more host-swap (H-swap) events are shown, either for all OTUs or only for OTUs that are correlated with host phylogeny. Out of the 350 bacterial lineages correlated with host phylogeny and present in at least four hosts, 313 harbour more co-speciation events than host-swaps. Non-speciﬁc (grey) lineages are lineages with a non-signiﬁcantly higher rate of co-speciation than the rate of host-swap. This higher observed rate may be due to overﬁtting to the host tree (see Methods). The Null Expectation bar represents the expected frequency of lineages harbouring more co-speciation than host-swaps by chance (see Methods). (b) Example of a co-speciating bacterial lineage belonging to the Clostridiales order. A blue line indicates the presence of a symbiont in a host. (c) OTUs correlated to host phylogeny harbour higher co-speciation rates than OTUs that are not. Co-speciation rate per OTU is deﬁned as the amount of co-speciation events relative to the number of host-swap events (two-tailed Wilcoxon’s rank-sum Observed bacterial lineages After controlling for overfitting Null expectation 0.0 Frequency 1.0 Co-spe > H-swap Non-specific Co-spe ≤ H-swap a All OTUs OTUs correlating with host phylogeny Observed bacterial lineages After controlling for overfitting Null expectation 0.5 Gazelle B. Rhinoceros Okapi Zebra Giraffe Urial Sheep Springbok Horse V. Warty Pig Bighorn Sheep Clost_1 Clost_2 Clost_3 Clost_4 Clost_6 Clost_5 Clost_7 Clost_8 Clost_9 Clost_10 0 50 Ma Hosts Clostriales spp. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 By contrast, we reasoned that organisms speciﬁcally and tightly linked to host physiology (for example, via immunity) might be more likely to be restricted to their host. In support of this hypothesis, we ﬁnd that 13 out of the 20 bacterial lineages that inhabit humans and have high co-speciation rates (40.8) belong to ﬁve different genera (such as Subdoligranulum) that are strongly associated with a human immune disease, inﬂammatory bowel disease (IBD)43 (Fig. 4e and Supplementary Fig. 14). This enrichment in highly co-speciating OTUs among genera negatively associated with IBD in comparison with lowly co-speciating OTUs is strongly signiﬁcant (permutation test, P value ¼ 0.0015, see Supplementary Note 9). All ﬁve of these genera are strongly depleted in patients affected by both Crohn’s disease and ulcerative colitis43, perhaps suggesting a functional link between co-speciating bacteria and host immune function. It should be noted, however, that even among published IBD studies there can be differences between disease-associated taxa, Interestingly, we ﬁnd that a majority of bacterial clades (67%) harbour more co-speciation than host-swap events (Fig. 4a,b). To test whether this percentage is higher than one would expect by chance only, we evaluated the percentage of bacterial clades that would show more co-speciation that host swap under a null model in which phylogenetic relationships between hosts are disrupted. We observed that, by chance, we would obtain about 10% of OTUs with more co-speciation than host-swap events, much less than the estimates obtained with the observed biological data. g Even though OTUs may have more co-speciation than host-swap events, the co-speciation rate might not be signiﬁcantly higher than the host-swap rate. This might be due to insufﬁcient phylogenetic information in the 16S rRNA data. In addition, an OTU might have more co-speciation than host swap only because the reconciliation algorithm (ALE) overﬁtted the host tree when searching for the best scenario of bacterial evolution, leading to 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Out of the 350 bacterial lineages correlated with host phylogeny and present in at least four hosts, 313 harbour more co-speciation events than host-swaps. Non-speciﬁc (grey) lineages are lineages with a non-signiﬁcantly higher rate of co-speciation than the rate of host-swap. This higher observed rate may be due to overﬁtting to the host tree (see Methods). The Null Expectation bar represents the expected frequency of lineages harbouring more co-speciation than host-swaps by chance (see Methods). (b) Example of a co-speciating bacterial lineage belonging to the Clostridiales order. A blue line indicates the presence of a symbiont in a host. (c) OTUs correlated to host phylogeny harbour higher co-speciation rates than OTUs that are not. Co-speciation rate per OTU is deﬁned as the amount of co-speciation events relative to the number of host-swap events (two-tailed Wilcoxon’s rank-sum test, P valueo0.001). (d) Average co-speciation rate per bacterial genus. For a full list of genera, see Supplementary Fig. 13. (e) Subdoligranulum has high co-speciation rates and is correlated with IBD in humans (two-tailed Wilcoxon’s rank-sum test). CD, Crohn’s disease; UC, ulcerative colitis. Other genera with similar patterns are shown in Supplementary Fig. 14. so future studies are needed to conﬁrm these preliminary observations and to turn these intriguing correlations into proper demonstration of causation. lineages most promising for such detailed study which are not the bacterial groups that have been the focus of most prior work and suggest their relevance to human health and disease. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 b 0.01 b a n d Observed bacterial lineages for overfitting Null expectation 0.0 Average co-speciation rate per genus Mitsuokella Catonella-like Fabibacter-like Caldicoprobacter-like Geosporobacter-like Sporobacter-like Alkalitalea-like Rikenella-like Alloprevotella Sarcina Streptococcus Roseburia Clostridium XI Clostridium sensu stricto Bacteroides Peptococcus-like Cellulosilyticum Gemmiger-like 1.0 0.8 0.6 0.4 0.2 e e Subdoligranulum 0.00 0.05 Relative abundance 0.15 0.10 Non-IBD CD UC IBD * e e c 1.0 0.0 0.5 Co-speciation rate * OTUs correlate OTUs do not correlate With host phylogeny d c Relative abundance IBD Figure 4 \| Large-scale vertical inheritance of mammalian gut symbionts. (a) Frequencies of bacterial lineages harb Figure 4 \| Large-scale vertical inheritance of mammalian gut symbionts. (a) Frequencies of bacterial lineages harbouring either more co-speciation Figure 4 \| Large-scale vertical inheritance of mammalian gut symbionts. (a) Frequencies of bacterial lineages harbouring either more co-speciation (Co-spe.) or more host-swap (H-swap) events are shown, either for all OTUs or only for OTUs that are correlated with host phylogeny. Out of the 350 bacterial lineages correlated with host phylogeny and present in at least four hosts, 313 harbour more co-speciation events than host-swaps. Non-speciﬁc (grey) lineages are lineages with a non-signiﬁcantly higher rate of co-speciation than the rate of host-swap. This higher observed rate may be due to overﬁtting to the host tree (see Methods). The Null Expectation bar represents the expected frequency of lineages harbouring more co-speciation than host-swaps by chance (see Methods). (b) Example of a co-speciating bacterial lineage belonging to the Clostridiales order. A blue line indicates the presence of a symbiont in a host. (c) OTUs correlated to host phylogeny harbour higher co-speciation rates than OTUs that are not. Co-speciation rate per OTU is deﬁned as the amount of co-speciation events relative to the number of host-swap events (two-tailed Wilcoxon’s rank-sum test, *P valueo0.001). (d) Average co-speciation rate per bacterial genus. For a full list of genera, see Supplementary Fig. 13. (e) Subdoligranulum has high co-speciation rates and is correlated with IBD in humans (two-tailed Wilcoxon’s rank-sum test). CD, Crohn’s disease; UC, ulcerative colitis. Other genera with similar patterns are shown in Supplementary Fig. 14. Figure 4 \| Large-scale vertical inheritance of mammalian gut symbionts. (a) Frequencies of bacterial lineages harbouring either more co-speciation (Co-spe.) or more host-swap (H-swap) events are shown, either for all OTUs or only for OTUs that are correlated with host phylogeny. NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 dietary items (Invertebrate, Vertebrate (excluding ﬁshes), Rotting carcass, Fish, Unknown Vertebrate, Fruits, Nectar, Seed, other plant materials (for example, grass, ground vegetation, seedlings, weeds, lichen, moss, small plants, reeds, cultivated crops, forbs, vegetables, fungi, roots, tubers, legumes, bulbs, leaves, above ground vegetation, twigs, bark, shrubs, herbs, shoots, aquatic vegetation, aquatic plants)) are deﬁned and each species is assigned a percentage for each item depending on its diet. We used Euclidian distance to build the diet distance matrix. As our dietary categories represent fuzzy variables (proportions between 0 and 1), we also computed diet distances using the Manly index, implemented in the dist.ktab function in the ‘ade4’ R package53. This control revealed no impact on our results, so we only present results obtained with the Euclidian distance matrix. support and power to discriminate between alternative hypotheses in all our analyses. support and power to discriminate between alternative hypotheses in all our analyses. We used USEARCH v8.0.1517 (ref. 46) to dereplicate 16S sequences. Chimera sequences were removed from the data, using the UCHIME algorithm47 and the reference Gold database, containing 5,181 16S sequences. We subsequently used the de novo algorithm, also implemented in UCHIME, to further remove chimeras. We used the Ribosomal Database Project Classiﬁer48 to assign a taxonomy to each unique 16S sequence. We retained bacterial sequences with an assignment probability 40.8 at the phylum level (85% of all sequences). The ﬁnal data set comprises 44,444 unique bacterial sequences. Phylogenetic reconstruction. To use BDTT on our data, we reconstructed the phylogenetic tree of all 16S unique sequences. First, we added the V2 region of the 16S rDNA genes from 139 Cyanobacteria, 115 Rickettsiales, 21 Chlorobium and 24 Chromatiales to the set of 44,444 stool sequences. These external 16S sequences were used to incorporate calibrations when computing the divergence times (see below) and were subsequently removed for all our analyses. Measuring correlation proﬁles with diet and host phylogeny. For each time period, we correlated b-diversities to host dietary and phylogenetic distances. We used (i) Mantel test54 (with Pearson’s correlations, that is, a linear model) and (ii) a non-linear model (Generalized Dissimilarity Modeling (GDM) approach55) to measure correlation coefﬁcients between b-diversity and host phylogenetic or dietary distances. All Mantel tests were run with the MRM (Multiple Regression on distance Matrices) function from the ‘ecodist’ R package56. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 This function has the interesting property of allowing the user to run regression models with multiple predictors, a property that we used to measure the combined effect of host phylogeny and host diet in the prediction of gut community dissimilarities (see below in this section). The Mantel test (with Pearson correlations) assumes a linear model between predictor and response variables. To evaluate the robustness of our results to the potential nonlinearity of the relationship between compositional dissimilarity and host dietary or phylogenetic distances, we also computed correlation coefﬁcients with the GDM approach55 to relax the linear hypothesis. The use of GDM did not affect the overall correlation patterns and we noted that the explanatory only slightly increases in comparison with the linear Mantel approach (Supplementary Fig. 4). 2 We aligned all 16S reads with the sina program (version 1.2.11) using the Silva database49, producing an alignment of 751 sites. We then removed sites containing 495% of gaps from the alignment. The ﬁnal alignment contains 257 positions. To limit computational burden, we used FastTree50 to reconstruct the ML phylogenetic tree of all 44,444 unique stool sequences plus 299 external sequences. We used the GTR model and the default CAT approximation to model rate heterogeneity across sites. We used the RDP taxonomy to constrain the topology of the phylogeny, forcing all phyla and all classes within the Proteobacteria (alpha, beta, gamma, delta, epsilon) to be monophyletic. The phylogenetic tree was rooted either on the branch separating Actinobacteria or Firmicutes from the rest of the sequences. Finally, we used PATHd8 (ref. 51) to produce a cladogram from the ML tree reconstructed by FastTree (see ‘Phylogenetic reconstructions’ in the Supplementary Methods for further details). To test whether R2 values were signiﬁcantly higher than expected by chance, we used permutation tests on the distance matrices and computed the 95% credibility interval of correlations between community dissimilarities and both host dietary and phylogenetic distances at each phylogenetic slice, producing 95% credibility envelopes for both factors. At each slice, host names were randomly shufﬂed 100 times in dietary and phylogenetic distance matrices, and correlations with community dissimilarities (b-diversities) were re-computed for each replicate, yielding a distribution of 100 R2 coefﬁcients with respect to each factor. A correlation with either host diet or phylogeny is considered signiﬁcant when the R2 is higher than the upper bound of the 95% credibility interval. 57 b-diversity through time and phylogenetic clustering. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 As explained in the section ‘Phylogenetic decomposition of community dissimilarities’ in the Results, we computed compositional turnover (using Sørensen27 or Bray–Curtis52 metrics) between communities at different time periods along the bacterial phylogenetic timescale. However, BDTT can be used with any type of taxonomic diversity metrics, such as Jaccard or Jensen-Shannon. BDTT can be employed on communities of both microorganisms and macroorganisms, in order to study the phylogenetic/timescale-dependent effect of a given factor on the distribution of biological entities across host sites or spaces. Finally, variance partitioning57 was used to quantify the variance in microbiome composition explained by the joint effect of host diet and phylogeny. We ﬁrst measured the correlation coefﬁcient associated with the union of host phylogenetic and host dietary effects (that is, using both predictors in the same model). Then we computed the correlation coefﬁcient associated with the intersection effect of host phylogeny and diet (equation (3)): Validation of BDTT on simulated data. To test whether the BDTT approach is able to disentangle the effect of factors shaping community assembly at different phylogenetic scales, we carried out simulation experiments. The rationale is to assemble communities under known processes, here under two independent environmental ﬁlters. Species are ﬁltered out of communities according to their two ‘environmental preferences’ or ‘traits’ (each trait represents a particular environmental variable). We make these traits evolve with decreasing evolutionary rates over time under models that differ in the strength of the rate decrease (see below). More speciﬁcally, one trait displays phylogenetic signal towards the tip of the phylogeny while the second trait will carry phylogenetic signal in higher regions of the tree. If communities are ﬁltered according to these two traits at the same time, we expect BDTT to be able to discriminate the phylogenetic scale at which each environmental gradient primarily shapes community composition (for example, the effect of the environmental variable that harbours deep phylogenetic signal should be seen at high phylogenetic scales in the BDTT analysis). Intersection R2 ¼ R2 Phylogeny only ð Þ þ R2 Diet only ð Þ Union R2 Phylogeny þ Diet ð Þ ð3Þ Intersection R2 ¼ R2 Phylogeny only ð Þ þ R2 Diet only ð Þ Union R2 Phylogeny þ Diet ð Þ ð3Þ ð3Þ Distribution and niche of individual bacterial lineages. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 At all bacterial scales deﬁned by time or evolutionary distance, we measured the percentage of individual bacterial lineages (OTU) that have a distribution across mammalian hosts correlated to host phylogenetic and dietary distances, using permutation multivariate analysis of variance tests58 and a false discovery rate (FDR) approach to correct for multiple tests. The simulation experiment contains four steps: (i) we simulated a phylogeny of 200 species. (ii) We simulated traits (representing the environmental preference of species) along this phylogeny with different models that have scale-dependent rates of trait evolution, that is, producing traits with phylogenetic signals that are located at different phylogenetic scales. (iii) We assembled communities of species with two environmental gradients that ﬁlter species according to their traits ( ¼ environmental preferences). These two gradients are linked to two independent environmental preferences of species ( ¼ two traits) that show phylogenetic signal at different phylogenetic scales (as simulated in (ii)). (iv) We applied BDTT to these data sets and tested whether a phylogenetic-scale disparity was observed (for further details, see ‘Validation of BDTT on simulated data’ in the Supplementary Methods). The ecological niche of a species (here each bacterial lineage) can be described by its mean and breadth along an environmental gradient. To describe the niche of gut bacterial lineages with respect to host diet, we used the multivariate co-inertia analysis called Outlier Mean Index59. This technique enabled us to project both hosts and their diet-associated bacteria onto the same axes and to compute niche means and niche breadth for each bacterial lineage. We used this technique for two reasons. First, contrarily to canonical correspondence analysis or redundancy analysis, the Outlier Mean Index does not assume a particular response curve of the species to the environment (for example, unimodal or linear). Second, this ordination technique gives equal weight to all hosts, independently of their bacterial lineage richness. Reconstruction of ancestral communities. Mammals diverged 4100 Myr ago. We expect that saturation of the phylogenetic signal in OTU turnover leads to systematic biases in inference of ancestral community compositions. To overcome these issues, we used the Count program37 to estimate the ancestral gut community compositions in a phylogenetic and probabilistic framework. Count was originally designed to model the evolution of gene families through gene gain and loss. NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications Methods M li Our ﬁndings are relevant to the recent debate over the extent to which hosts and their associated microbial communities should be considered as holobionts, deﬁned as coherent genomic systems on which selection operates44,45. However, as was recently pointed out10, evidence for a tight association between a small number of taxa and their hosts does not necessarily generalize to the entire microbiome, and we ﬁnd that only a minority of lineages fully co-speciate with their hosts. Moreover, evidence for co-speciation does not necessarily imply co-evolution, which needs to be established through more in-depth and mechanistic studies. Nonetheless, our ﬁndings reveal the speciﬁc microbial Mammalian microbiome data set. We analysed a data set of mammalian gut microbiota containing 33 mammals11 that represent 10 mammalian orders and cover various diets (carnivorous, herbivorous and omnivorous)30. These data are ideal because they cover a large diversity of mammalian clades and were produced at the same time within a single study, avoiding biases introduced with variation in DNA extraction protocols, choice of 16S primers and sequencing platform. The data consist of amplicons of the V2 region of the 16S rRNA gene. As conspeciﬁc samples were originally available in this data set for seven host species, only one individual per species was retained for further analyses (and chosen at random) to focus on the evolution of gut microbiota at the interspecies level only. We also ran analyses including the other conspeciﬁc samples to control for the effect of intrahost species variability in gut microbiome compositions. The number of hosts in this data set (33 mammals) did not preclude us from obtaining strong statistical NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 presence/absence at each internal node of the mammalian host tree. We used a non-homogeneous version of the gain/loss model where gain and loss parameters are allowed to vary across the branches of the host tree. Variation of gain and loss rates across bacterial lineages is modeled using discrete Gamma distributions, each with four rate categories. All rates were estimated in a ML framework. With all ML rate estimates and the observed presence/absence matrix, we computed the posterior probability for each bacterial lineage (OTU) to be present at each internal node of the host tree. At a given node, the expected total number of OTUs was computed by summing all OTU-speciﬁc posterior probabilities of being present at this node. Phylosymbiosis along the host phylogeny. To measure phylosymbiosis, we used gut microbiota compositions deﬁned at a recent phylogenetic timescale, recent enough so that the correlation with host phylogeny is near maximal but deep enough in the phylogeny to ensure that bacterial lineages appear in a sufﬁcient number of hosts. The selected slice creates 1,484 bacterial lineages (OTUs) that are observed in Z2 hosts. On average, each OTU has an observed DNA similarity of B97%. We rareﬁed the OTU table to avoid reconstruction biases owing to unequal sequencing across samples. In its original deﬁnition4,5,61, phylosymbiosis has been described as the congruence between the host tree and the tree of communities, inferred using the compositional dissimilarities between gut microbiota. Previous attempts to detect and measure phylosymbiosis from gut microbiome data employed parsimony- and distance-based methods to compute the tree of communities (for example, Neighbor Joining62), without probabilistic modelling of OTU evolution. In the case of parsimony4,11, previous authors considered all OTUs independently and recoded OTU relative frequencies into ordered discrete states, from 0 to 6, to reﬂect log-unit differences in their occurrence. The tree minimizing the number of transitions between these ordered states was considered as the Maximum Parsimony tree of communities. For distance-based methods, a dissimilarity matrix computed with a b-diversity metric, such as Bray–Curtis or UniFrac, was used to compute a dendogram (that is, the tree of community dissimilarities) with a hierarchical clustering method5,61. However, over millions of years of host evolution, many OTU gain and loss events have occurred, possibly randomizing the genuine historical signal of compositional change along the mammalian phylogeny. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 In molecular phylogenetics, parsimony- and distance-based approaches are known to reconstruct less accurate topologies and to be more sensitive to substitution saturation than probabilistic approaches that employ evolutionary substitution models63,64. Here, at the scale of mammals, which diverged 4100 Myr ago, we also expect issues related to the saturation of the phylogenetic signal in OTU turnover, leading to systematic biases in community tree reconstruction. Using the mammalian data set that we re-analysed in this study, Muegge et al.11 used approaches that did not employ explicit modelling of OTU dynamics of gain and loss to search for patterns of phylosymbiosis. They concluded that mammalian community compositional dissimilarities did not mirror host distances, questioning the existence of long-term phylosymbiosis in mammals. Reconstruction of ancestral diets. We ﬁrst present methodological details for reconstructing ancestral diets using extant and ancestral microbiome compositions. Our microbiome-based method to predict ancestral diets uses the correlation between extant diet and extant gut bacterial community compositions. Assuming that the relationship between diet and bacterial composition has not varied since the last common ancestor of mammals, ancestral diet can be predicted from reconstructed ancestral community compositions (Fig. 2b). The effects of carnivory and herbivory on the distribution of bacterial lineages are not maximal at the same phylogenetic timescale (Fig. 1d). Thus we selected a time slice (300 Myr ago) allowing us to construct communities having both carnivorous- and herbivorous-speciﬁc bacterial lineages to build a regression model to predict diet. We converted the OTU table to presence/absence data and restricted our analysis to bacterial lineages having a distribution across hosts that signiﬁcantly correlates with diet (permutation multivariate analysis of variance tests, with FDR correction for multiple tests). We then used a principal component analysis (PCA) to project host species according to the composition of their microbiota. Host coordinates along the ﬁrst axes of the PCA are then used as independent (explanatory) variables in a multinomial logistic regression model, in which the dependent (predicted) variable is diet, discretized into three categories: carnivore, omnivore, and herbivore. The optimal number of independent variables (PCA axes) to include in the regression model can be determined with model selection criteria such as Akaike’s Information Criterion (Supplementary Fig. 10a). For each ancestor, its reconstructed community is then projected onto the PCA. Projection coordinates are then used to predict a corresponding diet with the multinomial logistic regression. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 As for the reconstruction of ancestral diets using extant diets encoded as discrete character traits: we considered diet as a discrete variable with three states (Carnivorous, Omnivorous and Herbivorous). We used the ARD (All-Rates-Different) model of trait evolution implemented in the ‘ace’ function from the ‘ape’ R package60 to estimate the ML transition rates across dietary categories and to infer posterior probabilities for each diet state at each ancestor. After measuring the magnitude of the phylosymbiosis signal, we investigated its pattern along the phylogeny of hosts. First, we tested whether the time span of microbiome turnover over millions of years of host evolution has an impact on phylosymbiosis, yielding to weaker phylosymbiosis signals for the most ancient mammalian clades and higher signals for the most recently diverged clades. We correlated the amount of phylosymbiosis (SES values) to crown ages of each focal mammalian ancestor (Fig. 3). Although we computed a R2 coefﬁcient, we did not test whether the correlation was signiﬁcant, because the data points are non- independent. Second, we tested whether dietary shifts during mammal evolution reduced the amount of bacterial lineages shared within mammalian clades and negatively impacted the signal of phylosymbiosis. We correlated the residuals of the linear regression between the amount of phylosymbiosis and time (SES–age) to a quantiﬁcation of dietary shifts. To measure these dietary shifts, we computed dietary distances between successive nodes in the mammalian phylogeny. We used ancestral diets reconstructed both with the trait-based approach using the dietary data of 1,534 mammals and with the microbiome-based approach. Each approach provides a probability distribution for each dietary category at each ancestral mammalian node. We computed the dietary distance between two consecutive nodes by computing a distance between the two probability distributions. When computing this distance, we accounted for the fact that omnivory is the union of carnivory and herbivory. Each node has three probabilities (summing to one) that correspond to the three diets (omnivory, carnivory and herbivory). We ﬁrst assigned one diet (omnivory, carnivory or herbivory) to each node by drawing a diet category from a multinomial distribution deﬁned with the three probabilities. Second, we recoded the diet as a two state variable, either ‘Carnivore’ or ‘Herbivore’: if the node was ﬁrst assigned ‘Omnivore’, the node is now assigned both Carnivore and Herbivore. Three conﬁgurations are possible: herbivore only, carnivore only, or both herbivore and carnivore. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 For each ancestor, the prediction provides a vector of probabilities for each diet category. To visually represent the evolution of diet along a linear gradient from carnivory to omnivory to herbivory, we transformed each vector of diet probabilities into a single value. We assigned 0 to carnivory, 0.5 to omnivory and 1 to herbivory and multiplied each probability by its corresponding weight and then summed the weighted probabilities. The new variable has values between 0 and 1, with 0 representing a probability of 1 to be a carnivorous and 1 a probability of 1 to be herbivorous. We also performed ancestral diet reconstruction at two other time slices to control for the inﬂuence of the phylogenetic scale at which community compositions are deﬁned on the ability to accurately infer ancestral diet. The 300 Myr ago time slice deﬁnes monophyletic clusters of 16S sequences (OTUs) with, on average, B94% similarity. We selected another time slice around 120 Myr ago creating OTUs with B97% 16S similarity. The last time slice used to reconstruct ancient diet is the slice at which the correlation between community dissimilarity and diet is maximal (see Fig. 1), around 600 Myr ago, building OTUs with B91% 16S similarity. Here we extend the original deﬁnition to apply the ‘phylosymbiosis’ concept to each individual host clade. Using ancestral community reconstruction with a model-based and probabilistic approach, we measured for each ancestor the amount of bacterial lineages that were ancestrally present and compared this number to a null expectation in which the observed relationships between hosts and bacteria are disrupted. To compute this null expectation, we ran Count under the same non-homogeneous gain/loss model (see ‘Reconstruction of ancestral communities’ in the Methods), with 100 random OTU tables created with the independent swap algorithm65 (50,000 iterations). This algorithm maintains OTU occurrence frequency across hosts and sample-speciﬁc richness during the randomization of the data and is widely used in ecology65. With these random distributions, we computed P values for each ancestor to detect those that have a signiﬁcantly higher number of present OTUs than randomly expected; these ancestors then deﬁne clades with mammalian species that possess gut microbiomes that share more bacterial lineages between each other than randomly expected. We computed the magnitude of the phylosymbiosis signal along the mammalian host tree, for each ancestor, by computing a SES per branch/node (see Results, equation (1)). NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Along host evolution, the composition of gut microbiota changes through gain or loss of OTUs, so probabilistic models implemented in Count are adequate to model gut microbiota evolution. Host phylogeny and dietary data. The 33 hosts in the data set11 represent 10 mammalian orders and cover various diets (carnivorous, herbivorous and omnivorous)30. Host phylogenetic time distances between our 33 mammals were deduced from a time-calibrated ultrametric phylogenetic tree reconstructed with 4,510 species30 and that was subsequently updated to include 5,020 species31. Without adding new mammalian species, we further updated the phylogenetic relationships and divergence times within the Carnivora clade with a highly resolved supertree that was recently published32. We used the EltonTraits 1.0 database to compute dietary distances23. EltonTraits compiles dietary attributes for a large number of mammals, including the 33 species present in our data set. Nine OTUs are considered as independent from each other. We used a probabilistic birth–death model of gain and loss of OTUs to compute probabilities of the 9 ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 distances between two nodes using the Sørensen metric. This procedure66 enables us to account for the fact that a carnivorous (or herbivorous) diet is a subset of an omnivorous diet. We repeated this procedure 100 times (draw samples from the multinomial distribution differ at each repetition) and we used the mean dietary distance between the two distributions. 2. Bennett, G. M. & Moran, N. A. Heritable symbiosis: the advantages and perils of an evolutionary rabbit hole. Proc. Natl Acad. Sci. USA 112, 10169–10176 (2015). 3. Sachs, J. L., Skophammer, R. G. & Regus, J. U. Evolutionary transitions in bacterial symbiosis. Proc. Natl Acad. Sci. USA 108(Suppl 2): 10800–10807 (2011). Detecting co-speciating bacterial lineages. We measured the amount of co-speciation and host-swap events per OTU at the same time slice that we used to measure the phylosymbiosis signal. We only conserved OTUs that are present in at least four hosts and we used the rareﬁed data matrix. We extracted the individual alignments for each OTU from the global alignment previously used to reconstruct the phylogenetic tree of all 16S sequences. Replicate sequences were treated as follows. When identical sequences were observed multiple times in a single host, only one sequence was retained in the alignment. Replicate sequences observed in multiple hosts were all conserved in the OTU alignment. 39 41 4. Ochman, H. et al. Evolutionary relationships of wild hominids recapitulated by gut microbial communities. PLoS Biol. 8, e1000546 (2010). g 5. Brucker, R. M. & Bordenstein, S. R. The hologenomic basis of speciation: 5. Brucker, R. M. & Bordenstein, S. R. The hologenomic basis of speciation: gut bacteria cause hybrid lethality in the genus Nasonia. Science 341, 667–669 (2013). 6. Sanders, J. G. et al. Stability and phylogenetic correlation in gut microbiota: lessons from ants and apes. Mol. Ecol. 23, 1268–1283 (2014). 7. Moeller, A. H. et al. Cospeciation of gut microbiota with hominids. Science 353, 380–382 (2016). p g We used the ALE algorithm39–41 to detect the co-speciation and host-shift events along the phylogenetic tree of 16S sequences. ALE employs a probabilistic and event-based approach, reconciling the symbiont phylogenetic tree with the mammalian host tree using a probabilistic model of co-speciation, host-swap, intrahost speciation and extinction of symbionts within the host tree. ARTICLE De Filippo, C. et al. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc. Natl Acad. Sci. USA 107, 14691–14696 (2010). 17. Degnan, P. H. et al. Factors associated with the diversiﬁcation of the gutmicrobial communities within chimpanzees from Gombe National Park. Proc. Natl Acad. Sci. USA 109, 13034–13039 (2012). g We then compared the amount of OTUs that harbour more co-speciation than host swap with a null expectation. This null model allowed us to measure the amount of OTUs that were observed to have more co-speciations than host swaps owing to chance or to the overﬁtting to the host tree, during ALE reconciliations. We proceeded as follows. We shufﬂed the names of the host tree to break up the initial host phylogenetic relationships and re-ran ALE to measure the amount of co-speciation and host swaps under this null expectation. We performed 100 replicates of the null model per OTU. For each OTU, we computed the number Nnull of null replicates for which the amount of co-speciation events is equal to or higher than the number of host-swap events. We computed a P value for each OTU by dividing Nnull by the number of replicates þ 1. The null expectation of the number of OTUs harbouring more co-speciation than host-swap events is then simply obtained by summing the P values (null expectations are represented in Fig. 4a, ‘Null expectation’ bars). 18. Turnbaugh, P. J. et al. A core gut microbiome in obese and lean twins. Nature 457, 480–484 (2009). 19. Blekhman, R. et al. Host genetic variation impacts microbiome composition across human body sites. Genome Biol. 16, 191 (2014). 20. Goodrich, J. K. et al. Human genetics shape the gut microbiome. Cell 159, 789–799 (2014). 21. Vaishampayan, P. A. et al. Comparative metagenomics and population dynamics of the gut microbiota in mother and infant. Genome Biol. Evol. 2, 53–66 (2010). 22. Oh, P. L. et al. Diversiﬁcation of the gut symbiont Lactobacillus reuteri as a result of host-driven evolution. ISME J. 4, 377–387 (2010). 23. Wilman, H. et al. EltonTraits 1.0: species-level foraging attributes of the world’s birds and mammals. Ecology 95, 2027 (2014). 24. Bonder, M. J. et al. The effect of host genetics on the gut microbiome. Nat. Genet. 48, 1407–1412 (2016). ARTICLE To control for overﬁtting, we computed for each replicate r the difference Dr,null between the number of co-speciations and the number of host swap and compared this null difference with the observed difference Dr,obs for this OTU. We computed a P value for each OTU, deﬁned as the number of times Dr,null is ZDr,obs, divided by the number of replicates þ 1. After correcting for multiple tests with an FDR approach, if a P value is higher than the signiﬁcance threshold (here, 0.05), then we cannot reject the hypothesis that chance and/or overﬁtting to the host tree are the reasons for observing more co-speciations than host swaps. The proportion of OTUs that were observed to have more co-speciation than host swap owing to overﬁtting is represented in grey in Fig. 3c. 25. Martiny, J. B. H., Jones, S. E., Lennon, J. T. & Martiny, A. C. Microbiomes in light of traits: a phylogenetic perspective. Science 350, aac9323 (2015). 26. Whittaker, R. H. Vegetation of the Siskiyou mountains, Oregon and California. Ecol. Monogr. 30, 279–338 (1960). 27. Sørensen, T. A method of establishing groups of equal amplitude in plant sociology based on similarity of species and its application to analyses of the vegetation on Danish commons. Kongelige Danske Videnskabernes Selskab 5, 1–34 (1948). 28. Lozupone, C. & Knight, R. UniFrac: a new phylogenetic method for comparing microbial communities. Appl. Environ. Microbiol. 71, 8228–8235 (2005). Code availability. BDTT has been implemented in R. All codes needed to run BDTT, as well as illustrative examples, are available here: https://github.com/ FloMazel/BDTT. 29. Cavender-Bares, J. & Reich, P. B. Shocks to the system: community assembly of the oak savanna in a 40 year ﬁre frequency experiment. Ecology 93, S52–S69 (2012). Data availability. The data that support the ﬁndings of this study (the multiple sequence alignment of the processed 16S data that we used for all phylogenetic analyses, the OTU table of unique sequences, the calibrated and non-calibrated bacterial phylogenetic trees used in the BDTT analyses, the matrix of host phylogenetic distances and the matrix of host dietary distances) are available from: https://github.com/mgroussi/MammalianGuts 30. Bininda-Emonds, O. R. P. et al. The delayed rise of present-day mammals. Nature 446, 507–512 (2007). 31. Fritz, S. A., Bininda-Emonds, O. R. P. & Purvis, A. Geographical variation in predictors of mammalian extinction risk: big is bad, but only in the tropics. Ecol. Lett. 12, 538–549 (2009). 32. Nyakatura, K. ARTICLE Initially, the ALE algorithm has been designed to reconcile gene trees with species trees with a model of gene speciation, transfer, duplication and loss. In the case of reconciliation between a symbiont tree and a host tree, these events correspond to co-speciation, host-swap, intrahost speciation and extinction events, respectively. ALE has three key features: (i) it is implemented in a probabilistic framework, (ii) it makes use of time calibrations in the host tree to constrain in time the host swaps during symbiont evolution (host swaps cannot happen back in time), and (iii) it also accounts for uncertainties in the OTU tree reconstruction. In the following, only the relative amount of co-speciation versus host swap is considered, as these events allow us to measure the amount of vertical versus horizontal inheritance in the gut microbiota. 8. Hosokawa, T., Kikuchi, Y., Nikoh, N., Shimada, M. & Fukatsu, T. Strict host-symbiont cospeciation and reductive genome evolution in insect gut bacteria. PLoS Biol. 4, e337 (2006). 9. Chung, H. et al. Gut immune maturation depends on colonization with a host-speciﬁc microbiota. Cell 149, 1578–1593 (2012). 10. Moran, N. A. & Sloan, D. B. The hologenome concept: helpful or hollow? PLoS Biol. 13, e1002311 (2015). 11. Muegge, B. D. et al. Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans. Science 332, 970–974 (2011). 12. Delsuc, F. et al. Convergence of gut microbiomes in myrmecophagous mammals. Mol. Ecol. 23, 1301–1317 (2013). 13. Ley, R. E. et al. Evolution of mammals and their gut microbes. Science 320, 1647–1651 (2008). 14. Carmody, R. N. et al. Diet dominates host genotype in shaping the murine gut microbiota. Cell Host Microbe 17, 72–84 (2015). ALE uses the information in the alignment of sequences to search for the ML values of co-speciation and host-swap rates. When the associated ML symbiont tree has been found, ALE provides the ML reconciliation scenario including the numbers of co-speciation and host swap. ALE uses a distribution of symbiont trees to search for the best reconciliation scenario. We run Phylobayes67 to obtain a posterior distribution of OTU trees with a GTR model and a discrete gamma distribution with four categories to model rate variation across sites. We run Phylobayes for 10,000 generations, sampling at every generation with an initial burn-in of 1,000 generations. 15. Yatsunenko, T. et al. Human gut microbiome viewed across age and geography. Nature 486, 222–227 (2012). 16. ARTICLE & Bininda-Emonds, O. R. Updating the evolutionary history of Carnivora (Mammalia): a new species-level supertree complete with divergence time estimates. BMC Biol. 10, 12 (2012). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 Third, we computed dietary We compared the accuracy and precision of trait- and microbiome-based models as follows. To measure the predictive power of our microbiome-based method, we used cross-validation experiments to evaluate the accuracy of the inference of extant diets using extant microbiome compositions (see Supplementary Fig. 10b,c and ‘Accuracy of our microbiome-based method of diet prediction’ in Supplementary Note 7). To compute precision of ancestral diets, we compared the trait- and microbiota-based reconstructions on 33 species to the trait-based reconstruction based on 41,500 species because this large taxonomic sampling exhaustively samples mammalian diversity and provides diet inferences that are in agreement with the fossil record38. To compute the precision of inferences, we measured the Shannon entropy of each probability vector. The entropy is measured as follows, with I the space of possible diet states and iAI: Entropy ¼ X i pi log pi ð2Þ ð2Þ The entropy measures how spread a probability distribution is: if the probability vector is uniform, the entropy is maximal. However, if the distribution is concentrated on a single category (meaning that probabilities are low for other categories), the entropy is low. In our case, as we are making predictions, we want to obtain probability distributions with entropies as low as possible, to be able to assign with conﬁdence an ancestral diet to a given ancestor. 10 NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms14319 34. Price, S. A., Hopkins, S. S. B., Smith, K. K. & Roth, V. L. Tempo of trophic evolution and its impact on mammalian diversiﬁcation. Proc. Natl Acad. Sci. USA 109, 7008–7012 (2012). 61. Brucker, R. M. & Bordenstein, S. R. The roles of host evolutionary relationships (genus: Nasonia) and development in structuring microbial communities. Evolution 66, 349–362 (2012). 35. Sanders, J. G. et al. Baleen whales host a unique gut microbiome with similarities to both carnivores and herbivores. Nat. Commun. 6, 8285 (2015). 62. Saitou, N. & Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425 (1987). 63. Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 (2003). 36. Sperling, E. A. et al. Oxygen, ecology, and the Cambrian radiation of animals. Proc. Natl Acad. Sci. USA 110, 13446–13451 (2013). 37. Csu+ro¨s, M. Count: evolutionary analysis of phylogenetic proﬁles with parsimony and likelihood. Bioinformatics 26, 1910–1912 (2010). 64. Felsenstein, J. Inferring Phylogenies (Sinauer Associates Incorporated, 2004). parsimony and likelihood. Bioinformatics 26, 1910–1912 (2010) 38. Stevens, C. E. & Hume, I. D. Comparative Physiology of the Vertebrate Digestive System (Cambridge University Press, 2004). 65. Gotelli, N. J. Null model analysis of species co-occurrence patterns. Ecology 81, 2606–2621 (2008). 39. Szo¨llo˜si, G. J., Boussau, B., Abby, S. S., Tannier, E. & Daubin, V. Phylogenetic modeling of lateral gene transfer reconstructs the pattern and relative timing of speciations. Proc. Natl Acad. Sci. USA 109, 17513–17518 (2012). 66. Pavoine, S., Vallet, J., Dufour, A. B., Gachet, S. & Daniel, H. On the challenge of treating various types of variables: application for improving the measurement of functional diversity. Oikos 118, 391–402 (2009). 40. Szo¨llo˜si, G. J., Rosikiewicz, W., Boussau, B., Tannier, E. & Daubin, V. Efﬁcient exploration of the space of reconciled gene trees. Syst. Biol. 62, 901–912 (2013). 67. Lartillot, N., Lepage, T. & Blanquart, S. PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating. Bioinformatics 25, 2286–2288 (2009). 41. Szo¨llo˜si, G. J., Tannier, E., Lartillot, N. & Daubin, V. Lateral gene transfer from the dead. Syst. Biol. 62, 386–397 (2013). Acknowledgements 42. de Vienne, D. M. et al. Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution. New Phytol. 198, 347–385 (2013). This work was funded by The Center for Microbiome Informatics and Therapeutics and is also supported by the National Science Foundation under Grant No. 0821391. F.M., S.L. and W.T. received support from the European Research Council under the European Community’s Seven Framework Programme FP7/2007-2013 Grant Agreement no. 281422 (TEEMBIO). F.M., S.L. and W.T. belong to the Laboratoire d’E´cologie Alpine, which is part of Labex OSUG@2020 (ANR10 LABX56). Part of the computations was performed using the CiGRI platform of the CIMENT infrastructure (https://ciment.ujf- grenoble.fr), which is supported by the Rhoˆne-Alpes region (GRANT CPER07_13 CIRA) and France-Grille (http://www.france-grilles.fr). We are thankful to Laure Gallien for her guidance in the use of the VirtualCom R package. y 43. Papa, E. et al. Non-invasive mapping of the gastrointestinal microbiota identiﬁes children with inﬂammatory bowel disease. PLoS ONE 7, e39242 (2012). 44. Bordenstein, S. R. & Theis, K. R. Host biology in light of the microbiome: ten principles of holobionts and hologenomes. PLoS Biol. 13, e1002226 (2015). 45. Theis, K. R. et al. Getting the hologenome concept right: an eco-evolutionary framework for hosts and their microbiomes. mSystems 1, e00028–16 (2016). 46. Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010). f 47. Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C. & Knight, R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200 (2011). Author contributions M.G., F.M. and E.J.A. conceived and designed the study. M.G. and F.M. performed all analyses. M.G., F.M., C.S.S., J.G.S. and E.J.A. analysed the data. J.G.S., C.S.S., S.L. and W.T. contributed to the writing of the manuscript. M.G., F.M. and E.J.A. prepared the manuscript. 48. Wang, Q., Garrity, G. M., Tiedje, J. M. & Cole, J. R. Naive Bayesian classiﬁer for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267 (2007). 49. Pruesse, E., Peplies, J. & Glo¨ckner, F. O. SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28, 1823–1829 (2012). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. 51. Britton, T., Anderson, C. L., Jacquet, D., Lundqvist, S. & Bremer, K. Estimating divergence times in large phylogenetic trees. Syst. Biol. 56, 741–752 (2007). References 1. Moran, N. A., McCutcheon, J. P. & Nakabachi, A. Genomics and evolution of heritable bacterial symbionts. Annu. Rev. Genet. 42, 165–190 (2008). 33. Philippot, L. et al. The ecological coherence of high bacterial taxonomic ranks. Nat. Rev. Microbiol. 8, 523–529 (2010). 11 NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications Additional information 50. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: computing large minimum evolution trees with proﬁles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 52. Bray, J. R. & Curtis, J. T. An ordination of the upland forest communities of Southern Wisconsin. Ecol. Monogr. 27, 325–349 (1957). 53. Dray, S. & Dufour, A. B. The ade4 package: implementing the duality diagram for ecologists. J. Stat. Softw. 22, 1–20 (2007). How to cite this article: Groussin, M. et al. Unraveling the processes shaping mammalian gut microbiomes over evolutionary time. Nat. Commun. 8, 14319 doi: 10.1038/ncomms14319 (2017). 54. Mantel, N. The detection of disease clustering and a generalized regression approach. Cancer Res. 27, 209–220 (1967). 55. Ferrier, S., Manion, G., Elith, J. & Richardson, K. Using generalized dissimilarity modelling to analyse and predict patterns of beta diversity in regional biodiversity assessment. Divers. Distrib. 13, 252–264 (2007). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 56. Goslee, S. C. & Urban, D. L. The ecodist package for dissimilarity-based analysis of ecological data. J. Stat. Softw. 22, 1–19 (2007). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 57. Legendre, P. Studying beta diversity: ecological variation partitioning by multiple regression and canonical analysis. J. Plant Ecol. 1, 3–8 (2008). 58. Anderson, M. J. A new method for non-parametric multivariate analysis of variance. Austral. Ecol. 26, 32–46 (2001). 59. Doledec, S., Chessel, D. & Gimaret-Carpentier, C. Niche separation in community analysis: a new method. Ecology 81, 2914–2927 (2000). 60. Paradis, E., Claude, J. & Strimmer, K. APE: analyses of phylogenetics and evolution in R language. Bioinformatics 20, 289–290 (2004). r The Author(s) 2017 12 NATURE COMMUNICATIONS \| 8:14319 \| DOI: 10.1038/ncomms14319 \| www.nature.com/naturecommunications
https://openalex.org/W3133900926	https://hal.archives-ouvertes.fr/hal-03170575v2/file/fimmu-12-630096.pdf	English	null	Club Cell Loss as a Feature of Bronchiolization in ILD	Frontiers in immunology	2,021	cc-by	5,703	Club Cell Loss as a Feature of Bronchiolization in ILD Paul Reynaud, Engi Ahmed, Isabelle Serre, Lucie Knabe, Sébastien Bommart, Carey Suehs, Isabelle Vachier, Jean Philippe Berthet, Micaela Romagnoli, Charlotte Vernisse, et al. To cite this version: Paul Reynaud, Engi Ahmed, Isabelle Serre, Lucie Knabe, Sébastien Bommart, et al.. Club Cell Loss as a Feature of Bronchiolization in ILD. Frontiers in Immunology, 2021, 12, pp.630096. 10.3389/fimmu.2021.630096. hal-03170575v2 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03170575 https://hal.science/hal-03170575v2 Submitted on 18 May 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License ORIGINAL RESEARCH published: 26 February 2021 doi: 10.3389/ﬁmmu.2021.630096 Club Cell Loss as a Feature of Bronchiolization in ILD Paul Reynaud 1, Engi Ahmed 1,2, Isabelle Serre 3, Lucie Knabe 1, Sébastien Bommart 4, Carey Suehs 1, Isabelle Vachier 1, Jean Philippe Berthet 5, Micaela Romagnoli 6, Charlotte Vernisse 1,2, Jean Pierre Mallet 1, Anne Sophie Gamez 1 and Arnaud Bourdin 1,2* Paul Reynaud 1, Engi Ahmed 1,2, Isabelle Serre 3, Lucie Knabe 1, Sébastien Bommart 4, Carey Suehs 1, Isabelle Vachier 1, Jean Philippe Berthet 5, Micaela Romagnoli 6, Charlotte Vernisse 1,2, Jean Pierre Mallet 1, Anne Sophie Gamez 1 and Arnaud Bourdin 1,2* 1 Department of Respiratory Diseases, Univ Montpellier, CHU Montpellier, Montpellier, France, 2 PhyMedExp, Univ Montpellier, CNRS, INSERM, CHU Montpellier, Montpellier, France, 3 Department of Pathology, Univ Montpellier, CHU Montpellier, Montpellier, France, 4 Department of Radiology, Univ Montpellier, CHU Montpellier, Montpellier, France, 5 Pulmonology Unit, Ospedale Ca’Foncello, AULSS2 Marca Trevigiana, Trevise, Italy, 6 Department of Cardiac, Thoracic and Vascular Surgery, Univ Nice, CHU Nice, Nice, France Background: Distal airway metaplasia may precede honeycombing in progressive ﬁbrosing interstitial lung disease (ILD). The SCGB1A1+ bronchiolar-speciﬁc club cell may play a role in this aberrant regenerative process. Keywords: club (clara) cell, idiopathic pulmonary ﬁbrosis, metaplasia, SCGB1A1, bronchiolization, interstitial lung disease Edited by: Antoine Froidure, Cliniques Universitaires Saint-Luc, Belgium Reviewed by: Arnaud Mailleux, Institut National de la Santé et de la Recherche Médicale (INSERM), France Charles Pilette, Catholic University of Louvain, Belgium Edited by: Antoine Froidure, Cliniques Universitaires Saint-Luc, Belgium Objective: To assess the presence of club cells in the small airways of patients suffering from ILD. Methods: Small airways (internal diameter <2 mm) in lung samples [surgical lung biopsy (SLB) and/or transbronchial lung cryobiopsy (TBLC)] from 14 patients suffering from ILD and 10 controls were morphologically assessed and stained for SCGB1A1. SCGB1A1 was weighted by epithelial height as a marker of airway generation (SCGB1A1/EH). Correlations between clinical, functional, and high-resolution CT (HRCT) prognostic factors and histomorphometry were assessed. Reviewed by: Arnaud Mailleux, Institut National de la Santé et de la Recherche Médicale (INSERM), France Charles Pilette, Catholic University of Louvain, Belgium Results: Small airways from samples with ILD patterns were signiﬁcantly less dense in terms of SCGB1A1+ cells [0.064 (0.020–0.172)] as compared to controls’ sample’s small airways [0.393 (0.082–0.698), p < 0.0001]. Usual interstitial pneumonia (UIP) patterns most frequently contained small airways with limited or absent SCGB1A1 expression (SCGB1A1/EH <0.025): UIP (18/33; 55%) as compared with non-UIP patterns (4/31; 13%) or controls (0/29; 0%): p < 0.0001. In addition, correlations with HRCT indicated a signiﬁcant negative relationship between SCGB1A1 and bronchiectasis as a feature of bronchiolization (Rho −0.63, p < 0.001) and a positive relationship with both forced vital capacity (FVC) and Hounsﬁeld unit (HU)-distribution pattern in kurtosis (Rho 0.38 and 0.50, respectively, both p < 0.001) as markers of ﬁbrotic changes. Correspondence: Arnaud Bourdin a-bourdin@chu-montpellier.fr Correspondence: Arnaud Bourdin a-bourdin@chu-montpellier.fr Specialty section: This article was submitted to Mucosal Immunity, a section of the journal Frontiers in Immunology Received: 16 November 2020 Accepted: 25 January 2021 Published: 26 February 2021 INTRODUCTION compared to traditional surgical lung biopsies (SLB), samples airway-centered, more proximal tissue, and this is likely to change not only the pathological patterns and eventually the ﬁnal pathological diagnosis but also the representation of the diﬀerent compartments. In particular, airways contained in TBLC samples are more likely to represent earlier generations than those taken in the more peripheral SLB samples (10). Intuitively, the small airways present in TBLC samples might then be less frequently aﬀected by bronchiolization. Given the relationship between this process and the ﬁbrotic changes aﬀecting the lung in progressing ILD, these pathological changes may have some diagnostic and/or prognostic value identiﬁable by clinical correlations in a cross- sectional study. Interstitial lung disease (ILD) are devastating aﬀections nearly constantly leading to respiratory failure and death. The early phases of the disease are hallmarked by progressive ﬁbrosis in the peripheral zones of the lung, which is associated with lung function decline (1). High-resolution CT (HRCT) has become the gold standard for diagnosing such ﬁbrotic changes and characterizing their progression (2). Notably, the presence of traction bronchiectasis (the enlargement of small airways by traction) is now considered a red ﬂag for subpleural ﬁbrotic changes. Interestingly, histomorphological assessments of these ﬁbrotic areas consistently report bronchiolar metaplasia of unclear origin (3). The latter “bronchiolization” may represent aberrant regeneration of the lung in a proﬁbrotic environment, where distal airway precursors never reach their potential as functional alveoli (4). The result is the presence of proximal features in the distal compartment currently detectable only via histopathology (5). The objective of this study was to assess the presence of club cells in the small airways of patients suﬀering from ILD by combining pathology, physiology, and HRCT data. We hypothesized that “protective” club cells characterized by their speciﬁc protein SCGB1A1 might be deﬁcient in ﬁbrotic diseases and particularly at the site where bronchiolar metaplasia occurs. Club cells are thought to be highly expressed at the small airway level (6), where they are supposed to play a protective role against noxious inhaled particles. Among the latter, cigarette smoke and occupational dust have also been implicated in the pathogenesis of idiopathic pulmonary ﬁbrosis (IPF) (7, 8). Interestingly, club cells were also attributed an accessory stem cell role at the small airway level, but this role has mostly been evidenced in animal models at the bronchoalveolar duct junction, where they are preferentially located (9). INTRODUCTION Due to recent advances in single-cell biology, it is now clear that these cells are heterogeneous and not all are playing the same role. Currently, they are considered as diﬀerentiation of basal cells, and the transition from basal to club cells has been ontogenically evidenced. Additionally, club cells are situated at the crossroads of the epithelial phenotype. Their natural trend is to progressively acquire features of goblet cells, but some can also move toward ciliogenesis. Whether or not appropriate transition pathways are failing during the bronchiolisation process remain unclear. Citation: Conclusion: Compared with controls, the small airways of patients with ILD more often lack SCGB1A1, especially so in UIP. Low densities of SCGB1A1-marked cells correlate with bronchiectasis and ﬁbrotic changes. Further research investigating SCGB1A1 staining as a pathological feature of the bronchiolization process is merited. Reynaud P, Ahmed E, Serre I, Knabe L, Bommart S, Suehs C, Vachier I, Berthet JP, Romagnoli M, Vernisse C, Mallet JP, Gamez AS and Bourdin A (2021) Club Cell Loss as a Feature of Bronchiolization in ILD. Front. Immunol. 12:630096. doi: 10.3389/ﬁmmu.2021.630096 February 2021 \| Volume 12 \| Article 630096 Frontiers in Immunology \| www.frontiersin.org Club Cells in ILD Reynaud et al. MATERIALS AND METHODS 7 0.0987 Weight (kg) 65.5 [60.65–81.75] 65 [62.25–83.75] 0.49 Height (cm) 160 [158.5–170] 166.5 [160–170] 0.44 Smoking history (p.y) 2.5 [0–28.75] 19 [1.25–30] 0.77 Smoking status (current-former-never) 1/5/8 1/5/4 Positive autoantibodies 0 0 PaO2 (mmHg) 84.2 [75.75–88.28] 88.65 [81.29–93.5] 0.15 FVC (l) 2.3 [1.74–3.31] 3.43 [2.75–4.07] 0.027* FVC (% pred values) 84.5 [68.5–2.77] 100 [92.25–107.5] 0.004* FEV1 (l) 2.1 [1.59–2.77] 2.39 [2.02–3.39] 0.077 FEV1 (% pred values) 84 [72.75–90] 95.5 [86–110.8] 0.010* TLC (l) 3.69 [3.55–4.49] 6.23 [6.16–6.59] 0.001* TLC (% pred values) 77.5 [66.25–84.75] 103 [101–106] 0.00005* RV (l) 1.57 [1.31–1.79] 2.64 [2.58–3.12] 0.0002* RV (% pred values) 89 [65–102] 116 [100–121] 0.0049* TLCO (% pred values) 56 [40–60] 76 [70–80] 0.0097* KCO 79 [73–96] 85 [78–103] 0.58 Kurtosis 6.33 [4.32–7.48] 14.77 [13.78–17.16] 0.0017* Skewness 2.29 [2.02–2.53] 3.59 [3.39–3.85] 0.00014* Whole lung (WL) 3.44 [2.79–4.12] 5.61 [3.54–6.17] 0.1095 % Normal lung (NL) 79.98 [71.14–82.04] 88.72 [88.2–90.25] 0.00045 Mean lung attenuation (Hounsﬁeld) −764 [−796 to −720] −838 [−859 to −819] 0.0012* Fibrotic score 15 [10–22.5] 0 [0–0] 0.00006* Ground glass opacity 5 [5–5] 0 [0–0] 0.059 Bronchiectasis score 6 [5.25–8] 0 [0–0] 0.00003* %NL, percentage of normal lung; BMI, body mass index; CT, computed tomography; DLCO, diffusing capacity of the lungs for carbon monoxide; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; ILD, interstial lung disease; IQR, interquartile range; KCO, transfer coefﬁcient of the lung for carbon monoxide; MLA, mean lung attenuation; N, number; PaO2, arterial oxygen pressure; RV, residual volume; SD, standard deviation; TLC, total lung capacity. *p < 0.05. Brieﬂy, lung samples were ﬁxed in 4% formalin at pH 7.4 and embedded in paraﬃn. Three-millimeter-thick sections were stained by H&E. Lung samples were anonymized and blindly assessed by an expert pathologist (TVC). Serial sections were used for SCGB1A1 immunostaining (dilution 1/10,000; Biovendor, Czech Republic). For some slides, a Bleu Alcian staining could also be performed. p Small airways (identiﬁed by an internal diameter of 2 mm or less) were assessed in all samples. Morphometry was assessed using the Cell P software (Olympus, Tokyo, Japan). Bronchiolization was referred to small airways with evidence of proximal features, including the presence of ciliated and/or goblet cells, as per the previously published standards (12, 13). Epithelial area, epithelial height (EH), and the percentage of SCGB1A1+ immune-stained area were determined for each small airway. MATERIALS AND METHODS CryoPID [NCT02763540] was a prospective trial evaluating the diagnostic concordance between TBLC and SLB, which were performed sequentially in the same patients between January 2016 and March 2018 at the Montpellier University Hospital (11). The study included patients with ILD requiring an SLB as per a multidisciplinary approach decision, given an undeﬁned HRCT pattern. For the current histology assessment, 14 CryoPID patients were compared with 10 control subjects randomly selected from the bio-bank of the Montpellier University Hospital. Pathological patterns reported from SLB and TBLC together by a pathologist involved in the present study were used. Controls had normal lung function, were free of chronic respiratory disease, and matched, as well as a possibility for smoking, in gender and age with the ILD population. They had undergone a lobar resection for a colic metastasis in the last 3 years. Lung parenchyma at a large distance from the metastasis was analyzed. New opportunities for lung tissue sampling are provided by the arrival of transbronchial lung cryobiopsies (TBLC). Beyond clinical and technical considerations, it seems that TBLC, as FIGURE 1 \| After SCGB1A1 staining, small airway epithelia were isolated via thresholding, perimeters, thickness/height, and by the area quantiﬁed. CGB1A1 staining, small airway epithelia were isolated via thresholding, perimeters, thickness/height, and by the area quantiﬁed. February 2021 \| Volume 12 \| Article 630096 2 Frontiers in Immunology \| www.frontiersin.org Reynaud et al. Club Cells in ILD TABLE 1 \| Population characteristics. ILD [Median (IQR25–75)] Controls [Median (IQR25–75)] p-value N 14 10 Age (years) 63 [43.5–65] 64 [57.5–67.5] 0.21 Gender (W/M) 10 vs. 04 3 vs. MATERIALS AND METHODS The SCGB1A1+/EH ratio was computed to adjust for variations known to aﬀect club cell density through airway generation (Figure 1). The small airway density for each sample was based on a semi-quantitative scale ranging from 0 (absence of small airways) to 5 (highest small airway density). The absence or near-absence of club cells was deﬁned by a SCGB1A1/EH ratio <0.025 by taking into account the background noise. Prognostic factors were recorded and included clinical (age, gender, smoking history), biological/laboratory [arterial oxygen pressure (PaO2), bronchoalveolar lavage cellularity (BAL)], and functional [forced vital capacity (FVC), diﬀusing capacity of the lungs for carbon monoxide (DLCO)] criteria. A visual tomodensitometric analysis scored ﬁbrotic changes (honeycombing and reticulations), intensity, and the spread of ground-glass opacity (GGO) (14), as well as the extent of bronchiectasis [ranking the severity of bronchiectasis by a lobe on a scale ranging from 0 to 3 (15) by acquiring consensus among two experienced thoracic senior radiologists (SB and GD)]. Automatic quantiﬁcation via the Thoracic VCAR software (GE HealthCare, USA) was also performed, classifying pixels from the whole lung (WL) volume into four groups as proposed by Shin et al. (16) (< −950 HU: “emphysema,” −950 to −700 HU “functional parenchyma,” −700 to −500 HU “interstitial disease,” > −500 HU “extra parenchymal”). Three prognostic variables were then derived: total lung capacity (TLC), percentage of normal lung (%NL), and mean lung attenuation (MLA) (17). Pulmonary attenuation distribution histograms were obtained for assessing skewness and kurtosis, previously reported as having the prognostic value (18). Statistical Analysis note, as these patients were deemed eligible for SLB, most had a mild functional impairment, suggesting that many were at an early stage of the disease. As previously reported (11), discrepancies in the pathological diagnosis between SLB and TBLC existed, potentially leading to diﬀerent ﬁnal diagnoses during a second multidisciplinary assessment (Table 2). For subsequent classiﬁcation as usual interstitial pneumonia (UIP) and non-UIP, only samples with a clear and conﬁrmed (high level of conﬁdence) UIP pattern were retained. Parametric and non-parametric tests were used to describe and compare normally and non-normally distributed data as appropriate. Spearman’s correlation coeﬃcients were computed for testing variable relationships. RESULTS Population Description Population Description Fourteen patients with ILD and 10 controls were studied (Table 1). As expected, patients with ILD had worse pulmonary function tests and higher HRCT semi-quantitative scores. Of Among patients with ILD, the mean SLB sample size was 49.5 mm [41.5–60] compared to 7 mm (6–8) (p < 0.00001) for TBLC. A signiﬁcant diﬀerence in the semi-quantitative scoring February 2021 \| Volume 12 \| Article 630096 Frontiers in Immunology \| www.frontiersin.org 3 Club Cells in ILD Reynaud et al. of small airway density between SLB and TBLC sample was found (Table 3). No diﬀerence was found between TBLC and SLB of small airway density between SLB and TBLC sample was found (Table 3). No diﬀerence was found between TBLC and SLB regarding club cell representation in the small airways. Fewer airways positively stained for SCGB1A1 in ILD, in particular, for those with a pathological UIP pattern, and in general, this staining was less intense (Figure 2A). This decrease persisted after adjusting on epithelial height as a surrogate marker of the generation of the airway assessed. Hence, SCGB1A1/EH was statistically lower in the small airways of samples from patients with ILD vs. controls (Figure 2B; the Kruskal-Wallis test, p = 7.598e-07). of small airway density between SLB and TBLC sample was found (Table 3). No diﬀerence was found between TBLC and SLB TABLE 2 \| Blinded histology and multidisciplinary diagnostic results for each patient. Patient Histology diagnosis based on TBLC Histology diagnosis based on SLB Diagnosis based on MDA 1 (before histology) Diagnosis based on MDA 2 (after histology) 1 UIP UIP Possible UIP CHP 2 Non- diagnostic PLCH DIP PLCH 3 UIP CHP NSIP IPF 4 UIP NSIP No classiﬁcation NSIP 5 RB-ILD RB-ILD RB-ILD RB-ILD 6 Non- diagnostic UIP NSIP IPF 7 PLCH UIP Possible UIP IPF 8 NSIP NSIP Fibrotic NSIP NOS 9 UIP UIP NSIP IPF 10 Non- diagnostic ALI NSIP NOS 11 NSIP LP Sarcoidosis CVID 12 UIP NSIP NSIP NSIP 13 Non- diagnostic UIP CHP IPF 14 UIP UIP Possible UIP IPF CHP, chronic hypersensitivity pneumonitis; CVID, common variable immune deﬁciency; DIP, desquamative interstitial pneumonia; HP, hypersensitivity pneumonitis; IPF, idiopathic pulmonary ﬁbrosis; LP, lymphoid process; MDA1, a ﬁrst multidisciplinary assessment; MDA2, the ﬁnal multidisciplinary assessment; NOS, not otherwise speciﬁed; NSIP, non speciﬁc interstitial pneumonia; OP organizing pneumonia; PLCH pulmonary TABLE 2 \| Blinded histology and multidisciplinary diagnostic results for each patient. Population Description Moreover, sparsely stained airways (deﬁned as SCGB1A1/EH <0.025) were signiﬁcantly more represented in the UIP pathological patterns [UIP 18/33 vs. non-UIP 4/31 (p = 0.0027)] (Table 2 and Figure 3). The tested prognostic correlations are provided in Figure 4. Interestingly, statistically signiﬁcant non-parametric correlations were found between SCGB1A1/EH and two surrogate markers of lung ﬁbrosis. First, a correlation was found with the kurtosis value of the histogram distribution of lung attenuation assessed at HRCT (Rho = 0.50, p < 0.001). The less the lung is involved by ﬁbrosis, the greater the club cell density found in the small airways, as the kurtosis value decreases with the extent of ﬁbrosis in ILD (18). Second, SCGB1A1/EH also positively correlated with FVC (Rho = 0.38, p < 0.001), as expected. The negative correlation found between SCGB1A1/EH and the bronchiectasis scores (Rho = −0.63, p < 0.001) is also represented in Figure 4. The latter indicates that fewer club cells were present in the small airways of patients with more intense bronchiectasis. CHP, chronic hypersensitivity pneumonitis; CVID, common variable immune deﬁciency; DIP, desquamative interstitial pneumonia; HP, hypersensitivity pneumonitis; IPF, idiopathic pulmonary ﬁbrosis; LP, lymphoid process; MDA1, a ﬁrst multidisciplinary assessment; MDA2, the ﬁnal multidisciplinary assessment; NOS, not otherwise speciﬁed; NSIP, non-speciﬁc interstitial pneumonia; OP, organizing pneumonia; PLCH, pulmonary Langerhans cell histiocytosis; RB-ILD, respiratory bronchiolitis-associated interstitial lung disease; SLB, surgical lung biopsy; TBLC, transbronchial lung cryobiopsy; UIP, usual interstitial pneumonia. § Deﬁned by SCGB1A1+/EH ratio < 0.025. Frontiers in Immunology \| www.frontiersin.org DISCUSSION With the aim of understanding the mechanisms of bronchiolization, we found that club cells are absent or nearly absent from these aberrant small airways in the most subpleural areas of ﬁbrotic lungs. This was particularly true in ILD where a UIP pattern could be identiﬁed. Bronchiolized areas seem to ﬁt with small airways saliently featuring proximal diﬀerentiation patterns very close to terminal airspaces. As expected, both TBLC and SLB could identify these patterns, even though the density of small airways in TBLC is largely lower. Interestingly, the correlation between these bronchiolization patterns (highlighted by the absence of club cells) and clinical prognostic markers (such as HRCT kurtosis and bronchiectasis), as well as physiology (FVC), was demonstrated. TABLE 3 \| Samples characteristics. SLB (controls) SLB (ILD) TBLC (ILD) p-value N 10 14 14 Number of small airways assessed 32 35 31 Small airway density [semi quantitative score (0 = minimal; 5 = maximal)] 2.1 ± 0.1 2.4 ± 0.6 0.4 ± 0.3 0.04 Epithelial area (mean ± SD per bronchiole. µm²) 20,818 ± 9,040 26,738 ± 11,733 19,638 ± 11,216 ns Epithelial height (mean ± SD per bronchiole. µm) 37.8 ± 7.8 39.5 ± 11.9 35.8 ± 11.4 ns SCGB1A1+ stained area 19.7 (4.2–27.7) 3.6 (1.6–7.4) 0.9 (0.3–2.9) <0.0001 SCGB1A1+/EH 0.5 (0.1–0.7) 0.1 (0.0–0.2) 0.0 (0.0–0.1) 0.02 % airway without club cells§ 0 (0/32) 14 (5/35) 55 (17/31) 0.01 § Deﬁned by SCGB1A1+/EH ratio < 0.025. TABLE 3 \| Samples characteristics. Most of our results were driven by a particularly high number of unstained or sparsely stained airways in lung samples from patients with a UIP pattern. Most of these pauci-SCGB1A1 small airways displayed features of bronchiolar metaplasia. Interestingly, they were referred to as “bronchiolizing areas” in the routine pathology report. Fukumoto et al. hypothesized that this phenomenon may indicate an impaired diﬀerentiation with aberrant proliferation (19). The latter hypothesis is supported by the morphologic appearance of these areas where proximal features are found where they should not be. The underexpression of the club cell-speciﬁc protein SCGB1A1 may be directly linked to the bronchiolization process, as already supposed by Jensen-Taubman (20). A blockade in the February 2021 \| Volume 12 \| Article 630096 4 Club Cells in ILD Reynaud et al. FIGURE 2 \| (A) Small airways are immune-stained for SCGB1A1 in controls, UIP, and non-UIP patterns. Upper left: (A) SLB from a control (no TBLC were available in this group). DISCUSSION Middle row: (B) Very limited staining in some (left lower part) but not all small airways sampled by SLB; Inserts are: Serial cut with a Blue Alcian staining suggestive of the presence of mucin-costaining and a higher magniﬁcation suggestive of goblet cell morphology for SCGB1A1 positively stained epithelial cells. Ectasic small airway from a TBLC UIP pattern with bronchiolar metaplasia nearly free of staining despite abundant mucostasis. Right: SCGB1A1 stained small airways obtained by SLB (D) and by TBLC (E) in non-UIP patterns. (B) Box plots demonstrating group differences (Control vs. ILD for SLB and TBLC) for SCGB1A1 staining variables. TBLC, transbronchial lung cryobiopsies; SLB, surgical lung biopsy; UIP, usual interstitial pneumonia; ILD, interstitial lung disease. FIGURE 2 \| (A) Small airways are immune-stained for SCGB1A1 in controls, UIP, and non-UIP patterns. Upper left: (A) SLB from a control (no TBLC were available in this group). Middle row: (B) Very limited staining in some (left lower part) but not all small airways sampled by SLB; Inserts are: Serial cut with a Blue Alcian staining suggestive of the presence of mucin-costaining and a higher magniﬁcation suggestive of goblet cell morphology for SCGB1A1 positively stained epithelial cells. Ectasic small airway from a TBLC UIP pattern with bronchiolar metaplasia nearly free of staining despite abundant mucostasis. Right: SCGB1A1 stained small airways obtained by SLB (D) and by TBLC (E) in non-UIP patterns. (B) Box plots demonstrating group differences (Control vs. ILD for SLB and TBLC) for SCGB1A1 staining variables. TBLC, transbronchial lung cryobiopsies; SLB, surgical lung biopsy; UIP, usual interstitial pneumonia; ILD, interstitial lung disease. FIGURE 2 \| (A) Small airways are immune-stained for SCGB1A1 in controls, UIP, and non-UIP patterns. Upper left: (A) SLB from a control (no TBLC were available in this group). Middle row: (B) Very limited staining in some (left lower part) but not all small airways sampled by SLB; Inserts are: Serial cut with a Blue Alcian staining suggestive of the presence of mucin-costaining and a higher magniﬁcation suggestive of goblet cell morphology for SCGB1A1 positively stained epithelial cells. Ectasic small airway from a TBLC UIP pattern with bronchiolar metaplasia nearly free of staining despite abundant mucostasis. Right: SCGB1A1 stained small airways obtained by SLB (D) and by TBLC (E) in non-UIP patterns. (B) Box plots demonstrating group differences (Control vs. ILD for SLB and TBLC) for SCGB1A1 staining variables. DISCUSSION TBLC, transbronchial lung cryobiopsies; SLB, surgical lung biopsy; UIP, usual interstitial pneumonia; ILD, interstitial lung disease. FIGURE 3 \| The proportion of samples with SCGB1A1/EH <0.025 was used to deﬁne the absence of positive staining [UIP 18/33 vs. non-UIP 4/31 (p = 0.0027)]. FIGURE 3 \| The proportion of samples with SCGB1A1/EH <0.025 was used to deﬁne the absence of positive staining [UIP 18/33 vs. non-UIP 4/31 (p = 0.0027)]. February 2021 \| Volume 12 \| Article 630096 Frontiers in Immunology \| www.frontiersin.org Club Cells in ILD Reynaud et al. FIGURE 4 \| Scatterplots demonstrating the relationships between selected pulmonary function variables [bronchiectasis score (A), % predicted forced vital capacity (FVC, % predicted) (B), and kurtosis of lung attenuation scores (C)] and % epithelial area of small airways that stains SCGB1B1+ weighted by epithelial height (EASCGB1B1+/EH). Smoothing is performed via a local loess estimator (in black). Spearman’s correlation statistics (Rho and FDR-corrected p-value) are provided. FIGURE 4 \| Scatterplots demonstrating the relationships between selected pulmonary function variables [bronchiectasis score (A), % predicted forced vital capacity (FVC, % predicted) (B), and kurtosis of lung attenuation scores (C)] and % epithelial area of small airways that stains SCGB1B1+ weighted by epithelial height (EASCGB1B1+/EH). Smoothing is performed via a local loess estimator (in black). Spearman’s correlation statistics (Rho and FDR-corrected p-value) are provided. TBLC and SLB in this regard. Correlations were tested at the individual level by expressing SCGB1A1 as a mean and our ﬁndings remained consistent. notch pathway might be involved in this ontogenically impaired process, as the notch is critically involved in basal to club cell transitioning (21). Wnt/β-catenin pathways were also shown to be involved. There are a few steps left to deﬁnitively demonstrate that bronchiolization precedes the ﬁbrotic process. Recently, by matching micro-CT imaging with histology, Verleden et al. showed that small airway involvement is the most striking ﬁnding in the early IPF stages, suggesting that the disease starts at this level (4). This mechanism may help accelerate the paradigm shift that is already in motion, where instead of envisioning bronchiolization and bronchiectasis as a consequence of ﬁbrosis, they are now understood to be an active part of the ﬁbrotic process [as again recently suggested (4)]. Our study also supports that the bronchiolization process is key in the course of the disease as we could ﬁnd correlations with two diﬀerent HRCT-derived parameters. DISCUSSION Kurtosis refers to the tailedness of the distribution histogram of lung attenuation, and this value was suggested to be a surrogate of interstitial changes (25). A signiﬁcant negative relationship between the expression of the club cell marker SCGB1A1 and the bronchiectasis score was also found; an intuitively-expected ﬁnding enhancing the concept that bronchiectasis found at HRCT is related to the bronchiolization process hallmarked by the absence of club cells. Convincingly, a signiﬁcant correlation was also found with FVC, a physiological marker more than relevant in interstitial lung diseases. Senescence may play a key role in the ﬁbrotic process characteristic of UIP (22) possibly via this bronchiolization. Aberrantly proliferating cells can lead to both replicative and premature senescence (23), possibly leading to these poorly diﬀerentiated airways. The secretion of senescence-associated secretory protein (SASP), mainly interleukin-8, has already been shown to be involved (24). Our study has several limits. Few patients were included due to the particular procedures used in the CryoPID study. Our patient group was so heterogeneous that artiﬁcially gathering patients from a “non-UIP” group was required. Moreover, staining for MUC5B, a validated bronchiolization marker, would have been of great interest to increase conﬁdence in the mechanism and further characterize the phenotype of the remaining club cells. As shown by Best et al. (25), a higher proportion of SCGB1A1+/MUC5B+ cells is observed in IPF, highly expressing genes related to mucins and chemoattractant cytokines for immune cells. Though we missed this opportunity, we nonetheless had attempted the Bleu Alcian staining in a few slides when some unused material was available; the latter suggested convergent ﬁndings and merits further investigation. In summary, we demonstrated that bronchiolization, a typical characteristic of ILD and of UIP in particular, is also hallmarked by the absence of club cells. This suggests that an impaired transition process aﬀected epithelial cell fate. Pauci-SCGB1A1 staining status merits further study as a potential marker of bronchiolization in ILD. Frontiers in Immunology \| www.frontiersin.org REFERENCES Plantier L, Crestani B, Wert SE, Dehoux M, Zweytick B, Guenther A, et al. Ectopic respiratory epithelial cell diﬀerentiation in bronchiolised distal airspaces in idiopathic pulmonary ﬁbrosis. Thorax. (2011) 66:651– 7. doi: 10.1136/thx.2010.151555 21. Gras D, Chanez P, Vachier I, Petit A, Bourdin A. Bronchial epithelium as a target for innovative treatments in asthma. Pharmacol Ther. (2013) 140:290– 305. doi: 10.1016/j.pharmthera.2013.07.008 6. Boers JE, Ambergen AW, Thunnissen FB. Number and proliferation of clara cells in normal human airway epithelium. Am J Respir Crit Care Med. (1999) 159:1585–91. doi: 10.1164/ajrccm.159.5.9806044 22. Alder JK, Chen JJ.-L, Lancaster L, DanoﬀS, Su S.-c, Cogan JD, et al. Short telomeres are a risk factor for idiopathic pulmonary ﬁbrosis. Proc Natl Acad Sci USA. (2008) 105:13051–6. doi: 10.1073/pnas.08042 80105 7. Akram KM, Lomas NJ, Spiteri MA, Forsyth NR. Club cells inhibit alveolar epithelial wound repair via TRAIL-dependent apoptosis. Euro Respir J. (2013) 41:683–94. doi: 10.1183/09031936.00213411 23. Adnot S, Amsellem V, Boyer L, Marcos E, Saker M, Houssaini A, et al. Telomere dysfunction and cell senescence in chronic lung diseases: therapeutic potential. Pharmacol Ther. (2015) 153:125–34. doi: 10.1016/j.pharmthera.2015.06.007 8. Singh G, Katyal SL. Clara cells and clara cell 10 kD protein (CC10). Am J Respir Cell Mol Biol. (1997) 17:141–3. doi: 10.1165/ajrcmb.17.2.f138 9. Rawlins EL, Okubo T, Xue Y, Brass DM, Auten RL, Hasegawa H, et al. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell. (2009) 4:525– 34. doi: 10.1016/j.stem.2009.04.002 24. Chilosi M, Carloni A, Rossi A, Poletti V. Premature lung aging and cellular senescence in the pathogenesis of idiopathic pulmonary ﬁbrosis and COPD/emphysema. Transl Res. (2013) 162:156–73. doi: 10.1016/j.trsl.2013.06.004 10. Colby TV, Tomassetti S, Cavazza A, Dubini A, Poletti V. Transbronchial cryobiopsy in diﬀuse lung disease: update for the pathologist. Arch Pathol Lab Med. (2017) 141:891–900. doi: 10.5858/arpa.2016-0233-RA 25. Best AC, Meng J, Lynch AM, Bozic CM, Miller D, Grunwald GK, et al. Idiopathic pulmonary ﬁbrosis: physiologic tests, quantitative CT indexes, and CT visual scores as predictors of mortality. Radiology. (2008) 246:935– 40. doi: 10.1148/radiol.2463062200 11. Romagnoli M, Colby TV, Berthet J.-P, Gamez AS, Mallet J.-P, Serre I, et al. Poor concordance between sequential transbronchial lung cryobiopsy and surgical lung biopsy in the diagnosis of diﬀuse interstitial lung diseases. Am J Respir Crit Care Med. (2019) 199:1249–56. DATA AVAILABILITY STATEMENT Of note, we decided to focus our analysis at the sample level (as opposed to the patient level) to take into account the regional heterogeneity of the ﬁbrotic process where zones of normal appearance are classically neighboring the involved tissue (6). This was also justiﬁed by the aim of comparing The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. February 2021 \| Volume 12 \| Article 630096 6 Club Cells in ILD Reynaud et al. REFERENCES 16. Shin KE, Chung MJ, Jung MP, Choe BK, Lee KS. Quantitative computed tomographic indexes in diﬀuse interstitial lung disease: correlation with physiologic tests and computed tomography visual scores. J Comput Assist Tomogr. (2011) 35:266–71. doi: 10.1097/RCT.0b013e31820ccf18 1. Olson AL, Giﬀord AH, Inase N, Fernández Pérez ER, Suda T. The epidemiology of idiopathic pulmonary ﬁbrosis and interstitial lung diseases at risk of a progressive-ﬁbrosing phenotype. Eur Respir Rev. (2018) 27:180077. doi: 10.1183/16000617.0077-2018 1. Olson AL, Giﬀord AH, Inase N, Fernández Pérez ER, Suda T. The epidemiology of idiopathic pulmonary ﬁbrosis and interstitial lung diseases at risk of a progressive-ﬁbrosing phenotype. Eur Respir Rev. (2018) 27:180077. doi: 10.1183/16000617.0077-2018 17. Ohkubo H, Kanemitsu Y, Uemura T, Takakuwa O, Takemura M, Maeno K, et al. Normal lung quantiﬁcation in usual interstitial pneumonia pattern: the impact of threshold-based volumetric CT analysis for the staging of idiopathic pulmonary ﬁbrosis. PLoS ONE. (2016) 11:e0152505. doi: 10.1371/journal.pone.0152505 2. Sverzellati N, Brillet P-Y. When Deep Blue ﬁrst defeated Kasparov: is a machine stronger than a radiologist at predicting prognosis in idiopathic pulmonary ﬁbrosis? Euro Respir J. (2017) 49:1602144. doi: 10.1183/13993003.02144-2016 2. Sverzellati N, Brillet P-Y. When Deep Blue ﬁrst defeated Kasparov: is a machine stronger than a radiologist at predicting prognosis in idiopathic pulmonary ﬁbrosis? Euro Respir J. (2017) 49:1602144. doi: 10.1183/13993003.02144-2016 18. Lynch DA. Quantitative CT of ﬁbrotic interstitial lung disease. Chest. (2007) 131:643–4. doi: 10.1378/chest.06-2955 3. Visscher DW, Myers JL. Histologic spectrum of idiopathic interstitial pneumonias. Proc Am Thorac Soc. (2006) 3:322– 9. doi: 10.1513/pats.200602-019TK 3. Visscher DW, Myers JL. Histologic spectrum of idiopathic interstitial pneumonias. Proc Am Thorac Soc. (2006) 3:322– 9. doi: 10.1513/pats.200602-019TK 19. Fukumoto J, Soundararajan R, Leung J, Cox R, Mahendrasah S, Muthavarapu N, et al. The role of club cell phenoconversion and migration in idiopathic pulmonary ﬁbrosis. Aging. (2016) 8:3091–105. doi: 10.18632/aging.1 01115 4. Verleden SE, Tanabe N, McDonough JE, Vasilescu DM, Xu F, Wuyts WA, et al. Small airways pathology in idiopathic pulmonary ﬁbrosis: a retrospective cohort study. Lancet Respir Med. (2020) 8:573–84. doi: 10.1016/S2213-2600(19)30356-X 4. Verleden SE, Tanabe N, McDonough JE, Vasilescu DM, Xu F, Wuyts WA, et al. Small airways pathology in idiopathic pulmonary ﬁbrosis: a retrospective cohort study. Lancet Respir Med. (2020) 8:573–84. doi: 10.1016/S2213-2600(19)30356-X 20. Jensen-Taubman SM, Steinberg SM, Linnoila RI. Bronchiolization of the alveoli in lung cancer: pathology, patterns of diﬀerentiation and oncogene expression. Int J Cancer. (1998) 75:489– 96. doi: 10.1002/(SICI)1097-0215(19980209)75:4<489::AID-IJC1>3.0.CO;2-P 5. ETHICS STATEMENT study, enrolled patients, performed biopsies, acquisition of data, immunostaining, morphometry, HRCT analysis, and the writing of the manuscript. EA participated to the study, enrolled patients, performed biopsies, acquisition of data, and the writing of the manuscript. IS participated to the study, assessed biopsies, and contributed to the immunostaining. LK, CV, CS, and IV participated to the study, interpretation of the data, and the writing of the manuscript. SB participated to HRCT analysis. MR, JB, JM, and AG participated to the study, enrolled patients, and performed biopsies. All authors contributed to the article and approved the submitted version. The studies involving human participants were reviewed and approved by CPP Ile de France. The patients/participants provided their written informed consent to participate in this study. AUTHOR CONTRIBUTIONS AB elaborated the study, enrolled patients, performed biopsies, participated to the interpretation of the data, and the writing of the manuscript. PR participated to the REFERENCES doi: 10.1164/rccm.201810-1947OC Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 12. Nettesheim P, Szakal AK. Morphogenesis of alveolar bronchiolization. Lab Invest. (1972) 26:210–9. 13. Allen TC. Pathology of small airways disease. Arch Pathol Lab Med. (2010) 134:702–18. doi: 10.5858/134.5.702 Copyright © 2021 Reynaud, Ahmed, Serre, Knabe, Bommart, Suehs, Vachier, Berthet, Romagnoli, Vernisse, Mallet, Gamez and Bourdin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 14. Shin KM, Lee KS, Chung MP, Han J, Bae YA, Kim TS, et al. Prognostic determinants among clinical, thin-section CT, and histopathologic ﬁndings for ﬁbrotic idiopathic interstitial pneumonias: tertiary hospital study. Radiology. (2008) 249:328–37. doi: 10.1148/radiol.2483071378 15. Edey AJ, Devaraj AA, Barker RP, Nicholson AG, Wells AU, Hansell DM. Fibrotic idiopathic interstitial pneumonias: HRCT ﬁndings that predict mortality. Eur Radiol. (2011) 21:1586–93. doi: 10.1007/s00330-011-2098-2 February 2021 \| Volume 12 \| Article 630096 Frontiers in Immunology \| www.frontiersin.org 7
https://openalex.org/W998241083	https://symphonya.unicusano.it/article/download/2015.2.08sola.scarso.scalabrini/10697	English	null	New Product Development and Disciplined Experimentation	Symphonya	2,015	cc-by	8,340	Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 Sola Davide, Scarso Borioli Giovanni, Scalabrini Gianfranco (2015) New Product Development and Disciplined Experimentation, Symphonya. Emerging Issues in Management (symphonya.unimib.it), n. 2, pp. 105 – 119. http://dx.doi.org/10.4468/2015.2.08sola.scarsoborioli.scalabrini * Associate Professor of Strategy, ESCP Europe, London (dsola@escpeurope.eu) Assistant Professor of Operation Management, ESCP Europe, London (gscarsoborioli@ecspeurope.eu) * Affiliate Professor of Business Operations, ESCP Europe, London (gscalabrini@escpeurope.eu) Abstract The process of developing new products always contains an element of uncertainty. This uncertainty translates into a significant risk for companies investing in the development of new products or services. The risk in new product development (NPD) can be based on ‘disciplined experimentation’: a structured process designed to rapidly identify the ‘vivid’ needs of the customer, test whether the main features of the new product or service will satisfy these needs (fast prototyping). ‘Disciplined experimentation’, in particular, addresses assumptions about value (how the initiative will produce outcomes that outweigh the effort involved), growth (how the initiative can be scaled up beyond the first group of customers) and sustainability (how quickly the organisation can adapt to the new initiative and how easily competitors will be able to replicate it). Keywords: Design Management; New Product Development; Product Engineering; Disciplined Experimentation; Fast Fashion Manufacturing; Heating Systems Manufacturing; Global Competition © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it http://dx.doi.org/10.4468/2015.2.08sola.scarsoborioli.scalabrini © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it To examine the issue of reducing uncertainty in new product development, we first undertook a review of the existing literature in order to establish the factors which determine success or failure in this area. New product development can be defined as the processes employed by a business to identify, design, create and bring to market new products or services (Brondoni 2009). But what processes should be used? And which of these are critical? Research into NPD sought to identify the processes involved in order to determine which, if any, contributed to success. If those success factors could be identified, then more business should be able to carry out NPD successfully. . Early work by Hustad (1977) adopted a broad perspective when defining the NPD process and included topics such as market planning, product strategy, product line extension, market forecasting, product abandonment and product liability. Other researchers were working to refine the definitions of the NPD process into more distinct factors. An early proponent of a managed approach to NPD was Morris (Morris 1990) who had spent forty years improving project management techniques and published his influential Management of Projects theory in 1990. In the 1970s Robert Cooper and his colleagues started looking at the difficulties businesses were encountering in bringing new products to successful fruition (Cooper, Kleinschmidt 1986; 1987; 1990; 1991). As a result, they developed the NewProd project which utilised a Stage/Gate approach. This breaks the process of developing a new product into a series of stages, separated by gates or hurdles which must be passed before progressing to the next stage. Cooper identified five main aspects of the NPD process; scoping, building the business case, development, testing and validating, and product launch (Cooper 2001). Loch (2000), while acknowledging that Stage-Gate is at the core of most NPD processes, argued that survival and growth ultimately depends on how well a company adapts to its specific environment. Davidson et al. (1999) reached a similar conclusion, emphasizing the need for flexibility so that the NPD process can be continually adjusted to an organisation’s changing needs and aims. According to Fixson (2009) most definitions of NPD include stages such as product opportunity identification, market and user analysis, idea generation, concept generation, concept refinement and selection, industrial design, prototyping, testing, financial evaluation and market introduction. 1. New Product Development Literature New product development (NPD) is an inherently uncertain process. Research shows that as many as 40% of new products fail to deliver anything approaching the promised objectives (Castellion, Markham 2013). This uncertainty results from many factors including the difficulty of identifying ‘vivid’ (strong and conscious) customer needs, the problem of correctly defining the right features and shaping how the user experiences the new product or service, identifying the most suitable route to market and, of course, getting the pricing right. All these challenges must be overcome in an environment where there is increasing competitive pressure to deliver cheaper, faster and better (Brondoni 2008). 105 Edited by: ISTEI – University of Milan-Bicocca 2. Critique of NPD Literature While all the NPD processes we reviewed have their uses in the quest to reduce risk, we noticed that they tend to share a number of pitfalls. First, all the NPD processes featured in our literature review require extensive time and commitment if they are to be used effectively, with speed being sacrificed in favour of quality of execution. The earlier approaches to NPD focused on the need to control all aspects of the process to ensure that the development plan was completed within budget. As the pace of technological and commercial development increased, some approaches sought to adapt keep pace with the changing environment, while others did not. For example, the Management of Projects method remained static while the Stage-Gate process developed by Cooper has re-invented itself to take on board the challenges of a modern, fast paced world where technology continually forces changes in design practice and design development. More recently, Agile Development has begun to be adopted by those outside the software development arena where it was born. The flexibility of the Agile approach allows the designers and developers to take on board the fickle demands of an ever more aware customer, enabling products to better meet the customers’ needs. Lean Start-up, a method proposed by Eric Reis in 2011, takes the involvement of the customer even further by encouraging continuous customer involvement from the very earliest stage of development, even as early as conception. Lean Start-up builds on the relationship with the customer to create an environment where a product could be launched well ahead of schedule, with upgrades being made available to extend the life of the product. Design Thinking and Design of Experiments offer a different view of the NPD process. These two approaches are useful in resolving problems using unconventional means and may offer innovative insights into the development of new products and services. However, except for the Stage-Gate process, all the other approaches assume that the right product has been selected and that the main emphasis should be on its development. And even in the case of the Stage-Gate method, one of the main problems is that businesses do not know how to ensure the gates work effectively to stop or allow progress. The tendency across all these approaches was to assume that once a particular product has entered the NPD process that this was the right product to develop. © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it Cormican and O’Sullivan (2004) saw strategy and leadership, culture and climate, planning and selection, structure and performance, and communication and collaboration as key factors. Kahn et al (2012) identified seven separate components of the NPD process; strategy (including portfolio management), process, research, project climate (including team organisation), company culture, commercialisation, metrics and performance evaluation. Amabile (1997), Smith & Reinertsen (1998) and DeCusatis (2008) analysed team characteristics and identified factors which can increase the creative ability of the team and help accelerate the NPD process. Thomke (2003) noted that team integration encouraged experimentation and prototyping, which Barczak, Griffin and Kahn (2009) also found was a factor of high-performing firms, suggesting this was a key factor in the NPD process. y p ‘Fail often to succeed sooner’ is reportedly one of the mottos of the successful product design firm IDEO (Kelley 2001), stressing the importance of being ready refine, cannibalise or even abandon previous ideas and assumptions. Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 ISSN: 1593-0319 106 © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it With the rapidly changing technological advances of the past decade there is a growing interest in the role of NPD processes that were created to handle uncertainty and changing customer needs and wants; among these new methods Agile NPD is leading the way. Agile originated in 2001 in the software development field and emphasises the importance of self managing, cross functional teams working quickly, flexibly and responsively. Edited by: ISTEI – University of Milan-Bicocca 2. Critique of NPD Literature There appeared to be an inability in any of the processes to consider outside influences such as an aggressive competitor launching a similar product more quickly. Even in those processes that encourage involvement with customers, such as Lean Start-up, there was no suggestion that a product could, Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 107 © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it potentially, be terminated. Rather the product would be adjusted to take on the comments of the potential customers. Another pitfall of the processes we reviewed was the lack of emphasis on encouraging ownership of the product being developed. The project team is simply presented with the product to be developed and told to get on with it. None of the processes reviewed provided much guidance on who should be involved in the ‘NPD team’, despite citing cross-functional teams as vital to success. The use of true cross-functional teams can improve ownership of the product being developed but this was not flagged as important. While the usefulness of experimentation is beginning to be acknowledged as a means of checking the viability of a product, it is not seen as central to the overall NPD process and, like the involvement of customers, is generally left till late in the NPD process when there is a physical product that can be handled. The Lean Start up and Design thinking approaches, and in particular Lean Start- up, have learned from the failings of previous approaches and introduced elements to overcome some of these pitfalls. Early experimentation through the construction of Minimum Viable Products (MVP’s) facilitates the cost efficient testing of just one or a few variables at a time. And the introduction of the Value and Growth hypothesis requires assumptions about why customers will want the product and how they will be accessed to be made explicit and repeatedly tested. Finally the concept of Pivot has reinforced the principle that when the working hypothesis supporting a new product is found to be flawed, the product should be reconsidered and amended or abandoned completely. Edited by: ISTEI – University of Milan-Bicocca 3. Research Question and Methodology Principles such as MVPs and the Value and Growth hypothesis represent a great advancement in the search for a robust theory of NPD. Nevertheless we believe that there are still some areas that need to be addressed. This conviction led to our decision to investigate the following research question and related hypothesis g g q yp Question: “Can disciplined experimentation reduce uncertainty in NPD in a fast changing environment?” g g Hypothesis: it can, if two conditions are fulfilled: First, that the experimentation is constructed in order to progressively test out and validate three variables, in particular First, that the experimentation is constructed in order to progressively test out and validate three variables, in particular - Value - how the new product or service will create value by resolving a ‘vivid’ need (as opposed to a latent one), producing outcomes that outweigh the effort required. - Growth - how the new product can be scaled up beyond the first group of pioneering customers, guaranteeing that the value created will also increase. - Sustainability - how easily the organisation will adapt to the changes required to implement the new product and how easily competitors can replicate it ISSN: 1593-0319 Edited by: ISTEI – University of Milan-Bicocca 108 © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it Second, that disciplined experimentation entails two mutually reinforcing dimensions - The use of creative experimentation techniques which exploit innovations in technology and process in order to maximise the number of experiments, while making them as inexpensive as possible through speed of execution - Use of experimental analysis approaches, such as Design of Experiments (DoE) that maximise the learning from each experiment performed - Use of experimental analysis approaches, such as Design of Experiments (DoE) that maximise the learning from each experiment performed In order to test the validity of our hypotheses the next step was to identify organisations that would be willing to participate in a long term study. We succeeded in engaging four organisations from industries as diverse as rail transportation (United Kingdom), animal health pharmaceuticals (United Kingdom), gas heating system manufacturing (Italy) and fashion (Italy) with whom we have worked for the last 18 months. This paper presents our preliminary findings. 4. The Progressive Hypothesis The success of a newly developed product, defined as its ability to contribute to the achievement or sustaining of competitive advantage of the firm, relies on a set of hypotheses which need to be validated. A hypothesis, in business as in science, is an explanation or a proposition made on the basis of limited evidence which serves as the starting point for further investigation. Until proven true, hypotheses are just statements. The only way to prove their validity is to test them. Testing will either prove or disprove the validity of a hypothesis, but it will also provide insights about how the hypothesis could be refined or even replaced by a better one. Just like a scientist in a laboratory, business executives should first make explicit the hypotheses behind the initiatives they wish to launch and then validate them through experiments. We propose, as stated before, that in the case of NPD there are three hypotheses that need to be validated progressively: first value, then growth and concluding with sustainability. As you move from each hypothesis to the next, the overall level of uncertainty reduces, but it is not until the new product has provided evidence for all three hypotheses that uncertainty will be significantly reduced, although never completely eliminated. The first hypothesis is about Value, which involves explaining how the new product will create value by producing outcomes that outweigh the effort involved. Typical questions that need to be addressed are: - What kind of problem does the new product solve? - Who are the people facing this problem? How aware are they of the problem? - Are they prepared to pay for someone to solve it? - Will the price they are prepared to pay for the new product be sufficient to cover the cost of its development? Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 109 © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it The second hypothesis is about Growth. Here we must think through how the initiative can be scaled up beyond the first group of customers, guaranteeing that the value created will also increase sufficiently. The typical questions to be addressed are: - Does the new product address the needs of a large enough group of people? - Will the new product need to be changed or adapted for this enlarged group? - How difficult and how costly would it be to scale-up to meet increased demand? - What is needed for the new product to appeal to this larger group? - Will the price need to be changed? - Will the price need to be changed? - How could we reach and engage a growing group of users? How much would this cost? - Will the increase in users be reflected in increasing value creation i.e. will the increase in outcomes outstrip the increase of the cost of achieving such growth? The last hypothesis is about Sustainability which has two mutually reinforcing facets. The first relates to the ease with which competitors can replicate the new product and the second concerns how easily the organisation itself will accept the changes required to implement the initiative. The typical questions for the first facet are: - How easily can the competition imitate the new product or substitute it with another product? How long would it take? - What kinds of barriers are there which will preserve the advantage? - What kinds of barriers are there which will preserve the - Are there other barriers we could create? How much would they cost? And for the second facet: - How will the organisation need to change to enable the new product to be implemented? - Will the organisation be able to cope with such changes? - Is there something about the initiative that we can adapt in order to make it more acceptable to the people and culture of the organisation? What impact would this adaptation have in terms of value creation? By addressing these questions executives engaged in NPD will be able to decide whether the new product merits being moved forward. Furthermore, this approach will help to identify potentially serious flaws which require fixing before the initiative can be progressed. Edited by: ISTEI – University of Milan-Bicocca © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it developed, but the most important thing is to select the ones that are the most appropriate for the particular circumstances. The fitness for purpose of any technique will depend on the time and resources available, but the ideal should be something which is simple, quick and inexpensive. developed, but the most important thing is to select the ones that are the most appropriate for the particular circumstances. The fitness for purpose of any technique will depend on the time and resources available, but the ideal should be something which is simple, quick and inexpensive. In any experiment about new product development, managers and engineers separate an independent variable (the ‘cause’) from a dependent variable (the ‘effect’) and then manipulate the former in order to observe changes in the latter. The manipulation, followed by careful observation and analysis, then gives rise to learning about relationships between cause and effect, which can be applied or tested in other settings. For example, the weight or shape of a product can be manipulated to examine its effect on how easy it is for customers to use. Fear of failure can also be an inhibiting factor in the use of experimentation and hypothesis testing. Because of this, the importance of a company culture which encourages transparency and appropriate risk is underlined. One of the key barriers to experimentation has always been the cost, since it has often been considered expensive in terms of the time involved and the effort expended. What has changed, particularly given the new technologies available, is that it is now possible to perform more experiments in an economically viable way while accelerating the drive towards a successful new product. To overcome the cost constriction barrier, managers have essentially two choices: - change the fundamental economics of experimentation through new creative processes and new innovative technologies - try to get more out of each experiment by employing sophisticated statistical methods, which help to manipulate multiple variables in a single experiment while maintaining integrity in its data analysis 5. The Two Facets of Experimentation To get maximum benefit from experimentation, there must be clarity about which tests will be carried out, in which order, and the metrics which will be used to judge the outcome and decide whether or not the hypothesis has been proven. Many experimentation tools and techniques are available and new ones can always be Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 110 5.2 Facet 2: Accelerating Learning through Experimentation Iteration time The time from planning the experiment to when the analysed results are available and used for planning another reiteration Capacity The number of same fidelity experiments that can be carried out per unit of time Strategy The extent to which the experiments are run in parallel or series Signal-to-noise ratio The extent to which the variable of interest is obscured by the ‘noise’ of too many other variables Type of experiment The degree of variable manipulation (incremental versus radical changes) Table 1: Thomke’s (2003) Factors that Affect the Learning by Experimentation able 1: Thomke’s (2003) Factors that Affect the Learning by Experimentation 5.1 Facet 1: Using Creative Processes and New Technologies to Increase the Number of Experiments New creative processes and new innovative technologies now enable more learning to be created more rapidly, and the outcomes can be incorporated in even more experiments at less expense. Examples can be found in: Customer usage simulations. This involves the building of simple mock-up user interfaces (e.g. a website) to see if customers are interested in a particular value proposition, including the description of the product features, the price and even how the product works. This particular type of process has been exploited by the Lean Start Up and Design thinking approaches. Computer modelling. Since 1945, when the first Monte Carlo based simulation was used to build a computer generated artificial world for the development of the hydrogen bomb, computer modelling has become an essential part of science. However, it is only with the dramatic increase in the availability of low cost computer power that computer modelling has become an everyday reality. Today, Artificial Intelligence (AI) packages or Computer Aided Design (CAD) can in many cases be used instead of physical experiments in design and market testing. 3D CAD has in many instances now eliminated almost completely the need for physical prototypes. Many organisations using ‘big data’ can use a computer simulation to assess the likely response of customers to, say, a change in price. Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 111 5.2 Facet 2: Accelerating Learning through Experimentation When all relevant variables are known, formal statistical techniques and protocols allow for the most efficient design and analysis of experiments. These techniques are used widely in many fields of process and product optimisation today and can be traced to the first half of the 21st century when the statistician and geneticist Sir Ronald Fisher first applied them to agricultural and biological science. The techniques he pioneered have become the foundation of what today we call Design of Experiments (DoE) Design of Experiments (DoE) is a statistical method of establishing which variables are important in a process, and the conditions under which these variables should work to optimize that process (Ilzarbe et al. 2008). Methods from the field of DoE have been applied to quality control problems in many engineering fields for several decades (Kuhn, Reilly 2002) and according to Ilzarbe et al (2008) many scientists and statisticians have contributed to DoE development and to its application in different fields. Thomke (2003) identifies seven factors (see Table 1) that affect the ability to learn through experimentation which are: fidelity, cost, iteration time, capacity, sequential and parallel strategies, signal to noise ratio and type of experiment. Table 1: Thomke’s (2003) Factors that Affect the Learning by Experimentation Factor Definition Fidelity of experiments The degree to which a model and its testing conditions represent a final product, process, or service under actual user conditions Cost of experiments The total cost of designing, building, running, and analysing an experiment, including expenses for prototypes, laboratory use, etc. Iteration time The time from planning the experiment to when the analysed results are available and used for planning another reiteration Capacity The number of same fidelity experiments that can be carried out per unit of time Strategy The extent to which the experiments are run in parallel or series Signal-to-noise ratio The extent to which the variable of interest is obscured by the ‘noise’ of too many other variables Type of experiment The degree of variable manipulation (incremental versus radical changes) Table 1: Thomke’s (2003) Factors that Affect the Learning by Experimentation Factor Definition Fidelity of experiments The degree to which a model and its testing conditions represent a final product, process, or service under actual user conditions Cost of experiments The total cost of designing, building, running, and analysing an experiment, including expenses for prototypes, laboratory use, etc. Edited by: ISTEI – University of Milan-Bicocca 6.1 Fashion House The fashion house is a fully integrated textile company with over a century of history and many innovations to its name. They were the first to introduce a certain type of dyeing, which allows fast and very economical production and to introduce large scale fully automated cutting plants. They were also among the first to launch successfully the ‘fast fashion’ business model made famous by the likes of ZARA, which quickly became the company’s main driver of growth and profitability. Unfortunately, in recent years the performance of the fast fashion division declined dramatically. A succession of management changes failed to reverse this decline. Late in 2013 a new management team, with considerable experience in turnarounds was called in. This new team invited us to work with the company, and in particular with the NPD team. They understood that the company’s core challenge, which would unlock the turnaround, was to return to its tradition of innovation. Their products suffered from two problems. First, they were coming to market much later than competitor’s products and and therefore, in the seasonal and rapidly changing world of fashion, it was impossible to command prices that would guarantee profitability (‘by the time the products were out it was time for the heavy discount sales’). Secondly, many of their products were not in line with customer tastes, leading to high levels of unwanted stock. The management decided to completely overhaul the NPD process by introducing a new system called Marketeyes, which introduced the discipline of experimentation to the heart of NPD. Marketeyes is a type of social workflow management system that allows an idea for a new product to go through a set of rapid experiments, which reduce the risk that the product will not succeed in the market. These experiments range from a simple and quick survey about the idea in the form of a professionally designed sketch of the garment with details of the price points and the type of material, that within 2 hours is sent to over 3000 shop assistants who give their view about the product, to a pre-ordering e-commerce facility that allows carefully selected ‘predictive’ customers (who historically have purchased garments that turned out to be successful) to view and pre-order products. © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it achieves volumes and revenue that are significant for the organisation, contributing to the bottom line and ultimately to the sustaining of their firm’s competitive advantage achieves volumes and revenue that are significant for the organisation, contributing to the bottom line and ultimately to the sustaining of their firm’s competitive advantage At the time of writing we have started work with all four multinationals, but because of the different stages of progress of the projects we have chosen, in this paper, to concentrate on just two of the cases: those of the fashion house and the heating systems manufacturer. 6. Early Evidence from Field Work As part of a 3 year study we have worked with a number of multinational organisations based in the UK and Italy all of which carry out a considerable part of their business outside their domestic markets. The sectors covered are fashion (production and retailing), heating systems (electric and gas), animal health pharmaceuticals and rail transportation. As part of the research we have been allowed to work alongside the NPD teams as they attempt to successfully launch new products. A successful launch is one where within a reasonable amount of time (always less than 24 months) the product Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 ISSN: 1593-0319 112 Edited by: ISTEI – University of Milan-Bicocca © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it or launched with a reduced range. Marketeyes is accessible and used by most of the divisions of the organisation from product design to supply chain, from management control to shop assistants, which enables the fashion house to spread knowledge about new products, as well as invite suggestions for new products. or launched with a reduced range. Marketeyes is accessible and used by most of the divisions of the organisation from product design to supply chain, from management control to shop assistants, which enables the fashion house to spread knowledge about new products, as well as invite suggestions for new products. Based on a recent spring collection, early evidence suggests that the products launched using Marketeyes perform twice as well, in terms of ‘in season sell’, compared with the products launched using the traditional NPD system. From the evidence gathered so far it seems that, thanks to fast experimentation and the learning gained from the analysis of the experimental results, Marketeyes provides a very efficient way to validate the value and growth hypothesis. It is too early to fully assess the sustainability hypothesis as only one aspect of sustainability – the ability of the company to adapt to the new product – can currently be examined. The competition has not yet reacted, because the size of the product portfolio produced using Marketeyes is still very small. 6.1 Fashion House The results of all the experiments are analysed through statistical techniques that provide insights about whether the product is likely to be successful and also the size of the potential success, in terms of volume and value of sales. Marketeyes keeps track of all the experiments and provides hard evidence of what does and what does not work as well as what can be changed to increase the chances of success. This has accelerated the process to the point where an informed decision as to whether to launch a new garment can be made within 7 working days. The process is not only speedy but also very discriminating, since many products that were initially thought to be potential blockbusters have been stopped Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 113 6.2 Gas and Electric Heating Systems The company we are working with is a multinational, serving over 50 countries and with production facilities in more than half a dozen countries, specialising in the design, manufacture, and distribution of electric and gas heating systems. It has a history of successfully developing good quality, mid-range, technically sophisticated products, all in its traditional core business. This has given the company the reputation of one of the best value for money brands in its industry in the world. The company has, over the last 15 years, installed a very large number of heating systems which will soon be due for replacement. The biggest challenge for the company is how to ensure that its own products will be chosen to replace or repair the aging systems rather than those supplied by their competitors. . This is complicated by the fact that its products are distributed mainly through independent installers and service centres and it is therefore these independent businesses who have the direct relationship with the users of the systems. However, most of the independent installers and service centres distribute and fit systems from a wide range of manufacturers and have proven to have very little long term loyalty to any of the brands they sell. Their choice of brand tends to hinge upon the current levels of commission and the likelihood of them being awarded a contract for maintaining the installations they carry out. Furthermore, in recent years new technology players such as Nest Labs with products like the ‘learning thermostat’, have entered the space from adjacent markets, introducing devices that are providing some of the features of traditional heating systems. Given this context, in early 2013 the company decided to launch a project to develop a device that would allow remote connectivity with heating systems, as well as a wide array of other features, such as automatic optimisation of temperature and safety alerts. Edited by: ISTEI – University of Milan-Bicocca © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it sort of ‘field laboratory’ to provide better information about, for instance, how its systems were performing and what parts needed replacement. sort of ‘field laboratory’ to provide better information about, for instance, how its systems were performing and what parts needed replacement. Initially, the project was driven by the R&D and engineering divisions who presented a business case to the Board which required a multi-million Euro investment for designing and deploying the product. The Board was not convinced. Several years before, a similar project (with a similar budget) not only failed dramatically but also caused difficulties with the company’s distribution network. In the light of this salutary experience the Board was reluctant to take another sizeable risk. As a result they decided to change their approach. They asked the marketing team to investigate how the level of risk could be reduced. g g The marketing team, working closely with our researchers, started by carefully identifying the main needs, or Job to Be Done, of each actor and the cost of the best currently available solution that would meet these ‘vivid’ needs (e.g. remotely controlled thermostat, plus a special maintenance contract). Based on this research the team produced a high quality brochure and a non-functional but graphically complete app for a device that presented the product as if it already existed, including information on price, key features and pictures of what the device would look like. Armed with the brochure and the app, the Company approached a number of installer-service centres selected on the basis of their openness to innovation. Each service centre was told that the product would be launched in 12 months but, as a valued partner, they were being given the opportunity to pre-order. Unlike most traditional market research approaches, the Company asked service centres who liked the product to actually sign an order, thereby capturing not just intention to buy, but actual sales. To their surprise, the first few sales meetings were a disaster. The potential customers were very negative about a number of aspects of the product including the design, the price and the main features. Undiscouraged, the team used this valuable, if somewhat painful, feedback to ‘pivot’: changing their working hypotheses to accommodate this new data. But they were not yet ready to go back to their R&D colleagues to commission the actual product. 6.2 Gas and Electric Heating Systems The purpose was to combine, in one device, the needs, or ‘Job to Be Done’, of three different actors or stakeholders; the end users needing to keep the temperature of their houses under (remote) control and have problems diagnosed by service centres , the service centres needing to streamline their operation by using remote diagnosis to tell them what needs to be done before the engineer makes a service call, and finally the Company itself which needed a Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 114 Edited by: ISTEI – University of Milan-Bicocca © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it Instead, guided by the insights generated by their research the team kept on using this approach to refine their understanding of what the product should be like. After another half-dozen iterations of the process: varying the features, price, and even design and listening carefully to the feedback, the team was leaving most of its sales meetings with sizeable pre-orders. They now knew that the product they visualised was one which would sell. The Board was presented with the findings of the team and the R&D department, after some persuasion, committed to create a prototype of a device which was simpler and easier to develop than originally envisaged. Although the marketing team had already significantly reduced the level of risk in creating and launching the new device, they knew that more could be done. They decided to run the same type of experiment directly with end customers: the people that would eventually have the device fitted in their homes. Working with service centres, the team put up display stands in major department stores which showcased the product to passing shoppers using pictures, leaflets and a very simple video cartoon. The comments received from prospective customers provided another rich source of information and triggered further ‘pivots’ and rapid design changes. For example, the team had hypothesised that they could rely on domestic Wi-Fi to transmit information from the device, given the high penetration of Wi-Fi in Italian households. However, their store-based research revealed that around half the Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 115 © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it people having Wi-Fi at home, switch it off overnight to save energy or because of worries about the health risks of electro-magnetic fields. As a result many people would prefer to buy a GPRS version. Using this sort of feedback changes were able to made to the price, features and presentation of the product at a stage when such alterations could be made quickly, cheaply and without embarrassment to the Company. The entire process took approximately 10 weeks from start to finish. It enabled the Company, at least theoretically, to avoid a costly failure with a product that would not have been well received. Instead, they now have a product that has a much greater chances of success. While this achievement alone would have justified the project, there were also a number of other benefits. Participating in the project allowed people across the organisation to understand the biases they had about what customers wanted and valued; biases that were often incorrect. The experience also had a strong impact on two aspects of how the company would work in future. First, it had the effect of accelerating the product development process as R&D staff had been forced to use unconventional methods such as 3D rendering to prototype and test their creations. Secondly, it led to the development of a ‘Go to Market’ model built on a clearer understanding of the tools and processes required by the salesforce to sell such an innovative product. Edited by: ISTEI – University of Milan-Bicocca 7. Discussion and Lessons Learned The three major lessons learned so far from this research are: - Companies should carry out as many experiments as possible at an early stage of the NPD process. This will reduce the overall time to market as well as the cost of designing and launching a new product. - Companies should carry out as many experiments as possible at an early stage of the NPD process. This will reduce the overall time to market as well as the cost of designing and launching a new product. g g g p - It is vital to have the courage to abandon or put on ice any new product if the evidence in support of the product from the experiments is not sufficiently strong. - There is a limit to the amount of uncertainty which can be removed through planned and deliberate experimentation. Even after the launch, it is best to consider all events and experiences as an extension of the research process and take note of the outcomes. The final lesson learned is also a note of caution. Many people mistakenly assume that experimentation is the same as running a pilot phase. However, we would argue that experimentation is very different from running a pilot for the following reasons: - Pilots are usually the prelaunch of an initiative that has already been developed, with the aim of the pilot being to prepare for full implementation. Experimentation, in contrast, is a process used to develop a new product and ensure that it meets the criteria of value, growth and sustainability. - In a pilot, the final or advanced version of the new product is tested, albeit on a small scale. This limits its usefulness as a means of testing because if it goes wrong it is hard to say which particular aspect led to the failure. As a result, areas for improvement cannot be accurately Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 ISSN: 1593-0319 116 © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it pinpointed. Experimentation is a progressive process, which scrutinises different aspects of a new product in turn, and therefore is much more likely to highlight exactly what is wrong, or right, about the new product. Although pilots are often carried out with the intention of smoothing the process of change within the organisation, if they are presented as a fait accompli - a predetermined new product from the top - they may have the opposite effect and build resistance. In contrast, experimentation can be done as a participative exercise, which allows staff to contribute to and understand the development of a new product, process or service. Involving staff in this way not only leads to a better developed initiative, it also means they are more likely to support its implementation 8. Limitations and Further Research The research reported in this paper has of necessity been limited to two case studies. From our experience so far the use of the framework suggested in this paper offers a means to improve the NPD process in terms of selecting appropriate new products that fully satisfy customer needs, speed of NPD delivery, and ultimately cost. Only through the continued application of the methods described can the framework be further tested, which will, of course, take time. However, the more people who apply the approach the sooner we will understand its impact more fully. As in the NPD approach we are recommending, we are looking for evidence to refine the framework. Applying the methods we have described to work in tandem with other methodological processes could offer improved performance of those methods and raise the success rate in selecting the right product for the right customer at the right time and at the right price. We would argue that disciplined experimentation could prove a useful addition to other NPD processes, provided it is conducted correctly. Bibliography Amabile Teresa M. (1997) Motivating Creativity in Organizations: On Doing What you Love and Loving What you Do, California Management Review, vol. 40, n. 1, pp. 39–58. http://dx.doi.org/10.2307/41165921 Barczak Gloria, Griffin Abbie, Kahn Kenneth B. (2009) Trends and Drivers of Success in Npd Practices: Results of the 2003 Pdma Best Practices Study, Journal of Product Innovation Management, vol. 26, n. 1, pp. 3–23. http://dx doi org/10 1111/j 1540 5885 2009 00331 x Bisgaard Søren (1992) Industrial Use of Statistically Designed Experiments: Case Study References and Some Historical Anecdotes, Quality Engineering, vol. 4, n. 4, pp. 547–562. http://dx.doi.org/10.1080/08982119208918936 Brondoni Silvio M. (2012) Innovation and Imitation: Corporate Strategies for Global Competition, Symphonya. Emerging Issues in Management (symphonya.unimib.it), n. 1, pp. 10-24 http://dx.doi.org/10.4468/2012.1.02brondoni Brondoni Silvio M. (2009) Market-Driven Management, Competitive Customer Value and Global Network, Symphonya. Emerging Issues in Management (symphonya.unimib.it), n. 1, pp. 8-25. http://dx.doi.org/10.4468/2009.1.02brondoni Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 117 dx.doi.org/10.1111/1540-5885.320071 Cooper Robert G., Kleinschmidt Elko J. (1987) New Products: What Separates Winners from Losers, Journal of Product Innovation Management, vol. 4, n. 3, pp. 169-184. http://dx.doi.org/10.1111/1540-5885.430169 Cooper Robert G., Kleinschmidt Elko J. (1990) New Products: The Key Factors in Succes,. American Marketing Assoc., Chicago. Cooper Robert G., Kleinschmidt Elko J. (1991) New Product Processes at Leading Industrial Firm, Industrial Marketing Management, vol. 10, n. 2, pp. 137-147. Cooper Robert G., Kleinschmidt Elko J. (1991) New Product Processes at Leading Industrial Firm, Industrial Marketing Management, vol. 10, n. 2, pp. 137-147. http://dx.doi.org/10.1016/0019-8501(91)90032-B p g Cooper Robert G., Kleinschmidt Elko J. (1993) Screening new products for potential winners, Long Range Planning, vol. 26, n. 6, pp. 74-81. http://dx.doi.org/10.1016/0024-6301(93)90208-W Cooper Robert G., Kleinschmidt Elko J. (1993) Screening new products for potential winners, Long Range Planning, vol. 26, n. 6, pp. 74-81. Cooper Robert G., Edgett Scott J., Kleinschmidt Elko J. (1998) Best Practices for Managing R&D Portfolios, Research - Technology-Management, vol. 41, n. 4, pp. 20-33. Cooper Robert G., Edgett Scott J., Kleinschmidt Elko J. (1997a) Portfolio Management in New Product Development: Lessons From the Leaders-Part I, Research - Technology Management, vol. 40, n. 5, pp. 16-28. Cooper Robert G., Edgett, Scott, & Kleinschmidt, Elko J. (1997b) Portfolio Management in New Product Development: Lessons from the Leaders-Part II, Research - Technology Management, vol. 40, n. 6, pp. 43-52. Cooper Robert G., Edgett Scott, Kleinschmidt Elko J. (2000) New problems, new solutions: Making portfolio management more effective, Research - Technology Management, vol. 43, n. 2, pp. 18- 33. Cooper Robert G. (2008) Perspective: The Stage-Gate Idea-to-Launch Process - Update, What's New, and NexGen Systems, The Journal of Product Innovation Management, vol. 25, n. 3, pp. 213-232. http://dx.doi.org/ 10.1111/j.1540-5885.2008.00296.x Cormican Kathryn, O’Sullivan David (2004) Auditing Best Practice for Effective Product Innovation Management, Technovation, vol. 24, n. 10, pp. 819–29. htt //d d i /10 1016/S0166 4972(03)00013 0 p g j Cormican Kathryn, O’Sullivan David (2004) Auditing Best Practice for Effective Product Innovation Management, Technovation, vol. 24, n. 10, pp. 819–29. http://dx.doi.org/10.1016/S0166-4972(03)00013-0 Davidson Jeffrey M., Clamen Allen, Karol Robin A. (1999) Learning from the Best New Product Developers, Research-Technology Management, vol. 42, n. 4, pp. 12–18. DeCusatis Casimer (2008) Creating, Growing and Sustaining Efficient Innovation Teams, Creativity and Innovation Management, vol. 17, n. 2, pp. 155–164. http://dx.doi.org/10.1111/j.1467-8691.2008.00478.x Ertas Atila, Jones Jesse C. (1996) The Engineering Design Process. 2nd ed. © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it Brondoni Silvio M. (2008) Ouverture de ‘Market-Driven Management and Global Markets - 2’, Symphonya. Emerging Issues in Management (symphonya.unimib.it), n. 2, pp. 1-12. http://dx.doi.org/10.4468/2008.2.01ouverture Buchanan Richard (1992) Wicked Problems in Design Thinking, Design Issues,vol. 8, n. 2, pp. 5-21. http://dx.doi.org/10.2307/1511637 Castellion George, Markham Stephen K. (2013) Perspective: New Product Failure Rates: Inﬂuence of Argumentum ad Populum and Self-Interest, Journal of Product Innovation Management, vol. 30, n. 5, pp. 976-979. tp://dx.doi.org/10.1111/j.1540-5885.2012.01009.x Cook Stephen C. (1989) Applying Critical Chain to Improve the Management of Uncertainty in Projects, MBA and MSc Thesis, Sloan School of Management and the Department of Electrical Engineering on May 9. Cooper Brant, Vlaskovits Patrick (2010) The Entrepreneur's Guide to Customer Development: A cheat sheet to The Four Steps to the Epiphany. s.l. Cooper-Vlaskovits. Cooper Robert G., Kleinschmidt Elko J. (1986) An lnvestigation into the New Product Process: Steps, Deficiencies and Impact, Journal of Product Innovation Management, vol. 3, n. 2, pp. 71- 85 dx.doi.org/10.1111/1540-5885.320071 New York, N.Y.: John Wiley & Sons, Inc. Fixson Sebastian K. (2009) Teaching Innovation through Interdisciplinary Courses and Programmes in Product Design and Development: An Analysis at 16 US Schools, Creativity and Innovation Management, vol. 18, n. 3, pp. 199-208. g pp http://dx.doi.org/10.1111/j.1467-8691.2009.00523.x Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 118 Edited by: ISTEI – University of Milan-Bicocca http://dx.doi.org/10.1007/s11518-012-5190-5 Highsmith Jim (2002) What is Agile Software Development? Crosstalk, Journal of the Defense Software Engineering, vol. 15, n. 10, pp. 4-9. Highsmith Jim (2004) Agile Project Management: Creating Innovative Products, Addison-Wesley Professional, Indiana. Hustad Thomas P. (1977) Approaches to the Teaching of Product Development and Management, Product Development Management Association, 113. Hustad Thomas P. (1977) Approaches to the Teaching of Product Development and Management, Hustad Thomas P. (1977) Approaches to the Teaching of Product Development and Management Product Development Management Association, 113. p g Ilzarbe Laura, Alvarez Maria J., Viles Elisabeth, Tanco Martin (2008) Practical Applications of Design of Experiments in the Field of Engineering: A Bibliographical Review, Quality and Reliability Engineering International, vol. 24, n. 4, pp. 417–428. http://dx.doi.org/ 10.1002/qre.909 http://dx.doi.org/ 10.1002/qre.909 Jenner Stephen (2007) Gates with Teeth: Implementing a Centre of Excellence for Investment Decisions, Paper Presented at the First International Stage-Gate Conference, February 20–21, St. Petersburg, FL. Kahn Kenneth B., Barczak Gloria, Nicholas John, Ledwith Ann, Perks Helen (2012) An Examination of New Product Development Best Practice, Journal of Product Innovation Management, vol. 29, n. 2, pp. 180-192. http://dx.doi.org/10.1111/j.1540-5885.2011.00888.x Kelley Tom (2001) The Art of Innovation: Lessons in Creativity from IDEO, America’s Leading Design Firm, Currency Books, New York. Kuhn D. Richard, Reilly Michael J. (2002) An Investigation of the Applicability of Design of Experiments to Software Testing, 27th NASA/IEEE Software Engineering Workshop, NASA Goddard Space Flight Centre, 4-6 December. © SYMPHONYA Emerging Issues in Management, n. 2, 2015 symphonya.unimib.it Goldratt Eliyahu H. (1997) Critical Chain, North River Press, Great Barrington. Hall Nicholas G. (2012) Project Management: Recent Developments and Research Opportunities, Journal of System Science and System Engineering, vol. 21, n. 2, pp. 129-143. http://dx.doi.org/10.1007/s11518-012-5190-5 http://dx.doi.org/10.1109/SEW.2002.1199454 Leach Larry P., (1999) Critical Chain Project Management Improves Project Management, Project Management Journal, vol. 30, n. 2, pp. 39-51. g pp Loch Christoph (2000) Tailoring Product Development to Strategy: Case of European Technology Manufacturer, European Management Journal, vol. 18, n. 3, pp. 246–58. p g Martin Roger L. (2009) The Design of Business: Why Design Thinking is the Next Competitive Advantage, Harvard Business Press, Boston. Morris Peter W. G. (1990) The Strategic Management of Projects, Technology in Society, vol. 12, n. 2, pp. 197-215. Morris Peter W. G. (1990) The Strategic Management of Projects, Technology in Society, vol. 12, n. 2, pp. 197-215. http://dx.doi.org/10.1016/0160-791X(90)90008-Z Mueller Roland M., Thoring Katja, (2012) Design Thinking versus Lean Startup: A Comparison of Two User-Driven Innovation Strategies, International Design Management Research Conference, August 8-9, Boston, USA f g Onyemar Vincent, Pesquera Martha R., Ali Abdul (2013) What Entrepreneurs Get Wrong, Harvard Business Review, May 2-7. Ri E i (2011) Th L S U H T d ' E U C i I i y Ries Eric (2011) The Lean Start-Up: How Today's Entrepreneurs Use Continuous Innovation to Create Radically Successful Businesses, Crown Business, New York. Rittel Horst W. J. (1972) On the Planning Crisis: Systems Analysis of the First And Second Generations, Bedriftsokonomen, vol. 8, pp. 390-396. f pp Simon Herbert (1969) The Sciences of the Artificial, MIT Press, Cambridge. Smith Preston G., Reinertsen Donald G. (1998) Developing Products in Half the Time – New Rules, New Tools, John Wiley & Sons, New York. Thomke Stefan H. (2003) Experimentation Matters – Unlocking the Potential of New Technologies for Innovation, Harvard Business School Press Boston. Edited by: ISTEI – University of Milan-Bicocca ISSN: 1593-0319 119
https://openalex.org/W4389345570	https://techrxiv.figshare.com/articles/preprint/Exploding_AI-Generated_Deepfakes_and_Misinformation_A_Threat_to_Global_Concern_in_the_21st_Century/24715605/1/files/43425978.pdf	English	null	Exploding AI-Generated Deepfakes and Misinformation: A Threat to Global Concern in the 21st Century	null	2,023	cc-by	4,320	This paper was downloaded from TechRxiv (https://www.techrxiv.org). LICENSE CC BY 4.0 SUBMISSION DATE / POSTED DATE 02-12-2023 / 05-12-2023 CITATION Singh, Dr. Pawan; Dhiman, Dr. Bharat (2023). Exploding AI-Generated Deepfakes and Misinformation: A Threat to Global Concern in the 21st Century. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.24715605.v1 10.36227/techrxiv.24715605.v1 10.36227/techrxiv.24715605.v1 Exploding AI-Generated Deepfakes and Misinformation: A Threat to Global Concern in the 21st Century Dr. Pawan Singh, Associate Professor, Department of Communication & Media Technology, J.C. Bose University of Science and Technology, YMCA, Faridabad, Haryana, India Dr. Bharat Dhiman, Assistant Professor, Department of Communication & Media Technology, J.C. Bose University of Science and Technology, YMCA, Faridabad, Haryana, India Dr. Bharat Dhiman, Assistant Professor, Department of Communication & Media Technology, J.C. Bose University of Science and Technology, YMCA, Faridabad, Haryana, India Their significance in spreading misinformation arises from several factors: Authenticity: Deepfakes appear genuine, making it challenging for people to discern between real and manipulated content. This authenticity allows false information to be disseminated widely without being easily recognized as fake. Virality: In today's digital age, information spreads rapidly across various online platforms. Deepfakes, due to their high-quality and attention-grabbing nature, have the potential to go viral quickly, reaching a vast audience before their falseness can be identified. Misrepresentation: Deepfakes can be used to misrepresent individuals, public figures, or events, altering perceptions and potentially damaging reputations or causing public unrest. By impersonating someone or altering their statements, deepfakes can deceive viewers and manipulate public opinion [1, 11]. Targeted Disinformation: They can be employed to spread targeted disinformation, amplifying existing tensions or conflicts by creating seemingly authentic content that reinforces certain narratives or biases. Deepfakes significance in spreading misinformation Deepfakes are highly realistic, AI-generated manipulations of audio, video, or images that convincingly depict events, situations, or individuals saying or doing things they never did. These creations are often so authentic-looking that they can mislead viewers into believing false information or fabricated scenarios [1, 17]. 2. Evolution of AI technology in generating realistic fake content The evolution of AI technology in generating realistic fake content, particularly through deep learning algorithms, has seen significant advancements over the past decade. Here's an overview of this evolution: Abstract Deepfakes the term was coined in 2018 by a Reddit user who created a Reddit forum dedicated to the creation and use of deep learning software for synthetically face swapping female celebrities into pornographic videos. According to Sumsub's research in 2023, the top-5 identity fraud types in 2023 are AI-powered fraud, money muling networks, fake IDs, account takeovers, and forced verification. The country most attacked by deepfakes is Spain; the most forged document worldwide is the UAE passport, whereas Latin America is the region where fraud has increased in every country. On November 24, 2023, the Union Government of India issued an advisory to social media intermediaries to identify misinformation and deepfakes. A deepfake refers to a specific kind of synthetic media where a person in an image or video is swapped with another person's likeness. AI-generated deepfakes have emerged as a complex and pervasive challenge in today's digital landscape, enabling the creation of remarkably convincing yet falsified multimedia content. This review paper examines the multifaceted landscape of deepfakes, encompassing their technological underpinnings, societal implications, detection methodologies, and ethical considerations. The review aggregates and synthesizes a broad array of scholarly articles, studies, and reports to elucidate the diverse typologies of deepfakes, including face-swapping, voice cloning, and synthetic media, while delineating the intricate methodologies employed in their fabrication. This review culminates in an overview of future directions and recommendations, advocating for proactive measures to counter the escalating threat posed by AI-generated deepfakes. Keywords: deepfakes, misinformation, generative AI, AI-generated misinformation, responsible AI, AI-generated deepfakes, deepfake detection, face-swapping, voice cloning, synthetic media, money muling networks Early Stages: Basic Generative Models: Initially, basic generative models like Restricted Boltzmann Machines (RBMs) and VariationalAutoencoders (VAEs) were used to generate synthetic data. These models had limited success in creating realistic content due to constraints in modeling complex data distributions. Introduction of Generative Adversarial Networks (GANs): The introduction of GANs by Ian Goodfellow and his team in 2014 marked a pivotal moment. GANs consist of two neural networks a generator and a discriminator engaged in a competitive process. The generator creates synthetic content while the discriminator learns to differentiate between real and fake data. This adversarial training enables GANs to produce more realistic and high-quality outputs across various domains, including images, videos, and text [2, 12]. Progression to Deep Neural Networks: The proliferation of deep neural networks, particularly convolutional neural networks (CNNs) and recurrent neural networks (RNNs), further enhanced the capabilities of AI in generating realistic content. CNNs excel in image-related tasks, enabling the creation of visually convincing deepfakes, while RNNs are effective in generating sequential data like text or audio. Technological Refinement and Accessibility: Over time, improvements in hardware capabilities and the availability of large datasets facilitated more sophisticated training of AI models. Additionally, the open-source nature of many deep learning frameworks allowed researchers and developers worldwide to contribute to the development of advanced algorithms, democratizing the creation of deepfake-generating tools [3]. Enhanced Realism and Multimodal Capabilities: Recent advancements in AI have led to the development of multimodal deepfakes, combining multiple modalities such as audio, video, and text to create more immersive and convincing fake content. This convergence of modalities enhances the realism of deepfakes, making them harder to detect. Concerns and Ethical Implications: The evolution of AI technology in generating realistic fake content has raised significant concerns regarding the potential misuse of deepfakes for spreading disinformation, manipulating public opinion, and violating privacy and consent. The Centre issued advisory to the significant social media intermediaries to The Centre issued advisory to the significant social media intermediaries to  Ensure that due diligence is exercised and reasonable efforts are made to identify misinformation and deepfakes, and in particular, information that violates the provisions of rules and regulations and/or user agreements.  Such cases are expeditiously actioned against, well within the timeframes stipulated under the IT Rules 2021.  Such cases are expeditiously actioned against, well within the timeframes stipulated under the IT Rules 2021.  Users are caused not to host such information/content/Deep Fakes.  Remove any such content when reported within 36 hours of such reporting.  Ensure expeditious action, well within the timeframes stipulated under the IT Rules 2021, and disable access to the content / information. The intermediaries were reminded that any failure to act as per the relevant provisions of the IT Act and Rules would attract Rule 7 of the IT Rules, 2021 and could render the organization liable to losing the protection available under Section 79(1) of the Information Technology Act, 2000 [22]. 3. Societal impact of deepfakes and the challenges: Erosion of Trust and Credibility: Deepfakes blur the line between truth and fiction, eroding trust in media, institutions, and even interpersonal relationships. When realistic but false content proliferates, it becomes increasingly challenging to discern authentic information from manipulated content [11]. Manipulation of Information and Misinformation: Deepfakes can be used to manipulate public opinion, spread false narratives, and create confusion around crucial issues. They can be leveraged for political propaganda, discrediting individuals, or inciting unrest by portraying fabricated events or statements as real. Privacy Concerns and Consent: Deepfakes raise significant privacy concerns as they can fabricate content that appears to feature individuals engaged in activities they never did. This challenges the notion of consent and can lead to the misuse of personal data. Potential for Exploitation and Blackmail: The creation of deepfakes poses the risk of exploitation, enabling malicious actors to create compromising content that could be used for extortion or blackmail. Impact on Reputation and Authenticity: Deepfakes can damage the reputation and authenticity of individuals or organizations. By falsifying information or portraying individuals engaging in inappropriate behavior, deepfakes can tarnish reputations irreparably. Challenges in Detection and Counteraction: The rapid advancement of deepfake technology makes it challenging to detect and counteract these manipulated media. Developing effective detection methods is crucial, but it's an ongoing race between the creation of fakes and the development of detection tools. Legal and Ethical Dilemmas: Addressing the legal and ethical implications of deepfakes is complex. Existing laws might not adequately cover the creation and dissemination of deepfakes, raising questions about accountability and liability. Impact on Journalism and Media Integrity: Deepfakes can undermine journalistic integrity, leading to a decline in public trust in media. It necessitates the implementation of stringent verification processes to maintain the credibility of information. 4. Union Government of India advisory to social media The Centre issued advisory to the significant social media intermediaries to 5. Types of Deepfakes: Deepfakes can be categorized into various types: Deepfakes can be categorized into various types: Face Swapping Deepfakes: These involve swapping faces in videos or images, replacing the original face with another person's face. Deep learning models, especially Generative Adversarial Networks (GANs), are often used to achieve highly realistic face swaps. Voice Cloning: This type of deepfake involves generating synthetic voice recordings that mimic someone else's voice. Neural network-based models can analyze and replicate a person's speech patterns and intonations, creating believable fake audio. Text-based Deepfakes: These involve generating written content, such as articles, social media posts, or comments, that mimic the writing style and content of a particular individual. Natural Language Processing (NLP) models can generate text that resembles a specific author's writing [4, 21]. Synthetic Media and Audiovisual Manipulation: This type combines various elements to create entirely fabricated content, including videos or audio recordings of events that never occurred or conversations that never took place. These deepfakes involve creating completely fictional scenarios or altering existing media to convey false information. Gesture and Behavior Manipulation: Some deepfakes focus on altering individuals' body movements, gestures, or behaviors in videos. These manipulations can change the meaning of the original content or create misleading impressions. Multimodal Deepfakes: These combine multiple modalities (audio, video, text) to create more immersive and convincing fake content. By synchronizing different modalities, these deepfakes become harder to detect and more persuasive [5]. 6. Impact of Deepfakes: The impact of deepfakes spans various domains and can profoundly affect individuals, society, and even global affairs. Here are some key impacts: The impact of deepfakes spans various domains and can profoundly affect individuals, society, and even global affairs. Here are some key impacts: Undermining Trust and Reality Perception: Deepfakes blur the distinction between truth and falsity, eroding trust in visual and audio evidence. This can lead to skepticism towards authentic media, making it challenging to discern real from manipulated content. Misinformation and Disinformation: Deepfakes are potent tools for spreading misinformation, manipulating narratives, and influencing public opinion. They can be used to fabricate events, statements, or behaviors, amplifying false information and sowing confusion. Political and Social Consequences: Deepfakes can influence elections, political discourse, and social dynamics. They may affect the credibility of public figures, manipulate public perceptions of policies, and even incite social unrest by fabricating inflammatory content. Privacy Violations and Personal Harm: Individuals can become victims of deepfakes, facing privacy breaches or reputational damage. Deepfakes can be used to create fake explicit content, defame individuals, or misrepresent their actions, leading to personal and emotional harm. Impact on Journalism and Media Integrity: The spread of deepfakes challenges the integrity of journalism and media. Journalistic credibility may suffer if deepfakes are mistaken for authentic sources, undermining the role of media as reliable purveyors of information. Impact on Journalism and Media Integrity: The spread of deepfakes challenges the integrity of journalism and media. Journalistic credibility may suffer if deepfakes are mistaken for authentic sources, undermining the role of media as reliable purveyors of information. Economic and Business Repercussions: Businesses and industries reliant on trust and authenticity may suffer economic setbacks due to deepfake-related scandals. For instance, financial fraud or manipulated corporate communications can lead to financial losses and reputational damage. Economic and Business Repercussions: Businesses and industries reliant on trust and authenticity may suffer economic setbacks due to deepfake-related scandals. For instance, financial fraud or manipulated corporate communications can lead to financial losses and reputational damage. Security Threats and National Security Concerns: Deepfakes pose security threats, including the potential for using fabricated content for cyberattacks, misinformation campaigns, or even in intelligence operations, undermining national security. Security Threats and National Security Concerns: Deepfakes pose security threats, including the potential for using fabricated content for cyberattacks, misinformation campaigns, or even in intelligence operations, undermining national security. 6. Impact of Deepfakes: Challenges in Law Enforcement and Legal Proceedings: Deepfakes present challenges for law enforcement and legal proceedings. Authenticating evidence becomes harder, and distinguishing real from manipulated content in legal contexts poses significant hurdles. 8. Ethical and Legal Implications: The proliferation of deepfakes raises significant ethical and legal concerns, touching upon various aspects of society, privacy, and human rights. Here are some key ethical and legal implications: 7. Detection and Mitigation Techniques: Detection and mitigation of deepfakes pose significant challenges due to their increasing sophistication. Several approaches and techniques are being developed to address this issue. Here are some common methods: Forensic Analysis: Experts use forensic techniques to analyze inconsistencies in deepfakes. This includes examining anomalies in facial movements, lighting, reflections, or inconsistencies in audiovisual elements that are not present in authentic media [1, 6, 19]. Digital Watermarking and Authentication: Some platforms use digital watermarks or cryptographic techniques to embed information into media files during creation. These watermarks can help verify the authenticity of the content. Machine Learning Algorithms: Counter AI algorithms are developed to detect anomalies in deepfake content. These algorithms leverage machine learning models trained on datasets of both real and synthetic media to identify patterns or artifacts specific to manipulated content. Face and Voice Recognition Technology: Advanced face and voice recognition technologies are utilized to identify discrepancies between real and fake elements in audiovisual content. These technologies compare facial features or voice patterns against known authentic data. Behavioral Analysis: Monitoring behavioral patterns, such as user engagement or interaction with content, can help detect anomalies associated with deepfake dissemination. Unusual behavioral patterns might signal the presence of manipulated content. Blockchain and Decentralized Verification:Blockchain technology is explored to create immutable records or timestamps for media content, allowing users to verify the authenticity and origin of media files. Collaborative Initiatives and Databases: Collaborative efforts involving researchers, tech companies, and governments aim to create comprehensive databases of deepfakes to train detection models and share knowledge and techniques for better detection. Policy and Legislation: Governments and regulatory bodies are exploring policies and legislation to address deepfake dissemination, establishing legal frameworks for prosecuting individuals involved in malicious creation and distribution of deepfakes. Ethical Implications: Informed Consent and Privacy: Deepfakes often use individuals' likenesses without their consent, infringing on privacy rights. The creation and dissemination of deepfakes without explicit consent raise ethical questions about respecting individuals' autonomy over their image and identity [7,8]. Manipulation and Misrepresentation: Deepfakes can manipulate and misrepresent individuals, leading to reputational harm, emotional distress, or damage to personal and professional relationships. This raises ethical concerns about the impact on an individual's dignity and well- being. Truth and Trust: Deepfakes blur the line between reality and fiction, eroding public trust in media and authentic information sources. Ethical considerations revolve around maintaining truthfulness, integrity, and transparency in communication and media representation. Societal Impact: Deepfakes can exacerbate societal divisions, manipulate public opinion, and distort historical records. Ethical considerations involve the broader impact on society, democracy, and the dissemination of accurate information. Legal Implications: Privacy Laws: Existing privacy laws might not adequately address the creation and distribution of deepfakes. Legislators are exploring amendments or new regulations to protect individuals' rights regarding their likeness and personal data. Intellectual Property and Copyright: Deepfakes may infringe upon intellectual property rights by using copyrighted material or individuals' likenesses without permission. Legal frameworks need to adapt to address these violations and enforce copyright protections. Defamation and Libel: Deepfakes can be used to defame or libel individuals by portraying them in false, damaging scenarios. Legal frameworks must delineate liabilities and address instances where deepfakes cause harm or damage reputations. Criminal Use and Fraud: Deepfakes have the potential for criminal use, including financial fraud, identity theft, or creating malicious content. Laws must be updated to prosecute individuals engaged in illegal activities using deepfake technology. Regulatory Approaches: Governments are considering regulatory approaches to mitigate the negative impacts of deepfakes, including labeling requirements for manipulated content or mandates for platforms to implement detection and removal protocols. 9. Public Perception and Awareness: Public perception and awareness regarding deepfakes are crucial in mitigating their impact and fostering resilience against misinformation. Here are some key aspects: Public perception and awareness regarding deepfakes are crucial in mitigating their impact and fostering resilience against misinformation. Here are some key aspects: Understanding the Existence of Deepfakes: Educating the public about the existence and capabilities of deepfake technology is essential. Many people may not be aware of how easily digital media can be manipulated, leading them to believe false information. Recognizing Signs of Manipulated Content: Teaching individuals to recognize signs of manipulated content, such as anomalies in facial expressions, unnatural movements, or inconsistencies in audiovisual elements, helps in discerning potential deepfakes. Media Literacy and Critical Thinking: Promoting media literacy programs that teach critical thinking skills can empower individuals to question and verify the authenticity of information they encounter online. This includes understanding biases, fact-checking methods, and verifying sources [8,19]. Responsibility of Content Sharing: Encouraging responsible behavior in content sharing is crucial. Educating the public about the impact of sharing potentially false or misleading information can help prevent the inadvertent dissemination of deepfakes. Role of Platforms and Technology Companies: Platforms and tech companies play a pivotal role in educating users about deepfakes and implementing measures to detect and label manipulated content. Providing tools for users to verify content authenticity can also aid in building awareness. Media and Journalism's Role: Media outlets and journalists can contribute by reporting on deepfakes, educating their audience about the risks, and demonstrating how to critically evaluate information sources. Community Engagement and Dialogue: Engaging communities in discussions about the implications of deepfakes fosters awareness and helps in understanding the potential consequences on society, democracy, and personal privacy. Continuous Updates and Adaptation: Given the rapid evolution of deepfake technology, continuous updates in educational programs and awareness campaigns are necessary to keep the public informed about emerging threats and detection methods [5,7,11]. 10. Future Directions and Recommendations: Future directions and recommendations concerning the landscape of deepfakes: Future directions and recommendations concerning the landscape of deepfakes: Technological Advancements: Invest in research and development of more sophisticated detection tools and algorithms capable of identifying increasingly realistic deepfakes. Explore AI-driven solutions that can adapt to evolving deepfake techniques. Technological Advancements: Invest in research and development of more sophisticated detection tools and algorithms capable of identifying increasingly realistic deepfakes. Explore AI-driven solutions that can adapt to evolving deepfake techniques. Collaborative Efforts: Foster collaboration among tech companies, researchers, policymakers, and civil society to create standardized protocols for detecting and combating deepfakes. Encourage data sharing and joint initiatives to tackle the issue collectively [20]. Education and Awareness: Implement comprehensive educational programs in schools, workplaces, and communities to enhance media literacy, critical thinking, and digital literacy. Equip individuals with the skills to recognize and respond to deepfakes. Education and Awareness: Implement comprehensive educational programs in schools, workplaces, and communities to enhance media literacy, critical thinking, and digital literacy. Equip individuals with the skills to recognize and respond to deepfakes. Regulatory Frameworks: Develop robust and adaptive legal frameworks that address the creation, distribution, and misuse of deepfakes. Consider amendments to privacy, defamation, and intellectual property laws to account for deepfake-related violations. Regulatory Frameworks: Develop robust and adaptive legal frameworks that address the creation, distribution, and misuse of deepfakes. Consider amendments to privacy, defamation, and intellectual property laws to account for deepfake-related violations. Platform Responsibility: Hold social media platforms and tech companies accountable for monitoring and regulating deepfake content on their platforms. Encourage the implementation of transparent policies and tools for users to report and verify content. Platform Responsibility: Hold social media platforms and tech companies accountable for monitoring and regulating deepfake content on their platforms. Encourage the implementation of transparent policies and tools for users to report and verify content. Ethical Guidelines: Establish ethical guidelines and industry standards for the responsible use of AI technology, including deepfake creation and dissemination. Promote ethical considerations in research, development, and deployment of AI systems. Global Cooperation: Encourage international collaboration and cooperation to address the transnational nature of deepfake dissemination. Facilitate information sharing and best practices among countries to combat the global spread of misinformation. Public-Private Partnerships: Foster partnerships between governments, academia, and private sectors to fund and support research initiatives aimed at understanding and countering the impact of deepfakes. Support Authentic Sources: Promote and trust content from reputable and verified sources. Encourage others to do the same, fostering a culture of credibility. Promote and trust content from reputable and verified sources. Encourage others to do the same, fostering a culture of credibility. 10. Future Directions and Recommendations: User Empowerment: Empower users with tools and resources to verify the authenticity of media content. Develop user-friendly verification tools and platforms that enable individuals to assess the credibility of information they encounter [9,20]. Continuous Monitoring and Adaptation: Stay vigilant and adaptive to emerging deepfake techniques. Continuously monitor and update strategies, technologies, and policies to stay ahead of evolving threats posed by manipulated media. Critical Thinking: Develop critical thinking skills to detect inconsistencies or suspicious elements within content. Be cautious of sensational or out-of-context information. Develop critical thinking skills to detect inconsistencies or suspicious elements within content. Be cautious of sensational or out-of-context information. Awareness and Education: Educate yourself and others about the existence and potential impact of deepfakes and misinformation. Understanding their capabilities and implications is crucial. Verification: Verify the authenticity of media content before sharing or believing it. Cross-reference information with reliable sources or fact-checking websites. Verify the authenticity of media content before sharing or believing it. Cross-reference information with reliable sources or fact-checking websites. Overreliance on AI Tools: While AI tools can aid in detecting deepfakes, avoid complete reliance on technology alone. Combine technological solutions with critical human judgment. Participation in Misinformation: Avoid engaging in the creation or propagation of misleading content. Participating in the dissemination of misinformation can have harmful consequences. Avoid engaging in the creation or propagation of misleading content. Participating in the dissemination of misinformation can have harmful consequences. Blind Belief: Refrain from blindly believing content solely based on its emotional appeal or alignment with pre-existing beliefs. Verify before accepting. Refrain from blindly believing content solely based on its emotional appeal or alignment with pre-existing beliefs. Verify before accepting. Ignoring Ethical Considerations: Don't overlook the ethical implications of deepfakes. Consider the potential harm or consequences before creating or sharing any manipulated content. Technological Solutions: Support the development and implementation of AI tools that detect and counter deepfakes, aiding in the identification of manipulated content. Conclusion: The rise of AI-generated deepfakes and the proliferation of misinformation pose significant threats on a global scale in the 21st century. These sophisticated technologies have the potential to disrupt democratic processes, manipulate public opinion, and undermine trust in media and institutions. The threat posed by AI-generated deepfakes and misinformation demand immediate attention and concerted action. Effective solutions must encompass a combination of technological innovations, regulatory measures, ethical considerations, and public awareness campaigns. Safeguarding the integrity of information and fostering a climate of trust and accountability in the digital realm are imperative for a resilient and informed society in the 21st century. Conflicts of Interest: The author declares no conflicts of interest. Funding: No funding was used in this work. Sharing Unverified Content: Avoid sharing unverified information or media without confirming its authenticity. Sharing misinformation inadvertently contributes to its spread. References: 1. Agarwal, Sakshi, and Lav R. Varshney, “Limits of Deepfake Detection: A Robust Estimation Viewpoint,” unpublished manuscript, arXiv:1905.03493, Version 1, May 9, 2019. 2. Ajder, Henry, Giorgio Patrini, Francesco Cavalli, and Laurence Cullen, The State of Deepfakes: Landscape, Threats and Impact, Amsterdam: Deeptrace, September 2019. 3. Atlantic Council’s Digital Forensic Research Lab, “#Stop the Steal: Timeline of Social Media and Extremist Activities Leading to 1/6 Insurrection,” Just Security, February 10, 2021. 4. Atlantic Council’s Digital Forensic Research Lab, “360/Digital Sherlocks,” webpage, undated. As of November 5, 2021: https://www.digitalsherlocks.org/360os- digitalsherlocks 5. Barari, Soubhik, Christopher Lucas, and Kevin Munger, “Political Deepfakes Are as Credible as Other Fake Media and (Sometimes) Real Media,” unpublished manuscript, OSF Preprints, last updated April 16, 2021. 6. Brown, Nina I., “Deepfakes and the Weaponization of Disinformation,” Virginia Journal of Law and Technology, Vol. 23, No. 1, 2020. 7. Changsha Shenduronghe Network Technology, ZAO, mobile app, Zao App APK, September 1, 2019. As of October 10, 2021: https://zaodownload.com 8. Chesney, Bobby, and Danielle Citron, “Deep Fakes: A Looming Challenge for Privacy, Democracy, and National Security,” California Law Review, Vol. 107, 2019, pp. 1753– 1820. 9. Clayton, Katherine, et al., “Real Solutions for Fake News? Measuring the Effectiveness of General Warnings and Fact‐Check Tags in Reducing Belief in False Stories on Social Media,” Political Behavior, Vol. 42, No. 2, 2020, pp. 1073–1095. 10. Cole, Samantha, “This Horrifying App Undresses a Photo of Any Woman with a Single Click,” Vice, June 26, 2019. 11. https://par.nsf.gov/servlets/purl/10233906#:~:text=in%20altering%20our%20beliefs%20 already,%2C%20humiliate%2C%20or%20harass%20victims. 12. https://www.frontiersin.org/articles/10.3389/fcomm.2023.1075654/full . https://www.frontiersin.org/articles/10.3389/fcomm.2023.1075654/full 13. https://www.europarl.europa.eu/RegData/etudes/STUD/2021/690039/EPRS_STU(2021)6 90039_EN.pdf 14. https://nbn-resolving.org/urn:nbn:de:0168-ssoar-86772-0 14. https://nbn-resolving.org/urn:nbn:de:0168-ssoar-86772-0 15. https://sumsub.com/fraud-report-2023/. 16. Merriam-Webster, “deepfake,” dictionary entry, undated-a. As of March 25, 2022: https://www.merriam-webster.com/dictionary/deepfake 16. Merriam-Webster, “deepfake,” dictionary entry, undated- https://www.merriam-webster.com/dictionary/deepfake 16. Merriam-Webster, “deepfake,” dictionary entry, undated-a. As of March 25, 2022: https://www.merriam-webster.com/dictionary/deepfake 17. Merriam-Webster, “disinformation,” dictionary entry, undated-b. As of April 25, 2022: https://www.merriam-webster.com/dictionary/disinformation 17. Merriam-Webster, “disinformation,” dictionary entry, undated-b. As of April 25, 2022: https://www.merriam-webster.com/dictionary/disinformation 18. Merriam-Webster, “misinformation,” dictionary entry, undated-c. As of April 25, 2022: https://www.merriam-webster.com/dictionary/misinformation 18. Merriam-Webster, “misinformation,” dictionary entry, undated-c. As of April 25, 2022: https://www.merriam-webster.com/dictionary/misinformation 19. https://www.mdpi.com/14248220/23/3/1708#:~:text=The%20detection%20of%20these% 20attacks,target%20defense%20(MTD)%20techniques. 19. https://www.mdpi.com/14248220/23/3/1708#:~:text=The%20detection%20of%20these% 20attacks,target%20defense%20(MTD)%20techniques. 20. https://www.sciencedirect.com/science/article/abs/pii/S0166497223000950 21. https://www.livelaw.in/law-firms/law-firm-articles-/deepfakes-personal-data-artificial- intelligence-machine-learning-ministry-of-electronics-and-information-technology- information-technology-act-242916 21. https://www.livelaw.in/law-firms/law-firm-articles-/deepfakes-personal-data-artificial- intelligence-machine-learning-ministry-of-electronics-and-information-technology- information-technology-act-242916 22. https://pib.gov.in/PressReleaseIframePage.aspx?PRID=1975445 22. https://pib.gov.in/PressReleaseIframePage.aspx?PRID=1975445 23. https://www.techtarget.com/whatis/definition/deepfake#:~:text=Deepfake%20AI%20is% 20a%20type,person%20is%20swapped%20for%20another. 23. https://www.techtarget.com/whatis/definition/deepfake#:~:text=Deepfake%20AI%20is% 20a%20type,person%20is%20swapped%20for%20another. 24. https://www.businesstoday.in/technology/news/story/what-are-the-different-types-of- deepfakes-and-how-you-can-spot-them-407118-2023-11-25 24. https://www.businesstoday.in/technology/news/story/what-are-the-different-types-of- deepfakes-and-how-you-can-spot-them-407118-2023-11-25
https://openalex.org/W1954113719	https://revistas.unal.edu.co/index.php/revsaludpublica/article/download/43490/46770	Spanish; Castilian	null	Prevalencia de bullying y factores relacionados en estudiantes de bachillerato de una institución educativa de Cali, Colombia, 2011	Revista de salud pública/Revista de salud publica	2,014	cc-by	5,440	Objective Determining the prevalence of bullying and its related factors amongst students from a high school in Cali, Colombia, 2011. RESUMEN Objetivo Determinar prevalencia de bullying y factores relacionados en estudiantes de bachillerato de una institución educativa de Cali, Colombia, 2011. Métodos Estudio de corte transversal en los estudiantes de sexto a noveno grado de una institución privada de la ciudad de Santiago de Cali. Para evaluar el bullying se usó la escala de Cisneros. Para controlar variables de confusión, se realizó un análisis multivariado mediante regresión logística no condicional. Resultados Se evaluaron 198 estudiantes, en quienes la edad promedio fue de 12,86 (DE=1,31) años. 50,5 % eran mujeres y 49,5 % hombres. La prevalencia de bullying fue 20,3 %. Se encontró asociación significativa entre ser víctima de bullying y disfunción familiar (leve y severa), así como con síntomas ansiosos. Conclusiones El bullying en estudiantes de sexto a noveno grado de esta institución educativa de Cali es un fenómeno frecuente y puede estar asociado a disfunción familiar leve y severa así como a síntomas de ansiedad con importancia clínica. Se requieren investigaciones adicionales para validar estos resultados. Palabras Clave: Bullying, prevalencia, estudiantes, instituciones académicas, estudios transversales, modelos logísticos (fuente: DeCS, BIREME). Bullying and its related factors amongst high school students From a school in Cali, Colombia Carlos A. Cassiani-Miranda1, Jennifer Gómez-Alhach2, Angela M. Cubides-Munévar1 y Mauricio Hernández-Carrillo1,3 1 Fundación Universitaria San Martín-Cali, Docente Ciencias básicas y clínicas Universidad Santiago de Cali seccional Palmira. kassio30@hotmail.com; angelacubides.epi@gmail.com 2 Departamento de Investigación de Clínica de Occidente. Cal, Colombia. jennifergomezalhach@ medicos.com 3 Escuela de Salud Pública, Universidad del Valle. Colombia. mauriciohc@gmail.com Recibido 27 Noviembre 2012/Enviado para Modificación 10 Julio 2013/Aceptado 16 Agosto 2013 Rev. salud pública. 16 (1): 14-26, 2014 Rev. salud pública. 16 (1): 14-26, 2014 Prevalencia de bullying y factores relacionados en estudiantes de bachillerato de una institución educativa de Cali, Colombia, 2011 Cassiani - Bullying escolar Material and Methods This was a cross-sectional study involving sixth to ninth grade students attending a private school in Santiago de Cali. Cisneros´ scale was used for assessing bullying in this population. Multivariate analysis was used with logistic regression to control confusing variables Results 198 students were included whose mean age was 12.86 (SD=1.31) years old; 50.5 % of the students were female (49.5 % male) and it was found that 20.3 % of the students had been bullied. A significant association was found amongst those being bullied and those suffering mild or severe family dysfunction and anxiety symptoms. Conclusions Bullying was frequent amongst sixth to ninth grade students from this school and it could have been related to the presence of mild or severe family dysfunction and clinically significant anxiety symptoms. Further research is needed to validate these results. Key Words: Bullying, prevalence, student, school, cross-sectional study, logistic model (source: MeSH, NLM). E l “Bullying” o acoso escolar (AE) es un fenómeno multicausal a nivel internacional en el ámbito escolar y constituye un problema de salud pública (1). Ocurre cuando un estudiante está expuesto repetidamente a acciones negativas que provienen de un compañero o de un grupo de personas en la escuela (2). Estas acciones negativas se pueden manifestar por medio del contacto físico, abuso verbal, gestos, rumores y exclusión de la víctima; además implica un desbalance entre la fuerza del agresor o bullies y la víctima (3). De acuerdo con datos del Health Behaviorin School-aged Children (HBSC) la prevalencia de victimización oscilaba entre 3 % y 33 % en los adolescentes de 11 a 15 años (4). Otros estudios señalan que la proporción de víctimas alcanza el 20,6 % en promedio con un rango entre 9 % y 54 % (5). En Estados Unidos se informan prevalencias globales de 29,9 %, 13,0 % para víctimas, 10,6 % para agresores o bullies y 6,3 % para ambos (6). En Uruguay, se reporta una prevalencia de bullying en hombres hasta del 43 % y de 17 % en mujeres (7). Otros datos en Latinoamérica muestran que en Chile la participación de estudiantes en el bullying osciló entre un 35 % a 55 % en el año 2005 (8). ABSTRACT Objective Determining the prevalence of bullying and its related factors amongst students from a high school in Cali, Colombia, 2011. 14 15 REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 16 consumo de sustancias adictivas como la nicotina (12) y alcohol (13). A estudios transversales y longitudinales han relacionado el hecho de ser víctima de bullying con trastornos psicopatológicos como depresión (14) y ansiedad (15). Existe evidencia sobre el rol familia (16) como factor de resiliencia en bullying. Por otro lado se ha encontrado asociación entre la exposición a violencia interparental física y el bullying especialmente en las mujeres (17). Abbotts et al y col (18) documentaron que la asistencia a la iglesia se puede considerar un factor protector de la agresión. El bullying es un fenómeno que está presente de manera reiterada en cualquier centro escolar y que causa problemas de salud (19). Por este motivo es importante conocer la prevalencia y dinámica del fenómeno así como sus factores relacionados en aras de diseñar intervenciones efectivas dirigidas a disminuir la incidencia de bullying y sus consecuencias. No obstante en el Valle del Cauca son pocos los estudios analíticos que exploren la prevalencia de bullying y sus factores relacionados. Por lo anterior, el objetivo de éste estudio es determinar la prevalencia de bullying y factores relacionados en estudiantes de bachillerato de una institución educativa privada ubicada de Cali durante el año 2011. Cassiani - Bullying escolar E En Colombia, según Chaux et al y col, 29,1 % de los estudiantes de quinto grado informaron ser victimizados, 21,9 % informó ser intimidador y 49,9 % reportaron ser observadores de bullying en los 2 últimos meses. (9). Un estudio realizado en Cali, mostró que el porcentaje de víctimas y agresores era de 24,7 % (10). Entre los factores que se han asociado al bullying se encuentran el sexo (ser hombre) (11), la edad (más frecuente entre 10 y 14 años) (5), y el REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 MATERIALES Y MÉTODOS Se realizó un estudio observacional de corte transversal. La población de estudio estuvo conformada por todos los estudiantes de básica secundaria (sexto a noveno grado) de una institución privada de la ciudad de Santiago de Cali, matriculados en el año lectivo 2011. Después de seleccionar los estudiantes, previo aval del Comité de Ética de la Fundación Universitaria San Martín, se solicitó permiso a las autoridades y se procedió aplicar las encuestas. En un primer momento se contactó al equipo directivo de la institución educativa con el propósito de explicarles los objetivos y el alcance de la investigación y proponerles su participación en el estudio. Al interior de cada grado se revisaron y actualizaron las listas de estudiantes. Se establecieron como criterios de inclusión: adolescentes matriculados y activos entre sexto y noveno de todas las edades presentes en estos grados que dieron su aprobación para participar en el estudio, mediante la firma de consentimiento informado por parte de padres, más la autorización de las directivas de la institución. Considerando los principios éticos para la investigación contenidos en las normas colombianas vigentes (20), los estudiantes participaron en forma voluntaria después de conocer los objetivos de la investigación. En el aula de clase, uno de los investigadores Cassiani - Bullying escolar 17 informó los objetivos del estudio y la forma de diligenciamiento del instrumento y se aclaró que no recibirán incentivo alguno por su participación. Para diligenciar apropiadamente los cuestionarios se necesitaron entre 40 y 50 minutos. La presencia de bullying fue la variable dependiente. El fenómeno de bullying se relacionó con variables independientes tales como consumo de tabaco y dependencia a la nicotina, Síntomas Depresivos con Importancia Clínica (SDIC), consumo problemático de alcohol, Síntomas de Ansiedad con Importancia Clínica (SAIC), disfunción familiar, actitud hacia el cristianismo; y variables sociodemográficas tales como sexo, edad y grado de escolaridad. Para determinar la existencia de bullying se usó la escala de Cisneros (21). Se trata de un cuestionario elaborado por Piñuel en el año 2000 (24) para sondear de manera periódica el estado y los índices de violencia en el entorno laboral, que luego se ha extendido a la evaluación del acoso en el ámbito escolar. REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 18 situaciones de intimidación descritas. En este informe para definir si un estudiante es víctima de intimidación escolar nos basamos en el IG pues nuestro objetivo era determinar la presencia o no de bullying. situaciones de intimidación descritas. En este informe para definir si un estudiante es víctima de intimidación escolar nos basamos en el IG pues nuestro objetivo era determinar la presencia o no de bullying. Para la medición de las variables independientes se utilizaron las siguientes escalas: Test de Etter para dependencia a nicotina (23) con 12 dígitos en quienes informaran consumo diario de cigarrillo, escala de depresión de Zung (ED-Z) (24), escala abreviada de actitud hacia el cristianismo de Francis (25), el Test para la identificación de trastornos relacionados con el alcohol (AUDIT) para evaluar consumo problemático de alcohol (26), APGAR familiar (27), escala de ansiedad de Zung versión abreviada de 5 ítems (28). Los datos fueron revisados manualmente y almacenados por duplicado para su posterior procesamiento en el paquete estadístico SPSS versión 17.0. Para las variables categóricas se calcularon porcentajes y para las variables cuantitativas, promedios y desviación estándar (DE), de acuerdo con la distribución de frecuencia de la variable. Para el porcentaje de individuos con diagnóstico de bullying se determinó el intervalo de confianza (IC) del 95 %. Para las variables categóricas se determinaron razones de oportunidad (OR) con IC95 %, y para las variables cuantitativas se aplicó la prueba de t de Student para determinar diferencia entre los individuos con presencia de bullying y los que no la informaban. Para controlar variables de confusión, se realizó un análisis multivariado mediante regresión logística no condicional y se calcularon los OR para las asociaciones entre bullying y las demás variables. Se tuvieron en cuenta aquellas variables relevantes que mostraron en el análisis bivariado una asociación con valor de probabilidad menor del 20 %. Para estimar el modelo final se siguieron las recomendaciones de Greenland (29) y se determinó la bondad del ajuste del mismo por medio del Hosmer Lemeshow (30). Para todas las pruebas estadísticas se aceptaron como significativos valores de probabilidad menores del 5 %. Se construyó un modelo explicativo para establecer la asociación entre bullying y las variables independientes. MATERIALES Y MÉTODOS La escala Cisneros evalúa 43 conductas de acoso solicitando a la persona que responde que valore en una escala de 0 (nunca) a 6 (todos los días) el grado periodicidad de las conductas descritas en cada uno de los enunciados de los ítems en el contexto escolar del estudiante, con una fiabilidad de 0,96. Esta escala fue validada por Cepeda en Bogotá, obteniéndose una versión de la encuesta constituida por 22 ítems con un adecuado desempeño psicométrico (22). Para la caracterización global de bullying se define una escala numérica en la que 3 corresponde a “Con frecuencia”; 2 a “A veces”; y 1 a “Nunca”, excepto en los ítems 7, 9, 19 y 26, donde a “nunca” se asignó 3, a “a veces” 2 y a “con frecuencia” 1. Así, en cada uno de los 22 ítems, 1 corresponde a un ambiente exento de la situación de intimidación, y 3 corresponde al caso donde la situación de bullying se presenta con frecuencia. A partir de esta escala numérica se definen tres índices para el estudio de bullying, basados en la propuesta presentada por Fidalgo y Piñuel (21). El primer índice, denominado índice global (IG), se calcula para cada estudiante y es definido como el promedio de la puntuación de sus ítems. Este índice toma valores en una escala continua entre 1 y 3, donde 1 indica que en el contexto escolar del estudiante no se presenta ninguna de las conductas de intimidación registradas en la encuesta y 3 indica que en el contexto escolar del estudiante se presentan “Con frecuencia” todas las REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 RESULTADOS La información se recogió a partir de 198 estudiantes de los grados sextos a noveno seleccionados por conveniencia. Ningún estudiante de los asistentes se negó a participar. La edad promedio del grupo fue de 12,86 (DE=1,31) años. En lo concerniente al género 97 (50,5 %) eran Cassiani - Bullying escolar 19 mujeres y 95 (49,5 %) eran hombres. En relación con la escolaridad, 59 (30,7 %) cursaban grado sexto, 48 (25,0 %) séptimo, 41 (21,4 %) octavo y 44 (22,9 %) noveno (Tabla 1). En relación con la ocurrencia de la intimidación, 39 (20,3 %) manifestaron ser víctimas de intimidación escolar según el índice global de la escala de Cisneros. Con respecto a la actitud hacia el cristianismo fue informada como mala en 45 (23,4 %) estudiantes. En relación con el funcionamiento familiar, 47 (24,5 %) estudiantes presentaron disfunción familiar leve, 24 (12,5 %) disfunción familiar moderada y 16 (8,3 %) disfunción familiar severa, según el APGAR familiar. Acerca del consumo de alcohol, fue clasificado como problemático en 11 (5,7 %) de los encuestados. Tabla 1. Características sociodemográficas de los estudiantes según la presencia de bullying en un colegio privado de Cali, Colombia. Año 2011 Variables sociodemográficas Escolares Intimidados Escolares no intimidados Total No. % No. % No. % Edad 11 12 13 14 15 16 7 10 13 5 3 1 17,90 25,60 33,30 12,80 7,70 2,60 27 39 30 37 19 1 17,60 25,50 19,60 24,20 12,40 0,70 34 49 43 42 22 2 17,70 25,50 22,40 21,90 11,50 1,00 Sexo F M 18 21 46,20 53,80 79 74 51,60 48,40 97 95 50,50 49,50 Grado escolar 6 7 8 9 10 14 12 3 25,60 35,90 30,80 7,70 49 34 29 41 32,00 22,20 19,00 26,80 59 48 41 44 30,70 25,00 21,40 22,90 Tabla 1. Características sociodemográficas de los estudiantes según la presencia de bullying en un colegio privado de Cali, Colombia. Año 2011 Tabla 2. Variables categóricas según sexo de los estudiantes con edades entre 11 y 16 años de un colegio privado de Cali, Colombia. Año 2011 11 y 16 años de un colegio privado de Cali, Colombia. Año 2011 Variable categórica Mujeres Hombres Total No. % No. % No. RESULTADOS % Víctima de intimidación escolar 18 18,60 21 22,10 39 20,30 Mala actitud hacía el cristianismo 22 22,70 23 24,20 45 23,40 Disfunción familiar Leve Moderada Severa 20 16 13 20,60 16,50 13,40 27 8 3 28,40 8,40 3,20 47 24 16 24,50 12,50 8,30 Consumo problemático de alcohol 9 9,30 2 2,10 11 5,70 Síntomas depresivos con importancia clínica 33 34,00 33 34,70 66 34,40 Síntomas de ansiedad con importancia clínica 4 4,10 4 4,20 8 4,20 REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 21 Cassiani - Bullying escolar Cassiani - Bullying escolar El análisis bivariado mostró una asociación estadísticamente significativa entre ser víctima de bullying y disfunción familiar leve (p=0,0001), disfunción familiar severa (p=0,0001) y síntomas de ansiedad con importancia clínica (p=0,002). La presencia de bullying fue independiente de la edad, sexo, actitud hacia el cristianismo, SDIC, disfunción familiar moderada y consumo problemático de alcohol (Tabla 3). En el análisis multivariado, después de realizar el ajuste del modelo mostró que se mantenía asociación significativa entre ser víctima de bullying y disfunción familiar (leve y severa) y los SAIC (Tabla 4 y 5). Tabla 5. Regresión logística ajustada por disfunción familiar y ansiedad para establecer la asociación con bullying en estudiantes de un colegio privado de Cali, Colombia. Año 2011 Variables en el modelo OR IC 95 % ρ Disfunción familiar leve 3,36 1,38 8,16 0,007 Disfunción familiar moderada 1,35 0,37 4,95 0,643 Disfunción familiar severa 6,6 1,98 22,00 0,002 SADIC 13,85 2,37 80,96 0,004 Bondad del ajuste de Hosmer-Lemeshow, X2 = 0,26;gl = 3, p = 0,9665 Tabla 5. Regresión logística ajustada por disfunción familiar y ansiedad para establecer la asociación con bullying en estudiantes de un colegio i d d C li C l bi Añ 2011 REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 20 En relación con la sintomatología depresiva, 66 (34,4 %) estudiantes presentaron puntajes de 20 o más en la escala de Zung. Para los síntomas ansiosos, se encontró que 8 (4,2 %) estudiantes informaron síntomas ansiosos de importancia clínica según la escala de ansiedad de Zung. En la Tabla 2 se presenta un análisis diferencial por sexo para estas variables categóricas. Tabla 3. Análisis bivariado de los factores asociados a bullying en estudiantes de un colegio privado de Cali, Colombia. Año 2011 Factores asociados Escolares Intimidados Escolares No Intimidados OR IC 95 % Valor ρ Sexo Femenino Masculino 79 74 18 21 1 1,24 (0,61-2,52) 0,54 Edad 15 y 16 14 13 12 11 20 37 30 39 27 4 5 13 10 7 1 0,67 2,16 1,28 1,29 (0,16-2,80) (0,61-7,60) (0,35-4,60) (0,33-5,03) 0,58 0,22 0,70 0,70 Actitud hacía al cristianismo Actitud buena Actitud menor 119 33 27 12 1 1,6 (0,73-3,50) 0,23 Funcionamiento familiar Buena función familiar Disfunción familiar leve Disfunción familiar moderada Disfunción familiar severa 92 33 19 8 12 14 5 8 1 3,25 2,01 7,66 (1,36-7,74) (0,63-6,39) (2,42-24,21) 0,008 0,23 0,001 Consumo alcohol Consumo no problemático Consumo problemático 143 10 38 1 1 0,37 (0,04 - 3,03) 0,35 Depresión No SDIC SDIC 101 52 25 14 1 1,08 (0,52-2,26) 0,82 Ansiedad No SAIC SAIC 150 2 33 6 1 13,6 (2,63-70,58) 0,002 Tabla 3. Análisis bivariado de los factores asociados a bullying en estudiantes de un colegio privado de Cali, Colombia. Año 2011 Tabla 4. Modelo saturado. Regresión logística ajustada por todas las variables medidas para los factores asociados a bullying en estudiantes p y g de un colegio privado de Cali, Colombia. Año 2011 de un colegio privado de Cali, Colombia. Año 2011 Variables en el modelo OR IC 95 % ρ Ser hombre 1,74 0,74 4,1 0,2 Tener 14 años 0,33 0,06 1,92 0,221 Tener 13 años 2,17 0,48 9,7 0,31 Tener 12 años 1,51 0,29 7,92 0,624 Tener 11 años 2,3 0,41 12,78 0,338 Menor actitud hacía el cristianismo 0,9 0,33 2,46 0,846 Disfunción familiar leve 4,59 1,72 12,22 0,002 Disfunción familiar moderada 2,34 0,58 9,36 0,229 Disfunción familiar severa 10,39 2,47 43,74 0,001 Consumo problemático de alcohol 0,4 0,03 4,23 0,447 SDIC 1,01 0,33 3,06 0,984 SAIC 22,59 3,15 161,99 0,002 Bondad del ajuste de Hosmer-Lemeshow, X2 = 8,63; gl = 8, p = 0,3742. REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 22 Existen varios modelos teóricos para explicar la asociación entre bullying y funcionamiento familiar que tienen que ver con el proceso de socialización de los niños. En este sentido, Barber (33) argumenta que existen tres procesos familiares críticos para el desarrollo de la socialización del niño. Estos procesos se relacionan con la creación de conexiones emocionales positivas con los cuidadores (conexión), la regulación de la conducta del niño en términos de supervisión, monitoría y otras formas de control en (regulación) y el desarrollo del niño de un sentido de identidad, eficacia y facilitación en un sentido de identidad, eficacia y dignidad (autonomía). Una explicación plausible del riesgo de ser víctima de bullying a partir de la disfunción familiar podría estar dado por el hecho que el estar expuesto a fenómenos violentos en el seno familiar haría que el niño aprenda a desarrollar una conducta sumisa ante el abuso de poder de los otros (32). Contrariamente a los hallazgos de nuestra investigación, otros estudios han encontrado falta de asociación entre bullying y el funcionamiento familiar. Veenstra et al y col (34) hallaron que variables familiares como el calor emocional, el rechazo y la sobreprotección no se asociaron con bullying. Adicionalmente, Rigby et al y col (35) encontraron una pobre influencia de las expectativas percibidas de los padres sobre el bullying. Es difícil concluir si las diferencias en estos resultados se deben a la influencia familiar en la dinámica del bullying o por diferencias metodológicas o la participación variables no consideradas. Esto enfatiza la necesidad de estudiar más la relación entre bullying y variables familiares. Nuestros resultados muestran una asociación significativa entre SAIC y ser víctima de bullying. Algunos estudios han establecido la relación entre bullying y ansiedad. Según Berry y Hunt (36) la bullying es un una experiencia estresante que al perpetuarse a través de los años predice la aparición de síntomas ansiosos. En esa misma dirección, Menesini et al, encontraron que ser víctimas de bullying se asociaba con padecer altos niveles de ansiedad (15). Otros estudios soportan el concepto que las víctimas de bullying manifiestan más síntomas ansiosos que los agresores (36). Dos de las teorías más consistentes para tratar de explicar la relación entre la victimización y los síntomas psicopatológicos como la ansiedad son la respuesta fisiológica al stress (37) y la distorsión cognitiva (38). DISCUSIÓN Los resultados de este estudio muestran una proporción de víctimas de bullying de 20,3 % similar a lo informado en otros estudios (4-10,22). Éstos resultados evidencian una asociación estadísticamente significativa entre bullying y la disfunción familiar (leve y severa) y los SAIC, aún después de ajustar por otras variables. Varios estudios muestran datos similares: Bowes y colaboradores (16) encontraron que una adecuada atmósfera familiar puede proteger a los adolescentes de las consecuencias negativas de la victimización. Así mismo, una encuesta transversal con 1 059 estudiantes italianos encontró que el bullying fue un predictor de exposición a violencia interparental y un grado variable de disfunción familiar especialmente en mujeres víctimas (17). En este sentido, Hernández y Gutiérrez, en un estudio de casos y controles encontraron que una de las variables que mejor explican el riesgo de sufrir bullying es pertenecer a una familia disfuncional y no compartir tiempo con la madre (31). Respecto del contexto familiar, se ha descrito que víctimas y agresores de bullying son sometidos, en mayor medida que otros menores, a un trato extremadamente coercitivo y hostil o están expuestos a contemplar conflictos o violencia adulta, aunque no siempre sean agredidos directamente en el seno familiar (32). REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 Así, se ha sugerido que los cambios fisiológicos en los sistemas de respuesta al stress como el eje hipotálamo-pituitario-adrenal median la Cassiani - Bullying escolar 23 asociación entre experiencias adversas tempranas y trastornos de ansiedad (37). La variabilidad individual en la reactividad al stress puede indicar que las víctimas de bullying lleguen a ser hiper o hiposensibles al stress explicando de esa manera porqué ellos desarrollan trastornos mentales tempranamente. Además, la experiencia de bullying en la adolescencia puede llevar a distorsión en la interpretación del entorno interpersonal, atribuyendo erróneamente connotaciones negativas a eventos vitales (38). En oposición, Bond et al y col, informaron que la victimización era predictiva de ansiedad en niñas (OR 2.60, IC95 % 1.2-2.5) pero no en niños (OR 1.36, IC95 % 0.6-3.0). Además en estudiantes de 9 grado no hubo asociación significativa entre síntomas ansiedad y bullying (OR 1.48 IC95 % 0.4-6.0) (39). Estas diferencias pueden estar relacionadas con el diferente comportamiento de esta asociación en diferentes contextos culturales o por diferencias sociales entre los grados escolares. De forma similar al nuestro, este estudio en adolescentes escolarizados no encontró asociación entre síntomas depresivos y bullying (OR 1.48 IC95 % 0.4-6.0) (39). Al Contrario, otros autores han encontrado asociación significativa entre el bullying, síntomas depresivos (14), consumo de alcohol (13) y de nicotina (12). Los estudios que han encontrado esta asociación entre bullying y tales factores se han realizado en muestras grandes hecho que podría explicar las diferencias con este trabajo cuya muestra puede ser insuficiente para encontrar diferencias significativas. Si bien este estudio no encontró asociación entre la actitud hacia el cristianismo y la victimización, no se encontraron estudios de asociación entre bullying y actitud hacia el cristianismo. Sin embargo, Sansone et al encontraron una asociación entre implicación en bullying durante la adolescencia y puntajes bajos en las escalas de espiritualidad en la adultez (40). No es posible establecer comparaciones adecuadas entre los hallazgos de nuestro estudio con este último debido a las diferencias en el diseño y la población estudiada. Se concluye que la frecuencia de bullying en los estudiantes de sexto a noveno grado de esta institución educativa de Cali es un fenómeno frecuente igual que en otras latitudes y se encuentra asociado a disfunción familiar (leve y severa) y los síntomas de ansiedad con importancia clínica. REFERENCIAS 1. Feder L. Bullying as a Public Health Issue. Int J Offender Ther Comp Criminol. 2007;51 (5):491-4. 2. Olweus D. Bullying at school. Basic facts and an effective intervention programme. Promot Educ. 1994;1 (4):27-31. 3. Trautmann A. Maltrato entre pares o “bullying” una visión actual. Rev Chil Pediatr. 2008;79(1):13-20. 4. Currie C, Nic Gabhainn S, Godeau E. International HBSC Network Coordinating Committee. The Health Behaviour in School-aged Children: WHO Collaborative Cross-National (HBSC) study: origins, concept, history and development 1982-2008. Int J Public Health. 2009;54Suppl 2:131-9. 5. Analitis F, Velderman MK, Ravens-Sieberer U, Detmar S, Erhart M, Herdman M, et al. Being bullied: associated factors in children and adolescents 8 to 18 years old in 11 European countries. Pediatrics. 2009;123 (2):569-77. p ( ) 6. Nansel TR, Overpeck M, Pilla RS, Ruan WJ, Simons-Morton B, Scheidt P. Bullying behaviors among US youth: prevalence and association with psychosocial adjustment. JAMA. 2001;285 (16):2094-100. 7. Cajigas de Segredo N, Kahan E, Luzardo M, Najson S, Ugo C, Zamalvide G. Agresión entre pares (Bullying) en un Centro Educativo de Montevideo: estudio de las frecuencias de los estudiantes de mayor riesgo. Rev Med Urug. 2006; 22(2): 143-151. y g g ( ) 8. Rudatsikira E, Muula AS, Siziya S. Prevalence and correlates of physical fighting among school-going adolescents in Santiago, Chile. Rev Bras Psiquiatr. 2008; 30(3):197-202. 9 Ch E M l A P dl k P S i i i li i l d i i l 9. Chaux E, Molano A, Podlesky P. Socio-economic, socio-political and socio-emotional variables explaining school bullying: a country-wide multilevel analysis. Aggress Behav. 2009;35(6):520-9. 10. Paredes MT, Álvarez MC, Lega LI. Estudio exploratorio sobre el fenómeno del “Bullying” en la ciudad de Cali, Colombia. Rev. latinoam. cienc. soc. niñez juv. 2008; 6(1): 295-317. 10. Paredes MT, Álvarez MC, Lega LI. Estudio exploratorio sobre el fenómeno del Bullying en la ciudad de Cali, Colombia. Rev. latinoam. cienc. soc. niñez juv. 2008; 6(1): 295-317. 11. Postigo Zegarra S, González Barrón R, Mateu Marqués C, Ferrero Berlanga J, Martorell PC. Behavioral gender differences in school relationships. Psicothema. 2009;21:453-8. 12 Vieno A Gini G Santinello M Different forms of bullying and their association to smoking 11. Postigo Zegarra S, González Barrón R, Mateu Marqués C, Ferrero Berlanga J, Martorell PC. Behavioral gender differences in school relationships. Psicothema. 2009;21:453-8. 11. Postigo Zegarra S, González Barrón R, Mateu Marqués C, Ferrero Berlanga J, Martorell PC. REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 24 diseñar estudios de seguimiento para validar los resultados de este trabajo y establecer la naturaleza de la asociación entre disfunción familiar, síntomas de ansiedad y bullying ■ diseñar estudios de seguimiento para validar los resultados de este trabajo y establecer la naturaleza de la asociación entre disfunción familiar, síntomas de ansiedad y bullying ■ Agradecimientos: María Camila Vargas por su aporte gramatical. Conflictos de interés: Ninguno. REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 Es necesario realizar investigaciones con un tamaño de muestra mayor en otras instituciones educativas de Cali y Colombia, además de REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 Cassiani - Bullying escolar 25 16. Bowes L, Maughan B, Caspi A, Moffitt TE, Arseneault L. Families promote emotional and behavioural resilience to bullying: evidence of an environmental effect. J Child Psychol Psychiatry. 2010; 51(7):809-17. 17. Baldry AC. Bullying in schools and exposure to domestic violence. Child Abuse Negl. 2003;27(7):713-32. 18. Abbotts JE, Williams RG, Sweeting HN, West PB. Is going to church good or bad for you? Denomination, attendance and mental health of children in West Scotland. Soc Sci Med. 2004; 58(3):645-56. 19. Sansone RA, Sansone LA. Bully victims: psychological and somatic aftermaths. Psychiatry (Edgmont). 2008;5(6):62-4. 20. Ministerio de Salud de Colombia. Resolución 008430 por la cual se establecen las normas científicas, técnicas y administrativas para la investigación en salud. Santafé de Bogotá; 1993. 21. Fidalgo A, Piñuel I. La escala Cisneros como herramienta de valoración del Bullying. Psicothema. 2004; 16(4): 615-624. 22. Cepeda-Cuervo E, Pacheco-Durán PN, García-Barco L, Piraquive-Peña CJ. Acoso Escolar a Estudiantes de Educación Básica y Media. Rev Salud Pública (Bogota).2008;10(4):517-28. 23. Etter JF, Le Houezec J, Perneger TV. A self-administered questionnaire to measure dependence on cigarettes: the cigarette dependence scale. Neuro psycho pharmacology. 2003; 28(2):359-70. 24. Díaz LA, Campo A, Rueda GE, Barros JA. Propuesta de una versión abreviada de la escala de Zung para depresión. Colomb Med. 2005;36(3):168-172. 25. Campo-Arias A, Oviedo HC, Cogollo Z. Internal consistency of a five-item form of the Francis scale toward Christianity among adolescent students. J Soc Psychol.2009;149(2): 258-62. 26. Alvarado ME, Garmendia ML, Acuña G, Santis R, Arteaga O. Validez y confiabilidad de la versión chilena del Alcohol Use Disorders Identification Test (AUDIT). Rev Méd Chile. 2009;137 (11):1463-8. 27. Forero LM, Mónica Cristina Avendaño MC, Duarte ZJ, Campo-Arias A. Consistencia interna y análisis de factores de la escala APGAR para evaluar el funcionamiento familiar en estudiantes de básica secundaria. Rev Col Psiquiatría. 2006;35(1): 23-29. 28. de la Ossa S, Martinez Y, Herazo E, Campo A. Estudio de la consistencia interna y estructura factorial de tres versiones de la escala de Zung para ansiedad. Colomb Med. 2009; 40(1): 71-7. 29. Greenland S. Modeling and variable selection in epidemiologic analysis. Am J Public Health. 1989; 79(3):340-9. 30. Hosmer DW, Taber S, Lemeshow S. The importance of assessing the fit of logistic regression models: a case study. Am J PublicHealth. 1991; 81(12):1630-35. 31. Hernández M, Gutiérrez MI. REFERENCIAS Behavioral gender differences in school relationships. Psicothema. 2009;21:453-8. 12. Vieno A, Gini G, Santinello M. Different forms of bullying and their association to smoking and drinking behavior in Italian adolescents. J Sch Health. 2011;81(7):393-9. 12. Vieno A, Gini G, Santinello M. Different forms of bullying and their association to smoking and drinking behavior in Italian adolescents. J Sch Health. 2011;81(7):393-9. 13. Radliff KM, Wheaton JE, Robinson K, Morris J. Illuminating the relationship between bullying and substance use among middle and high school youth. Addict Behav. 2012;37(4):569-72. 14. Fleming LC, Jacobsen KH. Bullying and symptoms of depression in chilean middle school students. J Sch Health. 2009;79(3):130-7. 15. Menesini E, Modena M, Tani F. Bullying and victimization in adolescence: concurrent and stable roles and psychological health symptoms. J Genet Psychol. 2009;170(2):115-33. Cassiani - Bullying escolar Cassiani - Bullying escolar Factores de riesgo asociados a la intimidación escolar en instituciones educativas públicas de cuatro municipios del departamento del Valle del Cauca. Año 2009. Rev Col Psiquiatría. 2013; 42(3), 238-247 32. Bowes, L, Arseneault, L, Maughan, B, Taylor, A, Caspi, A, Moffitt, T. School, Neighborhood, and Family Factors Are Associated With Children’s Bullying Involvement: A Nationally Representative Longitudinal Study. J Am Acad Child Adolesc Psychiatry. 2009;48(5):545-53. 33. Barber BK, Olsen JA. Socialization in context: Connection, regulation, and autonomy in the family, school, and neighborhood, and with peers. J Adolesc Res. 1997; 12(2):287–315. 34. Veenstra R, Lindenberg S, Oldehinkel AJ, De Winter AF, Verhulst FC, Ormel J. Bullying and victimization in elementary schools: a comparison of bullies, victims, bully/ victims, and uninvolved preadolescents. Dev Psychol. 2005;41 (4):672-82. REVISTA DE SALUD PÚBLICA · Volumen 16 (1), Febrero 2014 26 35. Rigby K. Why do some children bully at school? The contributions of negative attitudes towards victims and the perceived expectations of friends, parents and teachers. School- Psychol-Int. 2005; 26(2):147–61. 36. Berry K, Hunt CJ. Evaluation of an intervention program for anxious adolescent boys who are bullied at school. Adolescent Health. 2009;45 (4):376-82. 37. Heim C, Newport DJ, Heit S, Graham YP, Wilcox M, Bonsall R, et al. Pituitary-adrenal and autonomic responses to stress in women after sexual and physical abuse in childhood. JAMA. 2000; 284(5):592–7. 38. Kinderman P, Bentall RP. A new measure of causal locus: the internal, personal and situational attributions questionnaire. Personality and Individual Differences. 1996; 20(2):261–4. 39. Bond L, Carlin JB, Thomas L, Rubin K, Patton G. Does bullying cause emotional problems? A prospective study of young teenagers. BMJ. 2001; 323 (7311):480-4. 40. Sansone RA, Kelley AR, Forbis JS. Bullying in childhood and religious/spiritual status in adulthood among internal medicine outpatients. Int J Soc Psychiatry. 2013;59(8):739-44.
https://openalex.org/W2900642149	http://scielo.pt/pdf/aso/n227/n227a08.pdf	Portuguese	null	“Imãos de armas”: o CEP no cinema de propaganda da Primeira Guerra Mundial	Análise social/Análise social	2,018	cc-by	9,780	MARIA DO CARMO PIÇARRA https://doi.org/10.31447/as00032573.2018227.08 1 O portuense Alfredo Antunes da Mata fundou, em 1910, uma pequena empresa de pro dução de cinema que, em 1912, se chama Nunes de Matos & Cia – (Invicta Film). Inicialmente produz documentários de propaganda comercial e industrial além de atualidades filmadas, que exibe tanto nacional, como internacionalmente. O êxito dita que passe a fornecer atualidades às sociedades Pathé e Gaumont. Terá sido o caso da atualidade Manobras Navais Portuguesas, Manobras de Tancos, ambos de 1916, e Naufrágio do Veronese, de 1917. “Imãos de armas”: o cep no cinema de propaganda da Primeira Guerra Mundial Análise Social, liii (2.º), 2018 (n.º 227), pp. 438-457 https://doi.org/10.31447/as00032573.2018227.08 issn online 2182-2999 edição e propriedade Instituto de Ciências Sociais da Universidade de Lisboa. Av. Professor Aníbal de Bettencourt, 9 1600-189 Lisboa Portugal — analise.social@ics.ul.pt Análise Social, 227, liii (2.º), 2018, 438-457 Análise Social, 227, liii (2.º), 2018, 438-457 “Imãos de armas”: o cep no cinema de propaganda da Pri- meira Guerra Mundial. Embarques de tropas portuguesas, exercícios militares, a visita de Bernardino Machado à frente de batalha em Verdun e ao Reino Unido. No que se refere à participação na Primeira Guerra Mundial, resume-se a pouco mais do que isto o retrato dos “bravos soldados portugueses” no cinema. A exceção que confirma a regra é a do filme ama- dor alemão, Afundamento do Augusto Castilho, que documenta como a brutalidade deste primeiro conflito mundial não obs- tou a que ainda fosse sendo observado um código de honra militar e a bravura exigida aos combatentes portugueses para suprir a falta de meios de combate. palavras-chave: Primeira Guerra Mundial; propaganda; Portugal; cinema. “Brothers in Arms”: the Portuguese Expeditionary Corps in World War i propaganda films. We address the shipment of Portuguese troops, undertaking of military exercises, and the visits of Bernardino Machado to the Western Front (Verdun) and to the United Kingdom during the World War One. The cinematic portrait of the “brave Portuguese soldiers” is exam- ined. An alternative view is that of the German amateur film, Afundamento do Augusto Castilho (The Sinking of the Augusto Castilho), which documents how the brutality of the First World War coexisted with a code of military honor and the bravery required from Portuguese fighting men to make up for their lack of modern weaponry. keywords: World War One; propaganda; Portugal; cinema. https://doi.org/10.31447/as00032573.2018227.08 “Imãos de armas”: o cep no cinema de propaganda da Primeira Guerra Mundial Em Portugal, onde as primeiras sessões de cinema aconteceram meia dúzia de meses após a sessão histórica dos Lumière, em 1895, a natureza docu mental dos filmes foi preponderante no início da produção nacional. Só em 1911 estreia a primeira longa-metragem de ficção, Os Crimes de Diogo Alves, de gosto popular e inspirada pelo teatro. A ficção chega tarde, portanto, a um cinema que começou muito cedo, por iniciativa de fotógrafos curiosos, e elegeu como temas preferidos os da literatura de inspiração popular e melo dramática (Grilo, 2006). O investimento no cinema como forma de expressão artística é contemporâneo da República, sendo que a principal produtora de cinema do período, a Invicta Film – que veio a apostar na produção de fil mes ficcionais “tipicamente portugueses” –, é criada a par do novo regime e mantém uma atividade importante no campo das atualidades filmadas e dos documentários.1 Segundo Piçarra (2006), as atualidades filmadas nasceram com o cinema – fixadas pelos “caçadores de imagens” a soldo dos Lumière ou recriadas em estúdio por Méliès. Curtas-metragens mostravam acontecimentos políticos, económicos, desportivos, culturais, etc. e integravam os programas cinemato gráficos, sendo mostradas antes das longas-metragens de ficção. O aproveita mento do potencial propagandista das atualidades foi uma prática generalizada internacionalmente, sustentada por modelos mais ou menos informativos. Na conceção e finalidade eram comparáveis à imprensa escrita – constituiu-se MARIA DO CARMO PIÇARRA 440 uma imprensa cinematográfica que chegou a ser alvo de um estudo pela unesco em 1951 – e as atualidades eram, geralmente, editadas e mostradas semanal ou bissemanalmente. Porém, dado o poder do cinematógrafo, foram veículo recorrente de propaganda e é isso que explica que, além das majors – que optaram por manter este formato, caro e pouco rentável, por uma questão de prestígio –, muitos países tenham optado pelo apoio à produção de atuali dades estatais, que projetavam a nação através da divulgação das notícias do regime. A representação cinematográfica da participação portuguesa na Primeira Guerra Mundial devemo-la quase exclusivamente aos jornais de atualidades filmadas e aos documentários. E se é um facto que, como afirma Tiago Baptista (2011), a Primeira República não se interessou particularmente pelo cinema – o que é comprovável pela escassa produção legislativa e pela inexistência de apoios diretos ao cinema – há, porém, que assinalar o recurso ao poten cial propagandista do cinema para gerar unidade nacional em torno da par ticipação na guerra. 2 Ministro da Guerra de 22 de julho de 1915 a 8 dezembro de 1917 e responsável pela forma ção e instrução do cep. 3 Ferrão foi um dos sócios proprietários do Salão Central. Raul Lopes Freire, cinéfilo e dono do Salão Chiado, inaugurou este novo salão, no Palácio Foz, e, em 1908, constituiu a → 2 Ministro da Guerra de 22 de julho de 1915 a 8 dezembro de 1917 e responsável pela forma ção e instrução do cep. 3 Ferrão foi um dos sócios proprietários do Salão Central Raul Lopes Freire cinéfilo e “Imãos de armas”: o cep no cinema de propaganda da Primeira Guerra Mundial É certo que tal não foi uma originalidade da Primeira República Portuguesa. A sucessão de governos terá dificultado a conceção de um plano mais geral de apoio ao cinema e a atenção que lhe foi dada, através do seu uso, deliberado, como arma de propaganda, terá decorrido das neces sidades de legitimação por parte do regime e da produção de um consenso quanto à participação na guerra. Por outro lado, quer em Inglaterra quer em França aconteceram então os primeiros esforços organizados pelo Estado para a divulgação de doutrinas usando os noticiários cinematográficos, o que terá servido certamente de modelo. Importa, porém, analisar o uso que foi feito do cinema pela Primeira República durante a Primeira Guerra Mundial. Foi o ministro da Guerra Norton de Matos (1867-1955)2 o responsável pela criação da Secção Fotográfica e Cinematográfica do Exército (sfce), através de despa cho de 12 de janeiro de 1917, duas semanas antes da partida para a Flandres da 1.ª Brigada do cep. Dada a sua experiência cinematográfica e os serviços já prestados, será o capitão Carlos Nogueira Ferrão (1871-1938) – militar que em 1911 filmara, para a Lusa Film, Exercícios do Grupo de Baterias a Cavalo de Queluz, então já reformado, mas entretanto chamado ao serviço militar ativo por Norton de Matos – a organizar a secção criada por despacho, o que faz sob as ordens do tenente-coronel Desidério Bessa (1868-1920), e a produzir Embarque das Tropas Portuguesas.3 Posteriormente, já após o final da guerra, a 2 Ministro da Guerra de 22 de julho de 1915 a 8 dezembro de 1917 e responsável pela forma ção e instrução do cep.i 3 Ferrão foi um dos sócios proprietários do Salão Central. Raul Lopes Freire, cinéfilo e dono do Salão Chiado, inaugurou este novo salão, no Palácio Foz, e, em 1908, constituiu a → O CEP NO CINEMA DE PROPAGANDA DA PRIMEIRA GUERRA MUNDIAL 441 sfce é transformada na Direção dos Serviços Gráficos do Exército, através do Decreto n.º 5 935, de 28 de junho de 19194, e logrou sobreviver a duas mudan ças de regime: da Primeira República para o Estado Novo, na sequência do golpe militar de 1926, e deste para a democracia parlamentar, após a revolução de 25 de Abril de 1974. “Sociedade Animatográfica, Lda”, que geriu, mas da qual Ferrão foi, durante algum tempo, um dos associados. Quanto ao filme, Janeiro (2013) diz que fixa o primeiro embarque a 26 de janeiro, ou o seguinte a 23 de fevereiro. 4 Diário do Governo (1919), i Série (130), 4 de julho. 5 Segundo o Prontuário do Cinema Português (Matos-Cruz, Ferreira e Pina, 1989), este título é atribuído à Invicta Film. Porém, e dado que o realizador é Ernesto de Albuquerque, tal poderá ser engano. 5 Segundo o Prontuário do Cinema Português (Matos-Cruz, Ferreira e Pina, 1989), este título é atribuído à Invicta Film. Porém, e dado que o realizador é Ernesto de Albuquerque, tal poderá ser engano. “Sociedade Animatográfica, Lda”, que geriu, mas da qual Ferrão foi, durante algum tempo, um dos associados. Quanto ao filme, Janeiro (2013) diz que fixa o primeiro embarque a 26 de janeiro, ou o seguinte a 23 de fevereiro. 4 Diário do Governo (1919), i Série (130), 4 de julho. iário do Governo (1919), i Série (130), 4 de julho. O “MILAGRE” DA REPÚBLICA: PRELÚDIO DE UM CINEMA OFICIAL DE PROPAGANDA Antes da criação da sfce, o Ministério da Guerra subvencionou a produção de um filme de longa-metragem – 2500 metros de película, cerca de 90 minutos – que, com grande notoriedade, fixou a parada militar que decorreu em 22 de julho de 1916, em Montalvo, perto de Tancos. Parada da Divisão em Montalvo e Divisão da Instrução em Tancos, título com o qual foi distribuída uma ver são mais curta, de pouco mais de meia hora5 – que, por sua vez, foi relançada posteriormente, colorida com tintagens, com o título Exercícios de Infantaria, Cavalaria e Artilharia pela Divisão Militar de Tancos –, foi manivelado por Ernesto de Albuquerque (1883-1940). Albuquerque já era então conhecido por Cultura do Cacau (1909), filmado em S. Tomé e Príncipe, e realizado por si e pelo Capitão Ferrão (Piçarra, 2015). Estreada no Coliseu dos Recreios de Lisboa a 8 de agosto de 1916 e a 29 de agosto no Porto, esta produção consagrou a empreitada da criação e organiza ção, em pouco tempo, de uma unidade militar de grande dimensão – o Corpo Expedicionário Português (cep) –, à qual foi ministrada instrução no polígono de Tancos. O processo ficou conhecido – não obstante o laicismo da República – como “milagre de Tancos”. Apesar de Albuquerque ter sido o único operador com liberdade total para fixar a parada – da filmagem há um registo fotográfico de Arnaldo Garcez (1885-1964) –, assinale-se que a parada de 20 mil soldados também foi fil mada pela Invicta Film, resultando na produção A Mobilização Portuguesa em Tancos, com cerca de 40 minutos. Estreado ainda antes do filme oficial, a 3 de agosto, no cinema Passos Manuel, do Porto, este título terá sido visto por cen tenas de pessoas que esgotaram a sessão. Entusiasmado, o Primeiro de Janeiro “Sociedade Animatográfica, Lda”, que geriu, mas da qual Ferrão foi, durante algum tempo, um dos associados. Quanto ao filme, Janeiro (2013) diz que fixa o primeiro embarque a 26 de janeiro, ou o seguinte a 23 de fevereiro. 5 Segundo o Prontuário do Cinema Português (Matos-Cruz, Ferreira e Pina, 1989), este título é atribuído à Invicta Film. Porém, e dado que o realizador é Ernesto de Albuquerque, tal poderá ser engano. MARIA DO CARMO PIÇARRA 442 afirmou tratar-se do melhor filme português feito até então, mostrando que o soldado “de hoje” em “nada desmerece das tradições gloriosas do guerreiro antigo nosso”. 7 Curiosamente e apesar do título, as imagens são relativas a Tancos. 6 Visionável, mediante registo, em http://www.gaumontpathearchives.com. O “MILAGRE” DA REPÚBLICA: PRELÚDIO DE UM CINEMA OFICIAL DE PROPAGANDA Como refere Helena Pinto Janeiro (2013, p. 8) a propósito da estreia do documentário da Invicta, a imprensa local antecipou a estreia no Porto – no Salão da Trindade e no High Life (futuro Batalha) – do filme oficial, apresentado como: “A copia fiel das ultimas Manobras militares”, “admiravel ‘film’ feito por incumbencia especial do Ministerio da Guerra, dirigido e fiscalisado por um distincto official do Exercito […]. A sua divulgação foi autorizada pelo respectivo Ministro, como meio de propaganda dos progressos militares realisados pelos nossos exercitos” [O Primeiro de Janeiro, 11-08- -1916, p. 2]. Ambas as produções – estatal e privada – relativas às manobras de Tan cos estão perdidas. Do filme da dupla Ferrão/Albuquerque subsistem 1’ 41’’ de imagens que “migraram” para Contigent portugais, incluído no Journal Actualité distribuído pela Gaumont6, mais um minuto integrado na atualidade Lisbon Recruits das British Pathé News (1917, 35’’).7 Estes arquivos privados lograram preservar, pois, o que em Portugal não foi possível. Mostram-se sol dados a atravessar uma ponte em Vila Nova da Barquinha, marchando depois pelo planalto de Montalto. Fixa-se a construção de trincheiras, seguindo-se imagens da cavalaria, primeiro desfilando em formação e depois em exercícios no terreno. Finalmente, filmam-se a marcha para estação de comboio, acom panhada por populares, e a despedida na estação, em que os soldados acenam lenços brancos a partir das carruagens. Um intertítulo em inglês explica que “A jovem República está a dispensar a sua quota parte de homens aos exércitos Aliados e centenas de recrutas preparam-se para cumprir a sua missão”.i Portugal na Grande Guerra: Divisão Naval Portuguesa foi a produção ofi cial – a segunda, agora para o Ministério da Marinha – apresentada de seguida, tendo estreado no Salão Central de Lisboa, a 21 de setembro, e em dois cine mas do Porto, a 4 de outubro. Longa-metragem, em seis partes, da qual se desconhece o paradeiro de qualquer material, mostrava o quotidiano militar nos navios da marinha portuguesa, assim como exercícios navais tendo sido filmada por Albuquerque sob a direção de Ferrão.i Porém, ainda antes da criação da sfce e da encomenda da fixação quer do “milagre de Tancos”, quer da atividade da Divisão Naval, a documentação O CEP NO CINEMA DE PROPAGANDA DA PRIMEIRA GUERRA MUNDIAL 443 do esforço de guerra português já teria sido iniciada. 8 Nove conhecidos. Poderão ter existido mais. 9 Com pouco mais de 4’, é visionável em: http://www.cinemateca.pt/Cinemateca-Digital/Fic ha.aspx?obraid=4979&type=Video. 9 Com pouco mais de 4’, é visionável em: http://www.cinemateca.pt/Cinemateca-Digital/Fic ha.aspx?obraid=4979&type=Video. 8 Nove conhecidos. Poderão ter existido mais. O “MILAGRE” DA REPÚBLICA: PRELÚDIO DE UM CINEMA OFICIAL DE PROPAGANDA Não só para fazer a propaganda da ação da jovem República internamente junto ao público civil – que, como vimos, se interessou pela reorganização do exército – mas também para dispor de filmes que servissem para a propaganda junto das próprias tropas, que, através da projeção – cinematográfica, mas também de cada soldado na sua unidade –, eram animadas a cumprir o que a nação lhes MARIA DO CARMO PIÇARRA 444 “pedia”.10 Finalmente, os filmes serviam ainda, e talvez acima de tudo, para a projeção internacional do esforço português cujo novo regime, não obstante a sua recente imposição, apesar da sucessão de governos e das difíceis condições de vida em Portugal, mostrava assim ter força, legitimidade e capacidade para organizar um exército e participar na luta pela defesa das suas fronteiras e do seu estatuto de potência colonial.i Helena Pinto Janeiro (2013, p. 12) apurou que o filme da sfce sobre Tan cos foi exibido a individualidades estrangeiras, como o major-general Barnar diston (1858-1919), o chefe da missão militar franco-britânica que chegou a Portugal a 30 de agosto para negociar os termos da participação portuguesa no esforço de guerra da frente ocidental. Dois dias após uma sessão de projeção de cinema sobre a instrução ministrada em Tancos, a que assiste com Norton de Matos, Barnardiston escreve, num relatório oficial para o War Office, ter tido pouco tempo ainda para ter uma opinião definitiva sobre o exército por tuguês. Afirma, porém, que as impressões são favoráveis e que não vê motivo para o cep, após treinos, não ser de utilidade considerável. Acrescenta ainda que tanto ele como o colega francês estão bem impressionados com a energia e organização com que as autoridades militares portuguesas estão a investir no fortalecimento e desenvolvimento dos seus recursos. Os filmes Parada da Divisão em Montalvo e Portugal na Guerra – Divisão Naval Portuguesa não figuram sós na filmografia da participação portuguesa na Primeira Guerra Mundial. O jornal filmado francês Actualidades 14 incluiu um apontamento de cerca de dois minutos sobre Os Delegados de Portugal à Conferência dos Aliados, e também a Gaumont fixou O Embaixador de Portu gal em Paris, J. Chagas, na Conferência dos Aliados. Ernesto de Albuquerque filmou, provavelmente para o Ministério da Guerra, Partida dos Portugueses para a Guerra, sendo-lhe também atribuído Manobras Navais Portuguesas, para a Invicta. O “MILAGRE” DA REPÚBLICA: PRELÚDIO DE UM CINEMA OFICIAL DE PROPAGANDA Segundo a filmografia estabelecida, esta documentação foi feita predominantemente pela Invicta Films e pela Gaumont, embora haja títulos de filmes perdidos cuja produ ção não é conhecida e que podem ter uma origem não comercial, mas sim estatal. Assinale-se, em 1914, a produção de nove títulos8 cujo enfoque se reparte entre o registo da partida das tropas portuguesas para Angola, sobretudo, e Moçambique e a realização de exercícios militares. Destes títulos apenas dois – O Embarque de Tropas Expedicionárias para Angola e Moçambique e Exer cícios de Artilharia em 1914 – têm uma produtora conhecida: a Invicta Film. Desconhece-se quem assegurou a produção de As Expedições Portuguesas em África, Grandes Manobras de Artilharia da Serra do Pilar antes da sua Partida para Angola, Partida da Segunda Expedição Portuguesa para África, o ainda existente Partida do Regimento de Infantaria 19 de Chaves para Lisboa e Embarque para Angola9 [cf. Castelo de Chaves (1915)], Portugal na Guerra Europeia ou Os Portugueses e os Alemães em África e Tropas Portuguesas. Em 1915, o registo de partida de tropas para as colónias continua a dominar as atualidades sobre a participação portuguesa na guerra, sendo assegurado pela Invicta e pela Gaumont: O Castelo de Chaves – Formação do 19.º Regimento de Infantaria e a Cruz Vermelha (Gaumont) [cf. com Chegada do Regimento de Infantaria 19 a Chaves, de 1913, filmado por Alfredo Nunes de Matos, Invicta Film]; A Expedição a Angola (Invicta); Grandes Manobras de Tancos (Alfredo Nunes de Matos, Invicta); Navios de Guerra Escoltam as Tropas Destinadas a Angola e Moçambique (Gaumont) e Le Portugal envoie ses troupes en Angola envahie par les alemands (Gaumont). Como já foi assinalado, o ano em que a Alemanha declara guerra a Portugal, 1916, é aquele que, cinematograficamente, se destaca pela filmagem das manobras de Tancos e dos referidos exercícios navais. Sabe-se que pelo menos a primeira filmagem foi uma iniciativa de Norton de Matos, que quis que o cinema documentasse o acontecimento. Para quê? 10 Exercícios de Cavalaria em Torres Novas, Curso de 1911, estreado pela Empresa Portuguesa Cinematográfica em 18 de Setembro de 1911, foi exibido nas linhas franco-belgas em 1917. Consultar http://cvc.instituto-camoes.pt/cinema/cronologia/cro013.html. i Consultar http://cvc.instituto-camoes.pt/cinema/cronologia/cro013.html. 10 Exercícios de Cavalaria em Torres Novas, Curso de 1911, estreado pela Empresa Portugu p p g Cinematográfica em 18 de Setembro de 1911, foi exibido nas linhas franco-belgas em 19 11 Decreto n.º 4 214 de 13 de abril. Diário do Governo. i série (99) 8 de maio de 1918, p. 670. 12 Desconheço datas de nascimento e morte. Ter-se-á iniciado como realizador na sfce. Mais tarde, em 1929, ainda no âmbito da sua atividade no Exército, dirigiu a missão cinematográ fica a S. Tomé e Príncipe e Guiné, iniciativa do Agente Geral das Colónias Armando Cortesão. A sua filmografia conhecida inclui: Entrega da Bandeira da Cidade de Lisboa ao Cruzador “Vasco da Gama”, Escola de Aviação em Vila Nova da Rainha, Escolas de Oficiais Milicianos em Queluz, Provas Finais dos Alunos da Escola de Guerra, Transporte de Tropas para França, todos de 1917. Trechos de uma Exploração Agrícola em Borba (1927) e S. Tomé Agrícola e Industrial, Por Terras do Ébano e Guiné – aspectos Industriais e Agricultura (1929) – estes últimos três no âmbito da missão colonial promovida por Armando Cortesão – são os restantes títulos que lhe são atri buídos. O “MILAGRE” DA REPÚBLICA: PRELÚDIO DE UM CINEMA OFICIAL DE PROPAGANDA Também a Gaumont se interessou pela marinha portuguesa, produzindo – ou exibindo (era comum a migração de imagens através de intercâmbio entre produtoras ou cedência pela propaganda estatal. É possível que o Ministério da Marinha tenha cedido imagens de propaganda da divi são naval portuguesa em Marinha Portuguesa – Portugal após a Declaração de Guerra) – Partida de Marinheiros para Angola e África do Sul e Os Portugueses nas Forças Militares e Navais que partem para a África do Sul. Em suma, além das manobras do exército em Tancos cumpre destacar a fixação das manobras navais protagonizadas pela marinha portuguesa, em O CEP NO CINEMA DE PROPAGANDA DA PRIMEIRA GUERRA MUNDIAL 445 ambos os casos mandadas filmar pela República a Albuquerque. Quanto às iniciativas privadas, mantém-se os registos de partidas para África (Expedição Militar a Angola, Alfredo Nunes de Matos, Invicta Film; Partida de Marinhei ros para Angola e África do Sul e Os Portugueses nas Forças Militares e Navais que Partem para a África do Sul, produzidos pela Gaumont); e continuam os exercícios militares – Cavalaria Portuguesa em Treinos (Gaumont) – agora com a novidade de haver reportagens daqueles feitos fora de Portugal como A Escola de Instrução dos Oficiais Portugueses na Inglaterra (produção des conhecida). A Gaumont documenta Portugal após a Declaração de Guerra e, numa versão mais curta, de apenas 2 partes, Exército Português – Portugal após a Declaração de Guerra, além de Demonstração Patriótica dos Portugueses a favor dos Aliados, enquanto a Invicta filma o Corpo Expedicionário Português, assim referido pela primeira vez no título de um filme. SFCE E PRIVADOS FILMAM A “GUERRA POSSÍVEL” Com o início da atividade da sfce – cujo regulamento define ter como pro pósito “registar, para serem utilizados na projecção fixa e animada, todos os assuntos relativos à educação e preparação do exército, na paz e na guerra […]”11 –surgem vários filmes manivelados frequentemente pelo tenente Augusto Seara.12 A primeira produção conhecida da sfce é Festa no Instituto de Odivelas, obra curiosa, em que no colégio interno para filhas de militares se antecipam as comemorações da vitória aliada na Primeira Guerra Mundial. A “Feira da Vitória” aconteceu no 10 de junho, dia de Portugal, e, atestando a importância mobilizadora que lhe foi dada, contou com a presença do presi dente da República Bernardino Machado.i Entre os títulos filmados por Seara estão Provas Finais dos Alunos da Escola de Guerra, Participação de Portugal na Guerra (com pouco mais de três minu tos) e Lançamento da Canhoeira “Bengo”. Segundo Tiago Baptista (2014), um MARIA DO CARMO PIÇARRA 446 conjunto de filmes de Seara para a sfce estreou num evento em Lisboa, em 22 de outubro de 1917: Transporte de Tropas para França, Escola de Aviação em Vila Nova da Rainha, Escola de Oficiais Milicianos em Queluz e Entrega da Bandeira da Cidade de Lisboa ao Cruzador “Vasco da Gama”. Refira-se que, produzido pela Pathé, mas também com referência à sfce, conhece-se ainda Tropas Portuguesas no Front, estreado a 24 de julho de 1917 e promovido como tendo sido o primeiro a mostrar imagens das tropas portu guesas em França (Baptista, 2014). A Invicta documenta então Expedicionários em Campanhã e, entre outros títulos, destaquem-se os filmes, de produção francesa e inglesa, que documen tam a visita de Bernardino Machado ao Front. 13 Em linha em http://www.cinemateca.pt/CinematecaDigital/Ficha.aspx?obraid=2121&type =Video. SFCE E PRIVADOS FILMAM A “GUERRA POSSÍVEL” Em Participação de Portugal na Guerra – Regresso do Presidente da República da sua Viagem ao “Front” portu guez, a sfce limita-se a fixar o regresso do presidente da República, filmado sempre a grande distância, talvez para evidenciar a forte organização militar, a presença popular e a receção ordeira.13i Em 1918, a sfce estreou novos filmes de exercícios militares, Colégio Mili tar (também designado como Exercícios no Colégio Militar com a Assistência do Presidente da República) e um título que subsiste, Manobras do Campo Entrincheirado de Lisboa, uma produção da sfce e da Lusitânia Film, que só veio a estrear a 11 de janeiro de 1919 no Coliseu dos Recreios.ii O fim da guerra foi fixado pela secção em Como foi Recebida pela Popula ção de Lisboa a Notícia do Armistício, após o que é filmada Parada Militar de 5 de Dezembro de 1918 (também registada pela Gaumont), com que se pretende assinalar um ano da revolução sidonista, escassos dias antes do assassinato de Sidónio Pais. A sfce limitou-se a filmar em território nacional. Afirmou-se que terá sido a ditadura sidonista a impedir que Ernesto de Albuquerque se juntasse a Arnaldo Garcez para filmar, tendo este partido com o cep para documentar fotograficamente a participação portuguesa na guerra. Segundo a biografia de Albuquerque publicada pela Cine Revista a 15 de novembro de 1920, “Quando da nossa entrada na Grande Guerra, o general Norton de Mattos convidou-o a seguir para o front, e ei-lo prompto a seguir, para, embora arriscando a vida, fazer cinema! Mas o 5 de dezembro não deixou e Albuquerque não partiu” (1920, p. 2). Porém, esta justificação, avançada por uma revista de cinema, não me parece ter fundamento. Passa-se quase um ano desde a partida dos primei ros contingentes do cep – em Janeiro – até à ditadura presidida por Sidónio. Responsabilizá-lo pelo cancelamento da ida de Albuquerque não faz sentido, O CEP NO CINEMA DE PROPAGANDA DA PRIMEIRA GUERRA MUNDIAL 447 sobretudo depois do estadista ter mantido a sfce em atividade. Terão sido difi culdades técnicas e financeiras – os serviços de cinema do Exército eram muito recentes, por um lado, e, por outro, os custos da realização de filmes eram eleva dos – a obstar à documentação cinematográfica da presença do cep na frente? Ou terá sido a censura exercida pelos “Aliados” – que não permitiu a filmagem da guerra pelos serviços do exército português? 14 Decreto n.º 3354. SFCE E PRIVADOS FILMAM A “GUERRA POSSÍVEL” 448 MARIA DO CARMO PIÇARRA No caso português, a secção de fotografia dispunha de apenas um fotó grafo, Arnaldo Garcez, que documentou os exercícios de treino em Portugal e, posteriormente, o quotidiano do cep na frente de batalha. Dado que o cep estava sob o comando britânico, as fotografias tiradas por Garcez foram sujei tas à censura militar britânica, o que motivou reiteradas e mal sucedidas obje ções pelo Exército Português. Embora não sejam conhecidos documentos que o comprovem, é provável que a inexistência de filmes da sfce se deva também à sujeição à censura militar, britânica e também francesa.15 15 Após o Armistício, note-se que o filme mais relevante produzido pelo sfce e relativo ainda à Grande Guerra é Glorificação dos Soldados Desconhecidos Mortos na Grande Guerra, estreado a 21 de abril de 1921, que registou as cerimónias fúnebres dos dois soldados desconhecidos sepultados no Mosteiro da Batalha. SFCE E PRIVADOS FILMAM A “GUERRA POSSÍVEL” É preciso não esquecer que o cep esteve sob o comando britânico e o cap sob o comando francês. A Primeira Guerra Mundial reforçou o poder dos estados aliados quanto à imposição da censura, que andou de mão dada com a propaganda não só para manter as unidades nacionais, mas para um efetivo controlo da informação que poderia prejudicar a estratégia militar. Em Portugal, o estado excecional de guerra per mite ao Ministério da Guerra propor que Bernardino Machado decrete14, a 10 de setembro de 1917, a censura militar. Assim, fica assente que “Nenhuma fita cinematográfica, de qualquer natureza ou proveniência, que contenha assuntos militares, ou que direta ou indiretamente faça alusão aos exércitos beligerantes ou à grande guerra, poderá ser exibida nos territórios da República sem pre viamente ser sujeita à censura militar”. Os importadores ou proprietários de filmes passam, por isso, a ser obrigados a pedir o seu exame prévio ao Ministro da Guerra, prevendo-se sanções caso isso não seja observado. Outro dado a ter em atenção é que, estando o cep e o cap sob o comando dos exércitos inglês e francês, naturalmente que, em termos de registos das operações militares, estas divisões estariam sujeitas à censura severa estabe lecida nestes países. Com direito a designação – “Defense of the Realm Act (dora)”– no Reino Unido, entrou em vigor neste país logo em 8 de agosto de 1914, assumindo, entre outros propósitos, o de assegurar a segurança das forças e navios de Sua Majestade e prevenir a disseminação de falsos rumo res ou notícias passíveis de interferir com o êxito das operações militares. Já em França, a censura é instaurada logo a 30 de julho e prevê, como no caso inglês, a interdição de disseminação de informação que ponha em causa a rela ção com os países aliados ou neutros e de visar negativamente os oficiais ou reproduzir artigos saídos na imprensa estrangeira. Mesmo a disseminação de fotografias dos soldados franceses na frente de combate foi muito controlada e sabe-se que durante muitos meses não foram mostradas quaisquer imagens dos soldados ou publicadas notícias sobre como se vivia nas trincheiras (foram os soldados ou pessoal auxiliar de saúde que, progressivamente, divulgaram as difíceis condições em que fazia a guerra). Os civis eram mantidos na ignorân cia quanto ao que se passava e só no fim da guerra muitas das imagens conhe cidas foram sendo divulgadas. A PROJEÇÃO DOS SOLDADOS PORTUGUESES PELA PROPAGANDA ALIADA Que propaganda e censura andaram de mãos dadas no que respeita à informa ção veiculada sobre a Primeira Guerra Mundial é ponto assente. Tendo a noção que, genericamente, a imagem projetada das tropas portuguesas foi controlada (leia-se que foi proibido aos particulares, civis mas também militares, fotogra far e filmar a guerra) pelos Ministérios da Guerra Aliados – particularmente, o francês, o português, mas sobretudo o inglês – interessa então fixar qual a representação proposta. Esta, parece-me, terá correspondido a uma imagem e conteúdos definidos pelos Aliados, e sobretudo pelo Ministério da Guerra Britânico. O acervo disponibilizado online pelo Imperial War Museum (iwm) é o mais importante para fazer esta análise. Se o sfce não pôde acompanhar a visita de Bernardino Machado à frente de batalha em França, o certo é que o programa oficial foi registado pelo operador inglês autorizado para o efeito. The Visit of President Bernardino Machado of Portugal to the British and Portuguese Forces on the Western Front, October 1917 mostra o presidente português a sair do castelo de Montreuil, onde ficou alojado, perante uma guarda de honra britâ nica. Assinala-se que, às quatro da tarde de 11 de outubro, Machado e o Mare chal de Campo Haig inspecionam a Guarda de Honra da Rainha (Royal West Surrey Regiment) na estação de Lillers. O Brigadeiro-general John Charteris, responsável pelos serviços de informação de Haig, é um dos acompanhan tes da visita, que é registada pelo fotógrafo oficial do exército britânico, John Warwick Brooke. Esta atualidade mostra ainda a condecoração, no dia 13, de 4 oficiais portugueses, os primeiros a receberem a Cruz de Guerra (aos 3’ 45’’). 15 Após o Armistício, note-se que o filme mais relevante produzido pelo sfce e relativo ainda à Grande Guerra é Glorificação dos Soldados Desconhecidos Mortos na Grande Guerra, estreado a 21 de abril de 1921, que registou as cerimónias fúnebres dos dois soldados desconhecidos sepultados no Mosteiro da Batalha. O CEP NO CINEMA DE PROPAGANDA DA PRIMEIRA GUERRA MUNDIAL 449 Annales de la guerre, a principal série francesa de atualidades de produ ção estatal, preservada e disponibilizada pelo Établissement de Communica tion et Production Audiovisuelle de la Defense, também filmou, segundo um filme disponível no site do iwm, a visita de Machado a França, mas a sua edi ção n.º 31 centrou-se apenas na homenagem deste à determinação mostrada pelos franceses em Verdun. A PROJEÇÃO DOS SOLDADOS PORTUGUESES PELA PROPAGANDA ALIADA Neste registo, Machado inspeciona tropas antes de impor a Ordem Militar da Torre e da Espada ao presidente da câmara de Verdun. Segue-se uma viagem de carro pela cidade e arredores (Aisne, quinta Quennevières e ruínas de Chauny, do castelo de Ham e de Nesle). Assiste-se depois a um desfile de tropas. O filme mostra ainda aspetos da vida dos solda dos franceses nas trincheiras pós-batalha de Verdun.i O iwm tem em linha talvez o mais interessante filme sobre o cep em França: With the Portuguese Expeditionary Force in France. Produzido pela Topical Film Company para o War Office Cinema Committee, mostra o General Sir Henry Horne, comandante do Primeiro Exército, a ser saudado pelo General Tamagnini, comandante do cep, antes de inspecionar a guarda de honra, que depois desfila. De seguida mostra-se a deslocação de um bata lhão português, e o que transporta, e o treino de uma bateria de tiro, equipada com peças de artilharia francesas de 75 mm. Apresentam-se imagens, filma das em Junho de 1917, de um campo de treino em Marthes, onde os soldados aprendem a escavar trincheiras e como posicionar-se nelas com as armas. É nesse alinhamento que um soldado do 15.º Batalhão Português pousa para a câmara, mostrando o uniforme e o equipamento. Prossegue o registo de treinos com material militar, surgindo então um grupo de oficiais a pou sar para a câmara. Imagens de treinos com morteiros antecedem outras de oficiais ingleses a pousar com congéneres portugueses. Surgem imagens de formação ministrada, em que os instrutores ensinam a ler mapas e a dispa rar armas usando a mira telescópica. Uma segunda bobina mostra portu gueses na frente de batalha – o descritivo diz que é provável que se trate da 2.ª Divisão, a 21 de maio. Explica-se ainda que alguns dos soldados são do 22.º Batalhão. Apontamento ao gosto britânico (e da sua propaganda, tam bém): mostra-se o comandante do batalhão a tomar chá com os oficiais bri tânicos. Exercícios de fogo. É depois filmado o General Gomes da Costa, comandante da 2.ª Divisão, a falar com um general britânico. Mais exercí cios de fogo. Em ação: um vagão português de transporte puxado por mulas. O filme sustenta que o mais antigo aliado inglês é um excelente aliado. Segue- -se plano aproximado de soldado. Este pousa durante mais de 30’’. A PROJEÇÃO DOS SOLDADOS PORTUGUESES PELA PROPAGANDA ALIADA Ri-se e exibe-se, claramente a pedido.i Este filme, exemplo do que a propaganda britânica pretendia evidenciar, revela três preocupações: a de mostrar que os soldados portugueses estão MARIA DO CARMO PIÇARRA 450 equipados, que estão a ser treinados devidamente e que tudo é supervisio nado e mantido sob a ordem dos ingleses. A gentileza para com os portugueses – afinal velhos aliados porque bons aliados – quer mostrar à opinião pública equipados, que estão a ser treinados devidamente e que tudo é supervisio nado e mantido sob a ordem dos ingleses. A gentileza para com os portugueses i – afinal velhos aliados porque bons aliados – quer mostrar à opinião pública britânica, sobretudo, que esta aliança é benigna e contributiva para o bom sucesso do esforço de guerra.i Imagens raras são fixadas em A Portuguese Training Camp in England – Roffey Camp, Horsham. Com produção de H. C. Raymond e patrocínio do Ministério da Informação britânico, foi filmado a 18 de agosto de 1918 e mos tra portugueses em exercícios. Um descritivo do filme em linha informa que um excerto desta atualidade foi exibida em França. Começa com uma pose dos oficiais portugueses junto aos congéneres e instrutores britânicos. Fixa-se então um desfile da artilharia portuguesa. Passa-se à filmagem do treino dos soldados por um instrutor britânico. Finalmente, mostra-se a assistência, por civis e oficiais, aos exercícios. Entre eles está o ministro de Portugal no Reino Unido, Augusto de Vasconcellos. A imagem da assistência alterna depois com outras de uma corrida. Inclui-se um apontamento mais pitoresco, com a exi bição do Jogo do Pau16 (6’ 10’’ até aos 7’), antes de novo exercício. O filme termina com imagens do General Rosado, que, em julho de 1918, por decisão de Sidónio Pais (proclamado presidente da República a 9 de maio), substituíra o general Tamagnini no comando do cep.17 Em 1918, numa fase em que o prestígio do cep está bastante afetado devido à derrota em La Lys, a Topical Film Company volta a produzir um filme para o Ministério da Informação Britânico, sobre a artilharia pesada e a artilharia de campo portuguesa. O iwm escreve, no descritivo da obra, que Portuguese Troops in France 1918, inacessível em linha, terá sido filmado no verão desse ano. 16 O Jogo do Pau era um exercício da Cavalaria Portuguesa. Dragões de Moçambique (1934), filmado por Fernandes Tomás para a Agência Geral das Colónias, regista detalhadamente o Jogo do Pau, entre outros exercícios. 17 O decreto da sua nomeação é de 10 de julho e a 15, quando chegou a França, iniciou então negociações com os ingleses para melhorar as condições das forças que chegava para coman dar. Nessas negociações foi auxiliado por Vasconcellos. Não obstante a desconfiança britânica relativamente ao governo sidonista, conseguiu que o então Marechal Haig aprovasse um projeto de reorganização do cep, nomeadamente da 1.ª Divisão Portuguesa, nela agrupando os efetivos ainda existentes em França. É preciso lembrar que o prestígio do exército português estava então completamente afectado e Rosado queria organizar uma força de combate ativa, que afirmasse perante os aliados o prestígio português. 16 O Jogo do Pau era um exercício da Cavalaria Portuguesa. Dragões de Moçambique (1934), filmado por Fernandes Tomás para a Agência Geral das Colónias, regista detalhadamente o Jogo do Pau, entre outros exercícios. A PROJEÇÃO DOS SOLDADOS PORTUGUESES PELA PROPAGANDA ALIADA Vive-se então, como já referido, uma fase de mudança de comando das tropas portuguesas, marcada pelas negociações encetadas pelo General Rosado com o Reino Unido no sentido de reorganizar as tropas por tuguesas e voltar a ter uma força de combate ativa. 16 O Jogo do Pau era um exercício da Cavalaria Portuguesa. Dragões de Moçambique (1934), filmado por Fernandes Tomás para a Agência Geral das Colónias, regista detalhadamente o Jogo do Pau, entre outros exercícios. 17 O decreto da sua nomeação é de 10 de julho e a 15, quando chegou a França, iniciou então negociações com os ingleses para melhorar as condições das forças que chegava para coman dar. Nessas negociações foi auxiliado por Vasconcellos. Não obstante a desconfiança britânica relativamente ao governo sidonista, conseguiu que o então Marechal Haig aprovasse um projeto de reorganização do cep, nomeadamente da 1.ª Divisão Portuguesa, nela agrupando os efetivos ainda existentes em França. É preciso lembrar que o prestígio do exército português estava então completamente afectado e Rosado queria organizar uma força de combate ativa, que afirmasse perante os aliados o prestígio português. O CEP NO CINEMA DE PROPAGANDA DA PRIMEIRA GUERRA MUNDIAL 451 Outro arquivo, privado, fundamental numa filmografia da participação portuguesa na guerra é o da Gaumont Pathé. Embora muitos dos filmes não estejam acessíveis em linha, confirma-se, pelos descritivos disponíveis, que a Gaumont fixou desde cedo as partidas de tropas para Angola. Le Portugal intervient en Angola envahie par les troupes allemandes (1915, 55’’), mostraria um embarque, “repetido” em Le Portugal envoie des troupes pour sa colonie en Angola (192018, 5’), visionável, em que a câmara fixa soldados com fardamento “colonial” num cais de embarque, passando de seguida a fixar o seu embarque e, finalmente e demoradamente, o afastamento do navio “Portugal” no Tejo. Do navio, os soldados acenam lenços brancos. Antes do filme terminar mostram- -se imagens do desfile de soldados – os que embarcaram? Mais soldados? – num entroncamento de ruas, sob o olhar dos civis, pouco efusivos, e entre os quais há poucas mulheres. Além do já referido Contingent portugais (1917, 1’ 41’’), composto com imagens filmadas pelo Capitão Ferrão e por Ernesto de Albuquerque, o Journal Actualité mostrou, com o mesmo título, no mesmo ano e com menos de um minuto (1917, 52’’), o desembarque de tropas portuguesas em França. 18 Coloca-se a data que consta no site, relativa à primeira exibição. Este filme, ao contrário de outros existentes no arquivo, não tem a ficha de produção original e é possível que tenha sido fornecido por alguma produtora portuguesa. 19 Conforme ficha existente. O uso do castelhano terá que ver com a origem do material (é mais provável que tivesse sido a Invicta Film a fornecê-lo, na realidade)? Terá sido eventual mente distribuído em Espanha? Ou será mera confusão? A PROJEÇÃO DOS SOLDADOS PORTUGUESES PELA PROPAGANDA ALIADA Segundo a ficha do filme, trata-se de um novo contingente de tropas – provavelmente o de 23 de fevereiro. Após o desembarque dos soldados, fixa-se o desembarque de materiais de guerra e, finalmente, o desfile de tropas no cais. ii Castillo de Chavez19 (Portugal). Formation du 19eme regiment d’infanterie (1915, 35’’), não acessível, mostra a formação do regimento de infantaria 19. Com intertítulos em inglês, a atualidade Le president Machado en France (1917, 1’ 07’’) repete imagens de Annales de la guerre n.º 31. Outro arquivo privado com imagens únicas da participação portuguesa na guerra é o da British Pathé, com acesso livre do acervo em linha. Idênticos, Great Review of Portuguese Troops in England 1917 (54’’) e Portuguese Troops in England (50’’) mostram desfile das tropas portuguesas, planos de soldados com máscaras de gás, além da revista das tropas. Estas voltam a desfilar enquanto a câmara fixa um homem de barba e cabelo branco – quase seguramente Bernardino Machado, que assistiu à parada de Montalvo – que saúda as tropas tirando-lhes o chapéu. Esta atualidade pode ser um excerto sobrevivente de Parada da Divisão em Montalvo (1916), não só devido MARIA DO CARMO PIÇARRA 452 à presença de Bernardino Machado, mas por via do confronto das imagens em movimento com fotografias disponíveis no Arquivo Histórico Militar do Exército (a arborização do local parece idêntica numas e noutras). Portugal’s Army 1914-18 retoma o tema dos embarques/desembar ques. Inicia-se com imagens de soldados portugueses a desfilar num porto, seguindo-se sequências que fixam o desembarque de militares e de um canhão. Portuguese Horse Artillery 1914-1918 (s. d., 41’) é a versão mais curta de outra atualidade intitulada Portuguese Expedition – General Battery Firing 1914-1918 (s. d., 2’ 13’’). Abre com uma sequência mais geral, da artilharia e obuses puxados por mulas subindo uma encosta, seguindo-se sequên cias mostrando exercícios de fogo com peças de artilharia. Finalmente, a 48 segundos do fim, fixa-se novo movimento da artilharia a cavalo – agora em colina mais rochosa. Num campo, à vista de uma casa apalaçada, fixam-se mais exercícios de fogo com obuses. Não é prestada informação sobre o local das filmagens. É uma hipótese, a de que se tratem, também neste caso, de imagens dos filmes que fixaram o “milagre de Tancos”. A PROJEÇÃO DOS SOLDADOS PORTUGUESES PELA PROPAGANDA ALIADA Por via da compara ção com fotos existentes, e devido às características do terreno e arborização do mesmo, a hipótese é extensível a Portuguese Troops Training 1914-1918 (s. d., 56’). Na primeira sequência mostra soldados a puxar, de uma cons trução coberta, uma peça de artilharia a qual é depois preparada para dis parar. Segue-se uma panorâmica da assistência, predominantemente militar, em que pontuam alguns civis, entre os quais Bernardino Machado, e encerra com desfile de soldados. i Nota-se o esforço para, através dos filmes, mostrar a organização e moder nidade do exército português. Há até um apontamento britânico que sustenta que o soldado português e o tradicional aliado do Reino Unido são de con fiança. Pelo meio há uma outra sequência, mais pitoresca, que parece sublinhar a subalternização do exército português no seio dos Aliados, genericamente, e da velha aliança luso-britânica. Numa sessão de treinos, a que assistem o ministro de Portugal no Reino Unido, Augusto de Vasconcellos, e o general Rosado – apontado, durante o Sidonismo, para substituir o comandante da Primeira República, general Tamagnini –, exibe-se o Jogo do Pau. Tal sucede quando as tropas portuguesas, num derradeiro esforço organizativo, e após a derrota em La Lys, querem recuperar, sob o comando de Rosado, a dignidade militar de uma força de combate. O CEP NO CINEMA DE PROPAGANDA DA PRIMEIRA GUERRA MUNDIAL 453 20 Informação por e mail, a 27 10 2014. 21 Segundo Pimentel: “O Afundamento do Lugre Rio Cávado foi filmado em 2 de outubro de 1918: ‘navio afundado em 02-10-1918, pelo submarino u-139, classe Kreuzer, comandado por Lothar von Arnauld de la Perière’, aos comandos deste desde maio de 1918”. Pouco tempo depois, em 24 de novembro, o u-139 rendeu-se aos franceses. O Rio Cávado era um veleiro que transportava vinho do Porto para Bristol. “Chegaram às 11 horas da noite de ontem os tripu lantes do veleiro ‘Rio Cávado’. O navio naufragou às 6 horas da manhã do dia 2, a 290 milhas do Cabo Prior. Era a primeira viagem que fazia, tendo sido lançado ao mar em julho último e transportava vinho do Porto para Bristol.” O Século, de 7 de outubro de 1918. Existe uma cópia do filme na Cinemateca Portuguesa. Regista o afundamento do lugre e as tentativas para resgatar parte da carga de vinho do Porto. Há uma sequência relativa a um barco com cerca de duas dezenas de homens a bordo que poderá ser da tripulação do “Rio Cávado”, mas as imagens deste filme amador fixam sobretudo a alegria da tripulação do submarino alemão enquanto iça barricas de vinho do Porto para o convés. 20 Informação por e-mail, a 27-10-2014. OS “BRAVOS MARINHEIROS PORTUGUESES” NUM FILME AMADOR ALEMÃO Nas escassas imagens existentes analisadas, o único registo de guerra conhe cido é o do Afundamento do Augusto Castilho (1918, 5’ 20’’). Trata-se de um filme amador que não foi objeto de censura militar nem usado como instru mento de propaganda. Segundo informação de Joana Pimentel, da Cinema teca Portuguesa (2014)20, o registo chegou a Portugal nos anos 30, pela mão do armador Carlos Bensaúde, a quem terá sido oferecido pelo oficial alemão Kurt Von Pistor. Terá sido o próprio Von Pistor, capitão-tenente do submarino u139 que afundou o Augusto Castilho, a manivelar quer este filme, quer outro, também oferecido por si: O Afundamento do Lugre Rio Cávado21, depositado no anim. Pimentel teve acesso a esta informação através de “um antigo fun cionário do spn que mais tarde se tornou antiquário, o Sr. Alberto Cutileiro, com loja aberta na zona da Furnas em Benfica”.22 A lata do filme, depositado em 2000 pelo Ministério da Marinha, “que continha o elemento mais antigo que se conhece destas imagens, o internegativo em suporte de nitrato Zeiss Ikon Agfa”, descreve o conteúdo como: “Combate entre o submarino alemão u139 e o caça-minas Augusto de Castilho em 14 de outubro de 1918. Imagens colhidas pelo imediato do submarino Von Pistor. Informação proveniente do antigo rótulo do internegativo nitrato (31644)”. Em 20 de junho de 1936, na revista Animatógrafo, Fernando Fragoso refere a projeção deste filme, no Gimnásio, em Lisboa, num espetáculo em honra da Falange Espanhola, especificando tratar-se de uma obra pertença 22 Autor de O Combate do Caça-minas Augusto de Castilho com o Submarino Alemão u139 visto Através do Relatório do 1.º Oficial Encarregado da Artilharia, Capitão-Tenente Kurt von Pistor (1974) 22 Autor de O Combate do Caça-minas Augusto de Castilho com o Submarino Alemão u139 visto Através do Relatório do 1.º Oficial Encarregado da Artilharia, Capitão-Tenente Kurt von Pistor (1974). MARIA DO CARMO PIÇARRA 454 de um particular. Não se sabe como o filme ou o internegativo do mesmo foi para a posse da Marinha que, segundo Pimentel, “talvez nos anos 50”, pediu apoio à Cinemateca para tirar novas cópias do mesmo, o que terá sucedido efetivamente.23 Primeiro comentário: no filme fixa-se um episódio que atesta a obser vância do código de honra militar. OS “BRAVOS MARINHEIROS PORTUGUESES” NUM FILME AMADOR ALEMÃO O segundo comentário é que a versão alemã deste episódio da participação portuguesa na guerra documenta a his tória da coragem dos marinheiros que lutaram até à morte para cumprir a sua missão.i O filme mostra o Augusto de Castilho24 já após a rendição e a morte do capitão, Carvalho Araújo. O primeiro intertítulo explica: “O barco-patrulha português Augusto de Castilho derrotado após um longo bombardeamento pela artilharia”. Apresentam-se imagens da tripulação, sendo revistada por um oficial alemão. Este procura armas e documentos secretos. Surgem imagens de um salva-vidas, com um oficial alemão a bordo conversando com a tripulação portuguesa. O segundo intertítulo explica: “A tripulação é mandada embora”. Nova imagem do oficial alemão no salva-vidas com a tripulação portuguesa. O terceiro intertítulo assenta: “Os feridos são tratados pelo nosso médico de bordo”. Vê-se um médico militar a ligar a perna de um ferido. Noutro plano, um marinheiro português segura a perna do ferido enquanto o médico pros segue o tratamento. “O comandante vai a bordo do Augusto de Castilho. Está içada a bandeira branca”, lê-se no intertítulo seguinte. Sucedem-se planos do Augusto de Castilho, enquanto homens sobem a bordo, vindos de um bote. O quinto intertítulo descreve: “o nosso submarino ao lado do Augusto de Castilho. Atividade de artilharia das nossas armas de 15 cm”. As imagens ilustram as consequências do ataque ao navio português, evidenciando as O filme mostra o Augusto de Castilho24 já após a rendição e a morte do capitão, Carvalho Araújo. O primeiro intertítulo explica: “O barco-patrulha português Augusto de Castilho derrotado após um longo bombardeamento pela artilharia”. Apresentam-se imagens da tripulação, sendo revistada por um oficial alemão. Este procura armas e documentos secretos. Surgem imagens de um salva-vidas, com um oficial alemão a bordo conversando com a tripulação portuguesa. O segundo intertítulo explica: “A tripulação é mandada embora”. Nova imagem do oficial alemão no salva-vidas com a tripulação portuguesa. 23 No fundo da cp existem duas cópias em 35 mm e existiam duas em 16 mm, uma no Museu da Marinha (até 2000) e outra na Biblioteca Central da Marinha, tendo o primeiro organismo depositado no anim o respetivo internegativo em 2000. 24 Originalmente chamado Elite, este arrastão de pesca (a partir de 1909 foi usado na pesca do bacalhau) foi transformado em barco patrulha de alto mar. 23 No fundo da cp existem duas cópias em 35 mm e existiam duas em 16 mm, uma no Museu da Marinha (até 2000) e outra na Biblioteca Central da Marinha, tendo o primeiro organismo depositado no anim o respetivo internegativo em 2000. OS “BRAVOS MARINHEIROS PORTUGUESES” NUM FILME AMADOR ALEMÃO Em 8 de outubro de 1918 zarpou de Lisboa sob o comando do Capitão Carvalho de Araújo escoltando o vapor Beira até à Madeira, onde chegaram a 11. Recebeu então nova missão: a de escoltar o vapor S. Miguel, com destino a Ponta Delgada, nos Açores, o qual transportava mais de 200 passageiros. A 14 de outubro, o S. Miguel foi atacado pelo U-Boot alemão u-139, comandado por Lothar von Arnauld de la Perière. O Augusto de Castilho acabou por investir diretamente contra o submarino alemão, permitindo desde modo que o vapor de passageiros se afastasse. O Augusto de Castilho ren deu-se passadas duas horas de combate, quando perdera as comunicações, gastara quase todas as munições e, entre os feridos, se contava o capitão, que acabou por morrer. Os sobreviventes conseguiram chegar aos Açores no salva-vidas e bote do navio. Consultar http://www.momen tosdehistoria.com/MH_02_06_Marinha.htm. O CEP NO CINEMA DE PROPAGANDA DA PRIMEIRA GUERRA MUNDIAL 455 perfurações feitas pelo armamento alemão. Sexto intertítulo: “A arma na proa do caça-minas”, com breve imagem ilustrativa. De seguida, mostra-se “Provi sões e munições do Augusto de Castilho” sendo retirados do navio pelos mari nheiros alemães. Finalmente, o intertítulo nove e seguintes explicam como se processam os disparos contra o Augusto de Castilho sendo as imagens ilustra tivas.i Assinale-se que este filme amador não adota um registo de propaganda. O comandante do U-139, Von Arnauld de la Perière (1886-1941), era um herói da Marinha alemã, que veio a morrer num acidente de viação durante a ii Guerra Mundial. Responsável pelo afundamento de 194 navios, dois dos quais de guerra, dele dizia-se ser também um cavalheiro pois nunca afundava navios sem conhecer o manifesto de carga e colocar as tripulações a salvo ou dar-lhes meios para tal. O filme existente na Cinemateca Portuguesa confirma a fama de la Perière, mas atesta sobretudo a pobreza dos meios defensivos à disposição da Marinha Portuguesa e a coragem exigida aos marinheiros e sol dados portugueses para combater numa guerra que também foi, para a Repú blica, a da tentativa de afirmação de um poder que não tinha e de demonstração de recursos inexistentes.ii Que fique ainda assinalada a única longa-metragem ficcional inspirada na participação portuguesa na guerra. Foi preciso que outra guerra mundial eclodisse para que, agora num Portugal neutral e sob uma ditadura, estreasse a longa-metragem de ficção João Ratão (1940). OS “BRAVOS MARINHEIROS PORTUGUESES” NUM FILME AMADOR ALEMÃO Uma produção da Tobis Por tuguesa, teve realização de um dos realizadores mais reconhecidos de então e nacionalista assumido, Jorge Brum do Canto (1910-1994), que, posterior mente, veio a realizar Chaimite, um filme dedicado ao exército e sobre as cam panhas de África do final do século xix, em que foram “heróis” Mouzinho de Albuquerque e Paiva Couceiro. Quanto a João Ratão, inspirado numa opereta, foca sobretudo as intrigas em que se vê envolvido um soldado recém-chegado da Flandres, usando a guerra apenas como pano de fundo. De resto, embarques e desembarques de tropas portuguesas, exercícios militares – em Portugal, França ou Reino Unido. As visitas de Bernardino Machado à frente ocidental de batalha, junto a Verdun, e ao Reino Unido. A pouco mais do que isto se resume a fixação dos “bravos soldados portu gueses”, se usarmos o cinema como fonte exclusiva no que se refere à partici pação na Primeira Guerra Mundial. A exceção que confirma a regra é, como referido, a do filme amador alemão manivelado pelo Capitão Von Pistor, que documenta duas coisas: como a brutalidade deste primeiro conflito mun dial não obstou a que ainda fosse sendo observado, em certas situações, um código de honra militar – como a dupla Michael Powell (1905-1990) e Emeric Pressburger (1902-1988), fixou no extraordinário A Vida e a Morte MARIA DO CARMO PIÇARRA 456 do Coronel Blimp (1943) – que desapareceu por completo na Segunda Guerra Mundial; e a bravura exigida aos soldados portugueses para suprir a falta de meios de combate. Que fique escrito que outros arquivos, por explorar, mostrarão outras imagens dos soldados portugueses – porventura mesmo daqueles que foram feitos prisioneiros em La Lys. Dificuldades linguísticas, na pesquisa, e a moro sidade com que se têm digitalizado e disponibilizado os arquivos, o facto de haver documentos classificados, e que só em 2018 passarão a ser do domínio público, são alguns dos motivos para que esta seja uma filmografia provisória da participação portuguesa na Primeira Guerra Mundial. Recebido a 11-05-2017. Aceite para publicação a 20-04-2018. REFERÊNCIAS BIBLIOGRÁFICAS fontes Álbum de Ernesto de Albuquerque (http://www.cinemateca.pt/Cinemateca-Digital/Ficha.aspx ?obraid=31979&type=Imagem). Cine-revista, 7, 15-09-1917. Cine-revista, 8, 15-10-1917. Cine-revista, 30, 15-09-1919. Cine-revista, 49, 15-04-1921. Diário do Governo, i série, 99, 09-05-1918, p. 670. Diário do Governo, i série, 99, 09-05-1918, p. 670. Diário do Governo, i série, 130, 04-07-1919. Diário do Governo, i série, 130, 04-07-1919. “Films Nacionaes”. Cine-revista, 9, 15-11-1917. O Primeiro de Janeiro, 11-08-1916, p. 2. O Primeiro de Janeiro, 11-08-1916, p. 2. ✳ baptista, T. (2011), “Cinema e política na Primeira República”. In Actas do Colóquio A Vida Cultural em Lisboa no tempo da i República, Lisboa, Câmara Municipal de Lisboa, pp. 165- -186. baptista, T. (2011), “Cinema e política na Primeira República”. In Actas do Colóquio A Vida Cultural em Lisboa no tempo da i República, Lisboa, Câmara Municipal de Lisboa, pp. 165- -186. baptista, T. (2014), “Film/Cinema (Portugal)”. In U. Daniel, et al. (eds.) 1914-1918-online. International Encyclopedia of the First World War. Berlim, Freie Universität Berlin. Dispo nível em http://encyclopedia.1914-1918-online.net/article/filmcinema_portugal [consul tado em 10-05-2017]. baptista, T. (2014), “Film/Cinema (Portugal)”. In U. Daniel, et al. (eds.) 1914-1918-online. International Encyclopedia of the First World War. Berlim, Freie Universität Berlin. Dispo nível em http://encyclopedia.1914-1918-online.net/article/filmcinema_portugal [consul tado em 10-05-2017]. cutileiro A. (1976), O Combate do Caça-minas “Augusto de Castilho” com o Submarino Alemão “U. 139” visto através do Relatório do 1.º Oficial Encarregado da Artilharia, Capitão-tenente K t V Pi t Li b C t d E t d d M i h cutileiro A. (1976), O Combate do Caça-minas “Augusto de Castilho” com o Submarino Alemão “U. 139” visto através do Relatório do 1.º Oficial Encarregado da Artilharia, Capitão-tenente gomes de sousa, F. (1920), “Ernesto d’Albuquerque”. Cine-revista, 44, p. 2. gomes de sousa, F. (1920), “Ernesto d’Albuquerque”. Cine-revista, 44, p. 2. grilo, J. M. (2006), O Cinema da Não-ilusão: Histórias para o Cinema Português, Lisboa, Livros Horizonte grilo, J. M. (2006), O Cinema da Não-ilusão: Histórias para o Cinema Português, Lisboa, Livros Horizonte. o o te. janeiro, H. P. (2013), “The people in arms in the people’s entertainment: cinema and political janeiro, H. P. (2013), “The people in arms in the people’s entertainment: cinema and political piçarra, M. do C. (2018), “ ‘Irmãos de armas’: o cep no cinema de propaganda da Primeira Guerra Mun dial”. Análise Social, 227, liii (2.º), pp. 438-457. Maria do Carmo Piçarra » carmoramos@gmail.com » Centro de Estudos Comunicação e Sociedade, Uni versidade do Minho e Centre for Film Aesthetics and Cultures, University of Reading » Campus de Gualtar — 4710-057 Braga, Portugal. Recebido a 11-05-2017. Aceite para publicação a 20-04-2018. piçarra, M. do C. (2018), “ ‘Irmãos de armas’: o cep no cinema de propaganda da Primeira Guerra Mun dial”. Análise Social, 227, liii (2.º), pp. 438-457. Maria do Carmo Piçarra » carmoramos@gmail.com » Centro de Estudos Comunicação e Sociedade, Uni versidade do Minho e Centre for Film Aesthetics and Cultures, University of Reading » Campus de Gualtar — 4710-057 Braga, Portugal. Recebido a 11-05-2017. Aceite para publicação a 20-04-2018. piçarra, M. do C. (2018), “ ‘Irmãos de armas’: o cep no cinema de propaganda da Primeira Guerra Mun dial”. Análise Social, 227, liii (2.º), pp. 438-457. O CEP NO CINEMA DE PROPAGANDA DA PRIMEIRA GUERRA MUNDIAL 457 propaganda in Portugal (1916-1917)”. E-Journal of Portuguese History 11 (2), pp. 50-73. propaganda in Portugal (1916-1917)”. E-Journal of Portuguese History 11 (2), pp. 50-73. propaganda in Portugal (1916-1917)”. E-Journal of Portuguese History 11 (2), pp. 50-73. matos-cruz, J., ferreira, A. J., pina, L. (1989), Prontuário do Cinema Português, 1896-19 Lisboa, Cinemateca Portuguesa. matos-cruz, J., ferreira, A. J., pina, L. (1989), Prontuário do Cinema Português, 1896-1989, Lisboa, Cinemateca Portuguesa. piçarra, M. C. (2006), Salazar vai ao Cinema. O “Jornal Português” de Actualidades Filmadas, Coimbra, Minerva. piçarra, M. C. (2006), Salazar vai ao Cinema. O “Jornal Português” de Actualidades Filmadas, Coimbra, Minerva. piçarra, M. C. (2015), Azuis Ultramarinos. Propaganda Colonial e Censura no Cinema do Estado Novo, Lisboa, Edições 70. ribeiro, M. F. (1983), Filmes, Figuras e Factos da História do Cinema Português, 1896-1949, Lis boa, Cinemateca Portuguesa. Recebido a 11-05-2017. Aceite para publicação a 20-04-2018. piçarra, M. do C. (2018), “ ‘Irmãos de armas’: o cep no cinema de propaganda da Primeira Guerra Mun dial”. Análise Social, 227, liii (2.º), pp. 438-457. Maria do Carmo Piçarra » carmoramos@gmail.com » Centro de Estudos Comunicação e Sociedade, Uni versidade do Minho e Centre for Film Aesthetics and Cultures, University of Reading » Campus de Gualtar — 4710-057 Braga, Portugal. — 4710-057 Braga, Portugal.
https://openalex.org/W2036509718	https://europepmc.org/articles/pmc3742672?pdf=render	English	null	A Comparison of Methods for Clustering 16S rRNA Sequences into OTUs	PloS one	2,013	cc-by	8,240	Abstract Recent studies of 16S rRNA sequences through next-generation sequencing have revolutionized our understanding of the microbial community composition and structure. One common approach in using these data to explore the genetic diversity in a microbial community is to cluster the 16S rRNA sequences into Operational Taxonomic Units (OTUs) based on sequence similarities. The inferred OTUs can then be used to estimate species, diversity, composition, and richness. Although a number of methods have been developed and commonly used to cluster the sequences into OTUs, relatively little guidance is available on their relative performance and the choice of key parameters for each method. In this study, we conducted a comprehensive evaluation of ten existing OTU inference methods. We found that the appropriate dissimilarity value for defining distinct OTUs is not only related with a specific method but also related with the sample complexity. For data sets with low complexity, all the algorithms need a higher dissimilarity threshold to define OTUs. Some methods, such as, CROP and SLP, are more robust to the specific choice of the threshold than other methods, especially for shorter reads. For high-complexity data sets, hierarchical cluster methods need a more strict dissimilarity threshold to define OTUs because the commonly used dissimilarity threshold of 3% often leads to an under-estimation of the number of OTUs. In general, hierarchical clustering methods perform better at lower dissimilarity thresholds. Our results show that sequence abundance plays an important role in OTU inference. We conclude that care is needed to choose both a threshold for dissimilarity and abundance for OTU inference. Citation: Chen W, Zhang CK, Cheng Y, Zhang S, Zhao H (2013) A Comparison of Methods for Clustering 16S rRNA Sequences into OTUs. PLoS ONE 8(8): e70837. doi:10.1371/journal.pone.0070837 Editor: Maurizio Casiraghi, University of Milan-Bicocca, Italy Received April 13, 2013; Accepted June 24, 2013; Published August 13, 2013 Copyright: 2013 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Chen et al. This is an open-access article distributed under the terms of the Creative Commons unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported in part by NIH grants R01 GM59507 and UL1 RR024139, National Natural Science Foundation of China (No. 61170134), Aeronautical Science Foundation of China (No.20100853010) and Doctorate Foundation of Northwestern Polytechnical University (No. cx201017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hongyu.zhao@yale.edu A Comparison of Methods for Clustering 16S rRNA Sequences into OTUs Wei Chen1,2, Clarence K. Zhang3, Yongmei Cheng1, Shaowu Zhang1, Hongyu Zhao2,3* 1 College of Automation, Northwestern Polytechnical University, Xi’an, Shaanxi, China, 2 Department of Biostatistics, School of Public Health, Yale University, New Haven, Connecticut, United States of America, 3 Keck Biotechnology Laboratory, Biostatistics Resource, School of Medicine, Yale University, New Haven, Connecticut, United States of America August 2013 \| Volume 8 \| Issue 8 \| e70837 Introduction earthmicrobiome.org). Thousands of 16S rRNA sequence datasets have been generated through these community efforts as well as individual projects. Therefore, there is a critical need to develop and evaluate efficient and accurate computational algorithms to analyze these massive data collected from various biological and ecological environments. Microbes are estimated to have approximately 561030 cells on earth and more diverse than any other organisms [1]. They play a vital role in almost all biological processes in ecosystems from natural environments to human body [2–8]. Traditional culture- dependent microbial studies have limited our understanding of microbial communities, because only less than 1% of microbial organisms can be cultivated, identified, and characterized [9]. In recent years, with the development of next-generation sequencing technology, it is now possible to bypass the cultivated-based technology to sequence millions of sequences directly from relevant environments, e.g. human gut, soil, and salt lake. 16S rRNA sequences, the small unit of ribosomal RNA in prokaryotes, are the most widely used sequences for inferring the phylogenetic relations among microbial species [10–13]. The 16S rRNA based phylogenetic inference has revolutionized our view of microbial diversity and composition of many environments [14–17]. Many large-scale metagenomics projects have been undertaken to investigate various aspects of the microbial composition, e.g. Human Microbiome Project (http://commonfund.nih.gov/hmp), International Census of Marine Microbes (http://icomm.mbl. edu), and Earth Microbiome Project (http://www. Two approaches are commonly used to characterize microbial communities in the analysis of 16S rRNA sequences: taxonomy- dependent methods and OTU-based methods [18–22]. The taxonomy-dependent methods rely on the annotated sequences already deposited in the databases for taxonomic assignment of a query sequence by the best-matching sequence in the reference database. In the OTU-based methods, all the sequences are clustered into OTUs based on a distance matrix at a specified threshold. Although taxonomy-dependent methods can assign taxonomy to the query sequences based on previously character- ized microbes, lack of sufficient well-characterized microbes and reliable taxonomy often make it difficult to characterize novel sequences, and the robustness and accuracy of such methods are mainly dependent on the completeness of the annotated reference database [16,20,22,23]. Comparison of OTU-Based Clustering Methods Comparison of OTU-Based Clustering Methods the species level. In contrast, OTU-based methods are able to assign all sequences into OTUs without prior information of the reference taxonomy. Hence all sequences can be processed, including both microbes that have not been annotated in the databases as well as novel uncultured ones. The OTU-based methods are especially useful in analyzing less characterized microbial communities. Yet some issues that are unique to the OTU-based methods need to be addressed for their successful applications, such as the presence of sequencing errors which would result in an inflation of OTUs, the heterogeneous evolution rates in 16S rRNA which make it difficult to choose a consistent threshold to define OTUs, and biologically meaningful interpre- tations/annotations of the inferred OTUs. reduced biological accuracy, that is, there is a trade-off between complexity and accuracy [22,28] between the hierarchical clustering and heuristic clustering methods. In defining OTUs, some studies showed that it is difficult to use a consistent threshold because there is considerable overlap in the maximum intra-taxon distance between taxonomic levels [20]. Lastly, to avoid using a hard threshold value in clustering as implemented in hierarchical and heuristic methods, Hao et al [29] proposed a Gaussian mixture model-based clustering algorithm termed Clustering 16S rRNA for OTU Prediction (CROP). It adopts an unsupervised probabilistic Bayesian clustering algorithm and uses a soft threshold for defining OTUs. The CROP algorithm bypasses setting an often subjective hard cut-off threshold thus may effectively reduce the effects of PCR and sequencing errors in inferring OTUs. In general, the OTU-based methods can be categorized into hierarchical clustering, heuristic clustering and model-based clustering methods. In the hierarchical clustering category, a distance matrix measuring the difference between each pair of sequences is calculated first, and standard hierarchical clustering is then used to define OTUs at a specific level of sequence dissimilarity. Most of these methods have an O(N2) computational complexity, where N is the number of sequences, posing a significant computational bottleneck for processing large-scale sequencing datasets. The representative hierarchical clustering algorithm is Mothur [19], which is an improved version of Dotur [15]. Considering that the average-linkage method is relative conservative, Huse et al [24] proposed an improved single-linkage preclustering method (SLP) which can mitigate the effect of ‘‘noise’’ due to sequencing errors and the impact of abundant sequences so as to reduce the number of inflated OTUs. Comparison of OTU-Based Clustering Methods In order to reduce the complexity and memory requirement, Sun et al. [21] developed a new algorithm named ESPRIT, which adopts a k-mer distance to filter out large amount of unnecessary sequence pairs and store the reduced-distance with a sparse matrix. In addition to the filtering process, they introduced an hcluster algorithm to perform complete-linkage clustering, which reduces the computa- tional burden so that it can process millions of sequences at one time. However, this method still has a quadratic complexity. Recently, these authors proposed an improved version ESPRIT- Tree [22], a learning-based algorithm, which may achieve similar accuracy as ESPRIT but a quasi-linear computational complexity. Overall, the hierarchical clustering approaches may not be suitable for dealing with large-scale sequencing data because of their intrinsic computational complexity. As a result, greedy heuristic algorithms have been proposed to assign sequences into OTUs, which can substantially reduce the time and space complexity compared to a quasi-linear algorithm. The most commonly used heuristic clustering methods are CD-HIT [25] and Uclust [26]. They share many features while differ on how the sequences are sorted and mapped to existing cluster representative sequences. For a pre-defined threshold, these two algorithms first select an input sequence as a seed for the initial cluster, and then examine each input sequence sequentially. If the distance between the query sequence and representative sequences of the existing clusters is within the pre-defined threshold, the input sequence will be added to the corresponding cluster, otherwise a new cluster is created and the query sequence is stored as a new seed. Based on a grammar distance metric, Russell et al. [27] proposed a sequence clustering algorithm GramCluster, which has a memory complex- ity of O(LN), where N is the number of sequences and L is the average sequence length. Considering the computational efficiency and scalability, Ghodsi et.al [28] presented a greedy clustering algorithm named DNAClust which incorporates a novel k-mer filtering algorithm to avoid most pairwise alignments. Heuristic clustering algorithms achieve a lower complexity at the cost of With the availability of numerous OTU-based algorithms, it is a challenge for practitioners to choose an appropriate method for clustering their collected sequences into OTUs. Sun et al [21] studied the impact of alignment on OTU estimation. They found that pairwise alignments yielded a smaller distance than those from multiple alignments so that the use of pairwise alignment could reduce the inferred OTU number. Comparison of OTU-Based Clustering Methods Later, they illustrated the behavior of hierarchical and heuristic clustering algorithms in OTU construction, and concluded that hierarchical clustering algorithms are more accurate [17]. Huse et al [24] evaluated three different hierarchical clustering algorithms, and showed that the choice of clustering strategy could significantly affect the number of estimated OTUs. Overall, average-linkage clustering is more robust than complete-linkage clustering while single-linkage is rarely used because of its chaining effect [30]. Another study also showed that average-linkage clustering could provide better results and lower the effects of sequence errors in OTU estimation, yet it still overestimated the number of OTUs [31]. Schloss [32] examined some other factors such as distance calculation and sequence filtering methods which may affect the results in processing the 16S rRNA data. A more recent work by Schloss and Westcott [20] assessed the performance of four OTU-based algorithms on RDP-based benchmark datasets, and concluded that it is difficult to set a consistent threshold for defining OTU. Moreover, they introduced a novel heuristic clustering method (phylotype-OTU) with taxonomic information that can reduce the computation complexity without sacrificing the robustness of OTU assignments. This study is limited in the number of methods compared, and the choice of the RDP-based benchmark datasets that could not effectively reflect the nature of the data commonly used for OTU inference. For example, the 454 read lengths are often shorter than the 450 bps analyzed in the paper and sequencing errors were not explicitly considered. In order to investigate whether the secondary structure information would affect the estimation of OTUs, Wang et al., [33] used simulations to show that incorporating such information does not improve the OTU assignments. However, Schloss contested this point by more strict experiments [34]. Most recently, Sun et al [30] reported a large-scale benchmark study that evaluated seven OTU algorithms based on normalized mutual information (NMI) [35]. A bench- mark dataset was constructed based on RDP [36] and Taxcollec- tor [37]. Yet, it is not a strict criterion because the sequence divergence is not distributed evenly along the 16S rRNA gene and the retained sequences at a fixed 97% identity may only partially agree with the ground-truth. Meanwhile, some reads were annotated by multi-species, i.e. one-to-many mapping. However, the relative performance of different methods may depend on these processing steps to create the benchmark dataset. Introduction Another limitation of the taxonomy- dependent methods is that most existing reference databases are well-characterized only at the genus level or higher, rather than at August 2013 \| Volume 8 \| Issue 8 \| e70837 August 2013 \| Volume 8 \| Issue 8 \| e70837 1 PLOS ONE \| www.plosone.org Comparison of OTU-Based Clustering Methods Table 1. Details on the simulated datasets. Num. species Species length Speices similarity arrange (%) Total reads Initial Abundance ratio (%) Simclone10_1 10 226,252 70.00,93.00 69958 [6.25,3.89,5.06,11.31,7.74,29.77,8.04,12.50,5.95,9.52] Simclone10_2 10 218,255 70.59,92.94 148374 [6.13,8.02,4.25,9.91,9.43,14.62,10.38,12.26,11.79,13.21] Simclone15_1 15 59,71 50.68,83.04 63616 [3.57,1.78,1.78,5.35,3.57,17.86,3.57,7.14,1.79,1.79,5.36,3.57,10.71,14.29,17.86] Simclone15_2 15 59,82 50.68,82.54 134092 [7.58,5.30,12.12, 18.94, 3.79, 8.71, 17.05, 3.79, 8.71,14.02] Simclone20 20 64,261 25.58,94.19 115654 [2.87,1.97,2.12,4.54,4.84,9.53,6.05,6.05,3.02,2.42,3.18,5.45,3.18,7.57,8.62,6.81,5.45,4.84,8.62,2.87] Simclone30 30 212,241 69.72,94.44 128308 [4.41,5.26,1.07,4.21,2.98,0.96,1.73,3.07,4.56,5.45,1.12,4.92,4.76,2.66,3.93,1.02,2.10,4.90,4.56,4.48,3.46,0.85,4.62,4.77,3.31,4.01,3.55,2.25,3.87,1.14 Simclone50 50 210,242 69.86,95.85 152373 [1.22,3.10,2.78,0.68,0.58,1.70,3.50,1.46,2.19,0.90,3.22,1.09,1.87,3.08,3.41,3.72,2.18,0.68,0.81,0.97,3.21,0.99,3.15,1.10,4.06,1.27,0.85,1.19,2.20,1.86 1.66,3.53,2.16,1.94,3.72,1.19,3.21,3.39,1.42,2.07,0.44,0.38,2.38,2.95,3.93,0.62,2.47,1.91,0.21,1.38] Simclone100 100 212,276 67.03,95.93 248968 [0.14,0.94,1.55,0.72,1.44,0.89,0.62,0.91,1.29,0.97,0.37,1.76,0.70,0.43,1.02,0.48,0.32,1.12,1.25,0.81,1.04,0.36,0.94,0.42,0.57,0.60,0.32,1.44,0.45, 0.89,1.15,0.81,1.02,1.47,1.38,0.70,0.65,1.01,0.46,1.62,0.41,0.40,1.48,1.20,1.68,0.86,0.72,0.80,0.38,1.46, 1.80,1.02,1.25,1.77,1.29,1.09,1.54,0.80,1.74,1.62,1.06,1.70,1.28,1.56,1.13,1.50,0.38,1.58,0.57,1.01,1.13,0.68,1.27,1.76,0.99,0.75,0.37,1.39,0.34, 0.77,1.25,0.44,0.94,0.48,1.48,1.23,1.24,0.97,1.58,1.17,1.41,1.58,0.51,0.91,0.58,1.47,0.42,1.16,0.91,0.68] Simclone150 150 211,276 67.03,96.97 359153 [0.09,0.64,1.03,0.50,1.00,0.61,0.40,0.64,0.89,0.68,0.25,1.17,0.47,0.29,0.76,0.32,0.21,0.75,0.90,0.55,0.73,0.24,0.65,0.30,0.41,0.41,0.23,1.00, 0.31,0.62,0.82,0.57,0.71,1.01,0.97,0.52,0.47,0.69,0.33,1.09,0.28,0.27,1.02,0.82,1.11,0.61,0.51,0.56,0.28,0.97, 1.20,0.72,0.86,1.18,0.87,0.76,1.09,0.56,1.19,1.11,0.72,1.16,0.91,1.06,0.78,1.04,0.25,1.05,0.40,0.68,0.77,0.48,0.85,1.17,0.71,0.50,0.26, 0.92,0.21,0.53,0.83,0.30,0.65,0.33,0.99,0.84,0.86,0.69,1.07,0.77,0.93,1.06,0.34,0.62,0.43,0.99,0.29,0.82,0.64,0.48, 0.72,0.34,0.38,1.32,0.62,0.36,0.25,1.20,1.27,0.60,0.27,1.10,0.59,0.34,0.85,0.25,0.22,0.77,1.24,0.38,0.30,0.21,0.88,0.95,1.17,0.46,0.86, 0.27,0.32,0.65,0.72,0.32,0.91,0.70,0.75,0.88,0.31,0.84,1.20,0.60,0.29,0.24,0.19,0.54,0.41,0.55,0.86,0.82,0.49,0.65] Simclone200 200 190,276 64.26,96.97 484404 [0.06,0.47,0.77,0.37,0.75,0.46,0.31,0.48,0.65,0.50,0.19,0.87,0.35,0.22,0.54,0.24,0.16,0.57,0.65,0.42,0.54,0.19,0.49,0.22,0.29,0.30, 0.18,0.74,0.22,0.46,0.61,0.43,0.52,0.75,0.71,0.38,0.35,0.51,0.24,0.80,0.19,0.20,0.76,0.61,0.83,0.45,0.38,0.40,0.19,0.73, 0.88,0.52,0.63,0.86,0.64,0.55,0.82,0.40,0.86,0.82,0.53,0.86,0.65,0.78,0.58,0.76,0.19,0.79,0.30,0.51,0.58,0.35,0.63,0.87,0.53,0.38, 0.20,0.70,0.16,0.41,0.60,0.23,0.47,0.24,0.75,0.62,0.64,0.50,0.80,0.59,0.69,0.77,0.25,0.46,0.32,0.71,0.21,0.59,0.47,0.35,0.53, 0.26,0.28,0.98,0.47,0.26,0.18,0.88,0.93,0.44,0.18,0.81,0.42,0.25,0.61,0.18,0.17,0.59,0.92,0.28,0.23,0.14,0.66,0.70,0.87,0.33,0.63,0.19, 0.24,0.49,0.53,0.23,0.66,0.52,0.56,0.63,0.25,0.62,0.90,0.45,0.21,0.16,0.14,0.39,0.31,0.41,0.62,0.60,0.37,0.48,0.45,0.77,0.46, 0.43,0.56,0.56,0.16,0.27,0.47,0.83,0.34,0.65,0.16,0.14,0.62,0.68,0.43,0.51,0.46,0.76,0.62,0.31,0.91,0.48,0.40,0.41,0.86,0.56,0.51, 0.81,0.28,0.27,0.37,0.14,0.89,0.18,0.15,0.84,0.76,0.82,0.82,0.18,0.33,0.63,0.92,0.60,0.54,0.75,0.62,0.44] August 2013 \| Volume 8 \| Issue 8 \| e70837 PLOS ONE \| www.plosone.org 2 Clustering reads into OTU Despite many published studies discussed above on the developments and evaluations of various OTU inference methods, only several papers haven discussed how to choose an appropriate method for analyzing 16S rRNA sequences and more needs to be done to provide a comprehensive assessment of different methods. This is because many of these studies did not have ground truth, assumed error-free sequencing data, and did not consider the impact of sample complexity and abundance information on OTU inference. In this article, we compare ten OTU-based methods using both simulated and real data, where ground truth is known. We introduce errors in our simulated sequence reads to mimic real data settings. Sample complexity and abundance information is also explicitly considered in our comparisons. Our results suggest that when the default parameter settings are used, the commonly used methods tend to inaccurately estimate the number of OTUs. Therefore, it is important to choose proper parameters when applying a clustering algorithm for OTU inference. In addition, we note that the abundance of reads plays an important role on OTU clustering and the appropriate abundance thresholds depend on individual clustering algorithms. Our results may provide general guidelines on selecting an appropriate method for 16S rRNA data in microbial analysis. g We compared ten OTU-based methods from three broad classes: hierarchical clustering (Mothur [19], ESPRIT [21], ESPRIT-Tree [22], SLP [24], Muscle [41]+Mothur), heuristic clustering (CD-HIT [25], Uclust [26], GramCluster [27], DNAClust [28]) and model-based clustering methods (CROP [29]). For hierarchical clustering and heuristic clustering algo- rithms, we clustered reads into OTUs at or equal to dissimilarity thresholds ranging from 1% to 10% with an increment of 0.01 regardless of the definition of dissimilarity in different algorithms. For the model-based clustering method, CROP, reads were clustered into OTUs with a soft threshold, which ranged from 0.01 to 0.10. For the Muscle+Mothur method, Muscle is multiple- alignment program rather than a clustering algorithm. Hence we used Muscle (with the default parameters) to align the reads, and then the average neighbor algorithm in Mothur was used to perform the clustering (when we calculated the distance matrix for alignments from muscle, a gap was only penalized once, and terminal gaps were penalized). For Mothur, we used the pairwise.seqs command to obtain the distance matrix which adapted the Needleman-alignment algorithm to align sequences, and then the average neighbor algorithm was used to perform clustering. Assessment of clustering quality In order to generate robust statistical results, inspired by the concept of Q-CV test [42], we repeated the experiment 5 times for each simulated data set. For each iteration, 90% of the reads were randomly extracted from the simulated dataset. We evaluated the method performance with different metrics. We first examined the number of estimated OTUs, and then assessed the cluster quality using precision, recall and NID [43], respectively. Precision is defined by the number of reads that are both in class i and cluster j, divided by the number of the reads in cluster j, thus it measures the homogeneity of cluster j. Recall is defined as the proportion of reads from class i that present in cluster j, thus it measures the completeness. Precision and recall provide useful insight on local performance. NID is an information theoretic-based measurement that can assess the cluster globally with a nominal [0, 1] range. Real data (Clone43) The real data considered was described by Huse et al [39]. This study sequenced the V6 region of 16S rRNA from a community of 43 known microbial species. It consists of 202,340 reads with read length ranging from 57 to 145 bps. Comparison of OTU-Based Clustering Methods Comparison of OTU-Based Clustering Methods and relative abundance. Simclone15_1 and simclone15_2 were generated from 15 known species, simclone20 was generated from 20 known species sequences. likely to be generated from true sequences, yet the rare biosphere may not be as large as previously assumed [38] because many such units may be spurious, potentially from the accumulation of small sequence errors. Clustering reads into OTU To make relatively fair comparisons across different methods, we used the default parameter settings for all the OTU- based algorithms in our analysis. Data sets In our study, eleven datasets (both real and simulated) were used to evaluate ten existing OTU-based methods. Comparison of OTU-Based Clustering Methods In addition, it is recognized that abundance sequences are more August 2013 \| Volume 8 \| Issue 8 \| e70837 August 2013 \| Volume 8 \| Issue 8 \| e70837 PLOS ONE \| www.plosone.org 3 Simulated data Since it is difficult to evaluate the performance on a complex community because of the lack of ground-truth (e.g. the number of microbial species and their relative abundance), we used simulated datasets so that the species origin of each individual sequence read and the species abundance are known. To make the simulations more representative of nature samples, we considered simulations with different complexities, as reflected by the number of species (ranging from 10 to 200), the composition of the species (e.g. whether it is dominated by similar or distant species), and the read lengths (Table 1). To cover a wide range of scenarios, we generated ten 16S rRNA simulated datasets (simclone10_1, simclone10_2, simclone15_1, simclone15_2 and simclone20, simclone30, simclone50, simclone100, simclone150, simclone200) using the software 454Sim [40], which simulates 454 data using configurable statistical models that can accommodate different number of sequence reads, sequence lengths, errors rates, and abundance. Table 1 provides the detailed information for the simulated datasets, including the number and the length of species, the minimum and maximum distance among these species, simulated cluster size and other information. For example, Simclone10_1 and simclone10_2 were generated from 10 known species sequences but have a different distribution of reads length Now we define these measures more formally. Assume that there are N reads from m species (S1,S2,:::,Sm)and they are clustered into n clusters (C1,C2,:::,Cn) at specific dissimilarity threshold by a given clustering algorithm. Let \|Si\| denote the true number of reads from species i, \|Cj\| denote the number of reads from cluster j, and aij denote the number of reads from species i and categorized into cluster j. Using the above notations, precision and recall are defined as: Using the above notations, precision and recall are defined as: pij~ aij DCjD rij~ aij DSiD August 2013 \| Volume 8 \| Issue 8 \| e70837 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 4 Comparison of OTU-Based Clustering Methods NID is defined as: Table 2. Numbers of inferred OTUs from different dissimilarity thresholds for different algorithms. NID~1{ I(S,C) max(H(S),H(C)) Smaller NID values imply better clustering results. It stratifies both normalization and metric properties and has a tighter bound than other measures such as NVI, NMIjoint [43] and thus is a useful measure for cluster validation. Simulated data Clone43 Simclone15_1 Expected OTUs Inferred* OTUs(2%) inferred OTUs(3%) inferred OTUs(4%) Expected OTUs inferred OTUs(2%) inferred OTUs(3%) inferred OTUs(4%) Mothur 43 1882 720 369 15 63 41 20 Muscle+Mothur 2478 1418 784 117 89 54 ESPRIT 4474 4397 1733 131 131 55 ESPRIT-Tree 2301 1096 279 96 29 16 SLP 286 245 227 17 17 15 Uclust 2177 1883 597 80 75 51 CD-HIT 1473 1464 481 50 49 32 DNAClust 3768 3658 1103 239 225 53 GramCluster 2119 2071 2071 70 70 70 CROP 339 133 62 21 15 15 : all the listed numbers of OTU are the average numbers over xx simulations. August 2013 \| Volume 8 \| Issue 8 I(S,C)~ Xm i~1 Xn j~1 aij Nlog aij N jSijjCjj N2 , i~1,2,:::,m; j~1,2,:::,n H(S)~{ Xm i~1 Si Nlog Si N , H(C)~{ Xn j~1 Cj Nlog Cj N Inferred number of OTUs doi:10.1371/journal.pone.0070837.t002 PLOS ONE \| www.plosone.org 5 Comparison of OTU-Based Clustering Methods ilarities achieved by CD-HIT, Uclust and ESPRIT were 100%, 78% and 87.5%, respectively. It suggests that there is no consistent threshold for all the methods. For the low-complexity and short- length data set simclone15_1, CROP had the minimum NID (0) at 0.03 dissimilarity, SLP had the minimum NID (0) at 0.01 dissimilarity, Mothur had the minimum NID (0) at 0.04 and CD- HIT had the minimum NID (0.00034) at 0.07 dissimilarity. Most methods had low NID scores (, = 0.03) except Uclust (0.04) and ESPRIT (0.08). Meanwhile, SLP and CROP had a smaller average NID score with variation in the range from 0.01 to Dissimilar- ityminimumNID. Besides ESPRIT, Uclust and DNAClust, other algorithms had an average NID score smaller than 0.05. Similar results were found for the low-complexity data set simclone15_2. For the high-complexity data set simclone200, the threshold to achieve the minimum NID values skewed to the left across different algorithms and cluster qualities differed significantly for different dissimilarity thresholds. For examples, SLP, CROP and ESPRIT had the minimum NID value at 0.01 dissimilarity, whereas Muscle+Mothur and CD-HIT had the minimum NID at 0.02 dissimilarity. Similar trends were observed for simclone150, simclone100 and simclone50. These results suggest that a more stringent dissimilarity cut-off may be needed for high-complexity data sets. This is consistent with the fact when the samples consist of similar species, a higher dissimilarity threshold will incorrectly group the high-similarity species together. From the NID score, we can see that 1) Most of the current clustering algorithms could achieve a similar minimum NID value. It indicates that with a proper dissimilarity threshold for a specific algorithm, most of them have comparable results. Overall, ESPRIT, Uclust and DNAClust have poorer clustering results among these algorithms. 2) The optimal dissimilarity threshold for different methods should take the complexity of the data sets into account. For low-complexity and short-length read data, a higher dissimilarity is preferred to define OTU while for high-complexity and long-length read data, a lower threshold should be a better choice. As for the complexity of the datasets, it can be partly estimated by the distribution of the reads and the average distance among abundance reads. Inferred number of OTUs 3) For low- complexity and short-length read data, CROP and SLP may be preferred for their robustness to sequence errors, while for high- complexity reads, CROP should adopt smaller threshold (such as 1% for simclone200,simclone150), otherwise, it may lead to an under-estimation of number of OTUs for its over learning. 4) An interesting finding is that the NID curve for CD-HIT is similar to those of most hierarchical algorithms for high-complexity and long- length read. outperformed hierarchical clustering algorithms. This is partly due to the fact that these four data sets were generated from similar species populations. This suggests that if we choose the commonly used 3% dissimilarity to define OTUs at the species level, we may end up with an incorrectly estimated (overestimated or underes- timated) number of OTUs, thus a constant threshold (e.g. 3% dissimilarity) is not ideal. Therefore, the choice of dissimilarity for defining the taxonomy is both dependent on the cluster algorithms as well as on the complexity of the dataset. For comparisons between different algorithms, we found that SLP returned a smaller number of OTUs for the simulated datasets. This could be explained by the chaining results of SLP [30], which adapts a single-linkage pre-clustering (2%), thus, more sequencing errors can be tolerated with longer reads. Mothur inferred a smaller number of OTUs than Muscle+Mothur, consistent with the argument that pairwise distances tend to yield more biologically meaningful OTUs than those with multiple alignment distances [21]. Among the hierarchical clustering methods, SLP returned the smallest numbers of OTUs mainly due to the use of precluster which could reduce the spurious clusters generated by the erroneous sequences. Unfortunately, it often led to another problem. If the data sets are generated from near-clonal populations, it is sometimes difficult to differentiate similar species due to sequence errors. In the context of inferring the number of OTUs, CD-HIT performed much better than GramCluster and DNAClust regardless of the complexity of the simulated data sets. In general, at the same cluster scale referred to the cluster thresholds, the hierarchical clustering algorithms returned smaller numbers of OTUs than heuristic clustering methods. The exception is ESPRIT, which adapts a complete-linkage (default) clustering to group reads into OTUs. Inferred number of OTUs It applies a more stringent threshold so that no sequence can be added to an existing OTU unless the distances between the new sequence and the sequences already in the OTU are smaller than the threshold. For CD-HIT and Uclust, consistent with what was reported by Cai et al. [22], these two methods ran several orders of magnitude faster than the hierarchical clustering algorithms at the cost of accuracy. The Gaussian model-based clustering algorithm CROP achieved the best result in the inferred number of OTUs for lower complex data sets (such as data sets simclone10_1 and simclone10_2) but had worse performance for highly complex data sets (such as simclone200), where it under-estimated the numbers of OTUs. Relationship between the dissimilarity threshold and cluster algorithms From the above analysis, we found that it is difficult to use a constant threshold to define OTUs at a specific taxonomic level. To further compare the performance of different algorithms beyond the number of inferred OTUs, we calculate NID values for different dissimilarity cut-offs ranging from 0.01 to 0.1 with a step size of 0.01. Among these values, we chose the local minimum value of each algorithm and analyzed the mean and standard deviation of the NID covering the interval [0.01, DissimilarityminimumNID], where DissimilarityminimumNID is the dissimilarity level where it achieved the minimum NID, to compare these algorithms (Figure 1, Figure S1). As expected, the clustering qualities of different algorithms were dependent on the clustering dissimilarity, and the minimum NID scores for different algorithms were achieved at different dissimilarity values. This may be partly due to the fact that the dissimilarity definitions used in different methods are different. Some are alignment-based while others are alignment-free. Even for the alignment-based methods, they sometimes used different strategies to treat inserts, deletes and gaps. For example, for sequences S1 = ACGGTAT and S2 = ACGGGTATAC, the sim- Inferred number of OTUs For the real dataset Clone43 and the simulated dataset Simclone15_1, Table 2 summarizes the inferred numbers of OTUs with the dissimilarity threshold set at 2%, 3% and 4%, respectively, together with the true numbers of OTUs. The results for Simclone10_1, Simclone10_2, Simclone15_2, Simclone20, Simclone30, Simclone50, Simclone100, Simclone150 and Sim- clone200 are shown in Table S1. Although sequence divergence is not evenly distributed in the 16S rRNA region, 3% dissimilarity is often chosen in practice as the cutoff value to define bacteria species [2,21,32,39]. At this dissimilarity level, for the real dataset Clone43, ESPRIT gave the largest estimated number of OTUs (4,397), nearly 100 times larger than the expected 43 OTUs, while CROP yielded the smallest number of estimated OTU (133), still nearly three times of the true number. For the simulated dataset Simclone15_1, we found that CROP returned 15 OTUs, equal to the true number of OTUs, and SLP returned 17 OTUs. GramCluster yielded 225 clusters, the largest estimated number of OTUs among all the methods. Similar results were found for simclone15_2 which was simulated from similar species distribu- tion but different initial abundance with simclone15_1. For simulated datasets simclone10_1, simcloen10_2, and simclone30, these methods also tended to overestimate the number of OTUs at 3% dissimilarity except the SLP algorithm, yet, the extent of overestimation is smaller when it is compared with simclone15_1 and simclone15_2. This may be due to the fact that the length of template species generated for these three simulated data sets are longer and they share a strict inter-species similarity range, a smaller upper-limit but higher lower-limit similarity, so they can tolerant more sequence errors in clustering. The results for Simclone20 were similar to those of simclone30, which have similar species distribution but with an overall lower inter-species similarity. These results suggest that the upper-limit rather than lower limit of inter-species similarity plays an important role on clustering results. However, the results are different for the other four complex simulated datasets. When we inferred OTUs at 3% dissimilarity, most hierarchical algorithms underestimated the number of OTUs. For example, CROP returned 122 OTUs, SLP returned 144 OTUs and ESPIT-Tree returned 175 OTUs for the simclone200. A more interesting finding is CD-HIT, which inferred the true (or close to the true) number of OTUs, and : all the listed numbers of OTU are the average numbers over xx simulations. August 2013 \| Volume 8 \| Issue 8 \| e70837 Evaluation of different methods using Precision and Recall measures In addition to broader assessment of inferred OTU clusters in relation to the underlying community structure using the minimum NID score, we also calculated the precision and recall scores to obtain more detailed information how the sequences originating from the same species are clustered together and how the reads distribute in the clusters with different algorithms. We considered the simulated data sets where the corresponding template species for each read cloned from was known (Figure 2, Figure S2). For the dataset simclone15_1, CROP, CD-HIT, DNAClust inferred 15 OTUs with high precision and recall values which were close to 1, which suggest that these methods could cluster the reads accurately in this setting. All the other algorithms over-estimated the numbers of OTUs. The precision versus recall plots for this dataset show that some OTUs with a small cluster size and the reads are from the same species, in other words, reads from the same species were clustered into different groups. This August 2013 \| Volume 8 \| Issue 8 \| e70837 August 2013 \| Volume 8 \| Issue 8 \| e70837 PLOS ONE \| www.plosone.org 6 Comparison of OTU-Based Clustering Methods Figure 1. NID scores of ten algorithms based on the data set simclone15_1 and simclone_200. doi:10.1371/journal.pone.0070837.g001 Figure 1. NID scores of ten algorithms based on the data set simclone15_1 and simclone_200. doi:10.1371/journal.pone.0070837.g001 clone200). 1) With an appropriate choice of threshold, the inferred number of OTUs could match the true number for all the algorithms. 2) Many OTUs generated from Uclust and GramClus- ter contained more than one species. ESPRIT estimated a large number of OTUs, with most clusters having a small cluster size consisting of reads from the same species. 3) Mothur, CROP, SLP, Muscle+Mothur, ESPRIT-Tree and CD-HIT achieved better performance and clustered relatively small number of OTUs, but they sometimes led to inaccurate clusters by grouping reads from different species into one cluster. explained the observed points appear near (0, 1). There were also a few OTUs containing reads from different species and large proportion of reads were from a dominating species while others were from different species. It can explain the points closed to (0, 0). For example, GramCluster inferred 17 OTUs and there were 19 points having non-zero (precision, recall) values, 2 out of 19 were close (0, 0). Similar results were found in other low- and moderate complexity datasets (simclone10_1, simclone10_2, simclone15_2 and simclone20). Evaluation of different methods using Precision and Recall measures For the simulated datasets with a high complexity (such as more species and containing high- similarity species et al.), the trend became more obvious (simclone30, simclone50, simclone100, simclone150 and sim- Figure 2. A Precision versus Recall plot generated from data set simclone15_1. doi:10.1371/journal.pone.0070837.g002 Figure 2. A Precision versus Recall plot generated from data set simclone15_1. doi:10.1371/journal.pone.0070837.g002 August 2013 \| Volume 8 \| Issue 8 \| e70837 August 2013 \| Volume 8 \| Issue 8 \| e70837 PLOS ONE \| www.plosone.org 7 Comparison of OTU-Based Clustering Methods Table 3. Running time for different algorithms when cluster sequences into OTUs for dissimilarity thresholds ranging from 0.01 to 0.10 based on simclone20. input* Simclone20 Running time (minute) for sequences (wall time) Mothur UniqueSeq 469.00 Muscle+Mothur UniqueSeq 6.27 ESPRIT All 75.21 ESPRIT-Tree All 2.37 SLP UniqueSeq 586.55 Uclust All 0.87 CD-HIT All 3.85 DNAClust All 3.01 GramCluster All 36.85 CROP All 173.40 : UniqueSeq represented only the unique, unaligned sequences were takes as input, All represented all sequences including the identical sequences are taken as input. doi:10.1371/journal.pone.0070837.t003 Table 3. Running time for different algorithms when cluster sequences into OTUs for dissimilarity thresholds ranging from 0.01 to 0.10 based on simclone20. Table 3. Running time for different algorithms when cluster sequences into OTUs for dissimilarity thresholds ranging from 0.01 to 0.10 based on simclone20. : UniqueSeq represented only the unique, unaligned sequences were takes as input, All represented all sequences including the identical sequences are taken as input. doi:10.1371/journal.pone.0070837.t003 Comparison of OTU-Based Clustering Methods Comparison of OTU-Based Clustering Methods threshold, fewer OTUs were inferred. In addition, in order to recover the correct number of OTUs, different frequency thresholds may be needed according to the specific algorithms used. To illustrate these results, we analyzed five simulated datasets at different frequency thresholds and different dissimilar- ity thresholds to infer the numbers of OTUs (Figure S3 and Table S2). The results suggest that the abundance threshold plays an importance role in clustering reads into OTUs. For Simclone15_1 with an abundance threshold of 10, the ten algorithms inferred 30, 16, 22, 27, 15, 15, 20, 63, 20, and 21 OTUs, respectively. Similar results were obtained for Sim- clone15_2, with most algorithms identified the correct number of OTUs if we used 100 as the abundance threshold. Most true species were covered when we mapped OTUs to the original species. With an appropriate threshold, all algorithms can approximately recover the true number of OTUs (150 for ESPRIT, 10 for ESPRIT-Tree, SLP and CROP, and 50 for Mothur et al). Similar patterns were observed for other datasets (simclone10_1, simclone10_2 and simcloen20). It can be seen that the results from these simulated datasets were consistent with those observed with the real dataset Clone43 presented in Figure 2. some OTUs with a large number of reads and coming from different species. This suggests that for these OTUs which have a large and different size from surrounding OTUs, care is needed before downstream analysis. 3) The sequence abundance plays an important role in clustering sequences into OTUs. The estimated numbers of OTUs will be reduced and become more accurate by setting a proper frequency threshold to filter out sequences with low abundance. Existing OTU-based algorithms are sensitive to sequencing errors, it would be more effective to model sequencing errors and identify/remove ‘‘problematic’’ reads before clustering. This may be achieved by error calibration and modeling [44,45]. Our results suggest the importance of choosing a proper distance threshold for taxonomic definition because of the heterogeneity of the evolu- tionary rate of 16S rRNA genes [20]. In addition, the dissimilarity is correlated with the distance computing methods [32]. There- fore, it is desirable to establish a taxonomic specific distance threshold that can incorporate the information on diverse evolutionary rates for 16S rRNA, and distance computing methods into consideration. Our study has focused on the taxonomy independent clustering methods using the 454 sequence data. Acknowledgments We would like to thank the Yale University Biomedical High Performance Computer Center for computation support. We thank Alexander V. Alekseyenko and two anonymous reviewers for comments and discussions that improved the quality of the manuscript. Conclusion With rapid accumulation of 16S rRNA sequences and whole genome shotgun sequences, there is a great need to develop effective methods to analyze these data. In the past decade, taxonomy-dependent and OTU-based methods have been widely used to process the rRNA sequences. Because taxonomy- dependent algorithms depend on the completeness of existing databases while the majority of microbe species are unknown, the OTU-based methods will continue to play a vital role in microbial analysis. In this case, an accurate identification of OTU is critical for downstream analysis and biological interpretation. Although much work has been done to evaluate of the effect of the alignment, distance calculated methods, and the different variable region of 16S rRNA on OTU estimations [20,32], relatively little guidance has been provided on the choice of a proper method for OTU estimation. In this article, we used both real and simulated datasets to compare the performance of ten existing representative OTU-based algorithms. From these results, we conclude that: 1) Most of OTU-based algorithms may either overestimate or underestimate the number of OTUs if an improper dissimilarity is chosen. The appropriate dissimilarity cut-off for inferring OTUs is not only dependent on the specific methods but also related with the complexity of the datasets. For low-complexity and short- length read datasets, a higher dissimilarity can be used to define OTU. In this case, among the ten algorithms considered, SLP and CROP achieved a better performance on OTU estimation. For high-complexity and long-length read datasets, hierarchical cluster methods need a more stringent dissimilarity threshold. Further- more, CD-HIT has better performance similar as hierarchical clustering methods. 2) Most existing OTU-based algorithms tend to partition the samples from the same species into several sub- clusters and those OTUs sometimes have a small size, suggesting that we may reduce the overestimated number of OTUs through integrating small clusters. Unfortunately, even in the case of optimal cluster results with a minimum NID score, there also exist Comparison of OTU-Based Clustering Methods It is undoubtedly informative to relate each OTU to a biological entity. With the rapid accumulation of databases containing experimentally verified sequences, incorporating information of annotated sequences into OTU-based clustering methods will be critical yet few methods simultaneously analyze both annotated and unan- notated sequences. Last but not least, we have focused on the analysis of 454 data and it is worthwhile to evaluate the performance with sequences generated from other sequencing platforms, e.g. Illumina, where the whole genome information is available. These results suggest the importance of imposing an abundance threshold in OTU inference. As for the removed reads (non- abundant reads), we may use a single-clustering or heuristic clustering method to assign the read to specific OTUs that were produced from subset of abundant reads. Supporting Information Figure S1 The NID Scores of different algorithms. (TIFF) Figure S1 The NID Scores of different algorithms. (TIFF) Figure S2 Precision versus recall plots for different datasets. (TIFF) Figure S2 Precision versus recall plots for different datasets. (TIFF) Figure S3 The results of OTUs estimated with different frequency thresholds (/abundance) at different dissimilarity levels based on the dataset Clone43. (TIFF) Figure S3 The results of OTUs estimated with different frequency thresholds (/abundance) at different dissimilarity levels based on the dataset Clone43. (TIFF) Time cost for OTU-based methods Impact of abundance on clustering sequences into OTUs Impact of abundance on clustering sequences into OTUs Some research showed that abundant sequences were more likely to be generated from true species while the rare sphere may be an artifact due to accumulation of sequencing errors [38]. In order to test whether abundance has an effect on OTUs estimation, we used different frequency thresholds to define abundant data set and then clustered the abundant sequences into OTUs (Figure 3, Figure S3, and Table S2). For the data set Clone43, when we used 10 as a threshold, the coverage was 93.4% and the estimated number of OTUs by ESPRIT, ESPRIT-Tree, Mothur, Muscle+Mothur, SLP, CROP, CD-HIT, GramCluster, DNAClust and Uclust was 520, 55, 56, 87, 45, 44, 118, 313, 527, and 82, respectively. With a frequency threshold 100 covering 84.2% of the total sequences, the above algorithms identified 57, 39, 40, 47, 39, 38, 53, 47, 60 and 56 OTUs, respectively, at the 0.03 dissimilarity threshold. As expected, with a higher frequency We noted that time cost is a critical factor when we choose an appropriate method for analyzing large scale 16S rRNA datasets. Table 3 lists the running time for each method on the dataset simclone20. Uclust took the least time to complete the clustering (0.87 minute) when the dissimilarity threshold ranged from 0.01 to 0.1, followed by ESPRIT-Tree and DNAClust (2.37 minutes and 3.01 minutes). SLP took the longest time to complete clustering (,587 minutes). Among the heuristic clustering methods, GramCluster took the longest time to cluster sequences (36.85 minutes), due to the additional time needed to create grammar dictionary for defining dissimilarity. The running time of CROP (,173 minutes) was shorter than that of Mothur and SLP. In general, the hierarchical clustering and model-based clustering algorithms took more time to assign sequences into OTUs than those of heuristic clustering methods. Figure 3. The results of OTUs estimated with different frequency thresholds at different dissimilarity levels, from the data set Clone43. doi:10.1371/journal.pone.0070837.g003 Figure 3. The results of OTUs estimated with different frequency thresholds at different dissimilarity levels, from the data set Clone43. doi:10.1371/journal.pone.0070837.g003 August 2013 \| Volume 8 \| Issue 8 \| e70837 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 References Hao X, Jiang R, Chen T (2011) Clustering 16S rRNA for OTU prediction: a method of unsupervised Bayesian clustering. Bioinformatics, 27(5): 611–618. 8. Oakley BB, Fiedler TL, Marrazzo JM, Fredricks DN (2008) Diversity of human vaginal bacterial communities and associations with clinically defined bacterial vaginosis. Appl Environ Microbial, 74(15): 4898–4909. 30. Sun Y, Cai Y, Huse SM, Knight R, Farmerie WG, et al. (2012) A large-scale benchmark study of existing algorithms for taxonomy-independent microbial community analysis. Briefings in Bioinformatics, 13(1): 107–121. g pp ( ) 9. Kellenberger E (2001) Exploring the unknown: The silent revolution of microbiology. Embo Rep, 2(1): 5–7. 31. Quince C, Lanze´n A, Curtis TP, Davenport RJ, Hall N, et al. (2009) Accurate determination of microbial diversity from 454 pyrosequencing data. Nature methods, 6(9): 639–641. 10. Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, et al. (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA, 82(20): 6955–6959. 32. Schloss PD (2010) The effects of alignment quality, distance calculation method, sequence filtering, and region on the analysis of 16S rRNA gene-based studies. Plos computational Biology 2010, 6(7): e1000844. 11. Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science, 276(5313): 734–740. 33. Wang X, Cai Y, Sun Y, Knight R, Mai V (2011) Secondary structure information does not improve OTU assignment for partial 16s rRNA sequences. The ISME Journal. 12. Schloss PD, Handelsman J (2004) Status of the microbial census. Microbial Mol Biol Rev, 68(4): 686–691. 13. Sharpton TJ, Riesenfeld SJ, Kembel SW, Ladau J, O’Dwyer JP, et al. (2011) A High-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data. PLOS computational Biology, 7(1): e1001061. 34. Schloss PD (2013) Secondary structure improves OTU assignments of 16S rRNA gene sequences, The ISME Journal, 7: 457–460. 35. Studholme C, Hill DLG, Hawkes DJ (1999) An overlap invariant entropy measure of 3D medical image alignment. Pattern Recognition, 32(1): 71–86. ( ) 14. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, et al. (2005) Diversity of the human intestinal microbial flora. Science, 308(5728): 1635– 1638. 36. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiology, 73(16): 5261–5267. 15. Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. References 1. Whitman WB, Coleman DC, Wiebe WJ (1998) Prokaryotes: the unseen majority. Proc Natl Acad Sci USA, 95(12): 6578–6583. 22. Cai Y, Sun Y (2011) ESPRIT-Tree: hierarchical clustering analysis of millions of 16S rRNA pyrosequences in quasilinear computational time. Nucleic Acids Res, 39(14): e95. j y ( ) 2. Sogin ML, Morrison HG, Huber JA, Mark WD, Huse SM, et al. (2006) Microbial diversity in the deep sea and the underexplored ‘‘rare biosphere’’. Proc Natl Acad Sci USA, 103(32): 12115–12120. ( ) 23. Fabrice A, Didier R (2009) Exploring microbial diversity using 16S rRNA high- throughput methods. J Comput Sci Syst Biol, 2: 74–92. ( ) 3. Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, et al. (2005) Obesity alters gut microbial ecology. Proc Natl Acad Sci USA, 102(31): 11070– 11075. 24. Huse SM, Welch DM, Morrison HG, Sogin ML (2010) Ironing out the wrinkles in the rare biosphere through improved OTU clustering. Environ Microbial, 12(7): 1889–1898. 24. Huse SM, Welch DM, Morrison HG, Sogin ML (2010) Ironin 4. Ley RE, Turnbaugh PJ, Klein S, Gordon JI (2006) Microbial ecology: human gut microbes associated with obesity. Nature, 444(7122): 1022–1023. ( ) 25. Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics, 22(13): 1658–1659. 4. Ley RE, Turnbaugh PJ, Klein S, Gordon JI (2006) Microbial ecology: human gut microbes associated with obesity. Nature, 444(7122): 1022–1023. 5. Duncan KE, Gieg LM, Parisi VA, Tanner RS, Tringe SG, et al. (2009) Biocorrosive thermophilic microbial communities in Alaskan North Slope oil facilities. Environ Sci Technol, 43(20): 7977–7984. 26. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics, 26(19): 2460–2461. 27. Russell DJ, Way SF, Benson AK, Sayood K (2010) A grammar-based distance metric enables fast and accurate clustering of large sets of 16S sequences. BMC Bioinformatics, 11: 601. 6. Grice EA, Kong HH, Conlan S, Deming CB, Davis J, et al. (2009) Topographical and temporal diversity of the human skin microbiome. Science, , 324(5931): 1190–1192. 28. Ghodsi M, Liu B, Pop M (2011) DNACLUST: accurate and efficient clustering of phylogenetic marker genes. BMC Bioinformatics, 12: 271. ( ) 7. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, et al. (2009) A core gut microbiome in obese and lean twins. Nature, 457(7228): 480– 484. 29. Author Contributions Conceived and designed the experiments: WC HYZ. Performed the experiments: WC CKZ YMC. Analyzed the data: WC HYZ SWZ. Contributed reagents/materials/analysis tools: WC SWZ YMC CKZ. Wrote the paper: WC. Revised the manuscript critically: WC HYZ CKZ SWZ. August 2013 \| Volume 8 \| Issue 8 \| e70837 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9 Comparison of OTU-Based Clustering Methods Comparison of OTU-Based Clustering Methods References Appl Environ Microbial, 71(3): 1501–1506. 37. Giongo A, Crabb DB, Davis-Richardson AG, Chauliac D, Mobberley JM, et al (2010) PANGEA: pipeline for analysis of next generation amplicons. ISME, 4(7): 852–861. ( ) 16. Huse SM, Dethlefsen L, Huber JA, Mark WD, Relman DA, et al. (2008) Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing. PLoS Genet, 4(11): e1000255. 38. Reeder J, Knight R (2009) The ‘rare biosphere’: a reality check. Nature Methods, 6(9): 636–637. 39. Huse SM, Huber JA, Morrison HG, Sogin ML, Welch DM (2007) Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biol, 8(7): R143. 17. Sun Y, Cai Y, Mai V, Farmerie W, Yu F, et al. (2010) Advanced computational algorithms for microbial community analysis using massive 16S rRNA sequence data. Nucleic Acids Res, 38(22): e205. 40. Lysholm F, Andersson B, Persson B (2011) An efficient simulator of 454 data using configurable statistical models. BMC research notes, 4(1): 449. 18. Liu Z, DeSantis TZ, Andersen GL, Knight R (2008) Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrose- quencers. Nucleic Acids Res, 36(18): e120. 41. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res, 32(5): 1792–1797. 42. Baldi P, Brunak S, Chauvin Y, Andersen CAF (2000) Assessing the accuracy of prediction algorithms for classification: an overview. Bioinformatics 16, 412– 424. q ( ) 19. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, et al. (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbial, 75(23): 7537–7541. 43. van Rijsbergen CV (1979) Information retrieval. Boston: Butterworth. ( ) 20. Schloss PD, Westcott SL (2011) Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl. Environ. Microbial. 77(10): 3219–3226. 44. Margulies M, Egholm M, Altman WE, Attiya A, Bader JS, et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376– 380. 45. Richter DC, Ott F, Auch AF, Schmid R, Huson DH (2008) MetaSim – A Sequencing Simulator for Genomics and Metagenomics. PLoS ONE 3(10): e3373. 21. Sun Y, Cai Y, Liu L, Yu F, Farrell ML, et al. (2009) ESPRIT: estimating species richness using large collections of 16S rRNA pyrosequences. Nucleic Acids Res, 37(10): e76. August 2013 \| Volume 8 \| Issue 8 \| e70837 PLOS ONE \| www.plosone.org 10
https://openalex.org/W2410220274	https://www.scielo.br/j/ref/a/J8B8SQsRgDpYtQ3mD6rnFbv/?lang=pt&format=pdf	Portuguese	null	As mulheres praticando ciência no Brasil	Revista Estudos Feministas	2,016	cc-by	8,351	As mulheres praticando ciência As mulheres praticando ciência As mulheres praticando ciência As mulheres praticando ciência As mulheres praticando ciência no Brasil no Brasil no Brasil no Brasil no Brasil Resumo Resumo Resumo Resumo Resumo: As mulheres vêm aumentando sua participação em diferentes áreas da sociedade, mas ainda enfrentam obstáculos, inclusive para sua inserção no mundo da ciência. Buscando uma melhor compreensão sobre esse fenômeno, realizou-se uma pesquisa que mapeou a participação feminina no desenvolvimento de pesquisas no Brasil, a partir da análise dos currículos Lattes de 4.970 mulheres que defenderam suas teses de doutorado entre os anos de 2000 e 2013. Em relação aos procedimentos técnicos, foi utilizada uma pesquisa documental, cuja coleta de dados se deu no período de dezembro de 2013 a julho de 2014. Os resultados mostram que as mulheres conseguiram muitos avanços nessa área, mas que a desigualdade de papéis entre mulheres e homens ainda persiste dentro da ciência. Palavras-chave Palavras-chave Palavras-chave Palavras-chave Palavras-chave: mulheres; educação; ciências; pesquisas; doutorado. http://dx.doi.org/10.1590/1805-9584-2016v24n1p11 http://dx.doi.org/10.1590/1805-9584-2016v24n1p11 Shirley Doweslei Bernardes Borja Centro Federal de Educação Tecnológica de Minas Gerais, MG, Brasil Aline Moraes Lopes Centro Federal de Educação Tecnológica de Minas Gerais, MG, Brasil Aleixina Maria Lopes Andalécio Faculdade Novos Horizontes, Belo Horizonte, MG, Brasil Introdução Introdução Introdução Introdução Introdução Mesmo hoje, na sociedade da informação e do conhecimento, que, de acordo com Márcia Grossi,1 teve origem durante a Segunda Guerra Mundial como consequência da explosão informacional e documental, acelerando os processos de produção e de disseminação da informação, da revolução científica e técnica, as mulheres ainda não conquistaram seu empoderamento nas diferentes áreas da sociedade e continuam sofrendo com a Esta obra está sob licença Creative Commons. 1 GROSSI, 2008. Estudos Feministas, Florianópolis, 24(1): 406, janeiro-abril/2016 11 2 TAVARES, 2011, p. 8. 12 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 3 RISTOFF, 2006 (online). 4 ROSEMBERG e MADSEN, 2011. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO Nesse sentido, Fulvia Rosemberg e Nina Madsen4 explicam que, a partir de 2001, verifica-se uma preocu- pação governamental com o tema ‘Mulheres e gênero na educação’, demonstrada, principalmente, nas seguintes ações: criação da Secretaria Especial de Políticas para as Mulheres, em 2003; criação da Secretaria de Educação Continuada, Alfabetização e Diversidade, do Ministério da Educação (MEC); realização da 1ª Conferência Nacional de Políticas para as Mulheres (CNPM) e formulação do I Plano Nacional de Políticas para as Mulheres (PNPM), em 2004; 12 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL lançamento do Plano de Desenvolvimento da Educação; realização da 2ª CNPM e formulação do II PNPM, em 2007; lançamento do Plano Nacional da Educação (PNE) 2011- 2020, em 2010, que estabelece metas para a área da Educação nos próximos 10 anos. O PNE foi elaborado a partir da Conferência Nacional de Educação (CONAE), havendo propostas de várias organizações da sociedade civil, sempre com o objetivo de melhorar a qualidade da educação brasileira. Essas propostas foram enviadas para o Congresso Nacional. g Sobre a questão do gênero, o PNE lançou diretrizes que visam erradicar as discriminações históricas que as mulheres sofrem desde a infância e, assim, preparar as novas gerações para tratar com igualdade os homens e as mulheres, respeitando as diferenças e combatendo o machismo, ou seja, abolindo os estereótipos de gênero presentes na sociedade atual. Essa questão, no entanto, ainda é uma polêmica entre os parlamentares mais conservadores e os progressistas e permanece em estudo no Congresso. Outro ponto a ser destacado é uma pesquisa sobre os níveis de escolarização das mulheres entre os anos de 1998 a 2003, realizada pela Secretaria Especial de Políticas para as Mulheres (SPM), o Plano Nacional de Política para as Mulheres (PNPM), juntamente com o Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira (Inep), que apresenta dados que apontam que as mulheres estão se destacando nessa área:5 5 BRASIL, 2013. - Para o ensino médio: em 2003, as matrículas femininas atingiram um índice de 54%, enquanto que, para os homens, esse índice foi de 46%; - Na graduação: em 2003, as matrículas femininas ultrapassavam as masculinas em 12,8%; - Na graduação: em 2003, as matrículas femininas ultrapassavam as masculinas em 12,8%; - No nível de mestrado: o número de matrículas das mulheres aumentou, em 2003, 102,2%. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO desigualdade e com as violências moral, psicológica, sexual e física. Percebe-se, entretanto, que esse cenário vem sendo transformado ao longo dos anos. Como exemplo, pode-se tomar o campo da política, no qual as mulheres foram, durante muito tempo, excluídas de qualquer participação e, desde 2002, a maior parte do eleitorado no Brasil é composto por mulheres, como divulgado pelo Tribunal Superior Eleitoral, que informou que 52% dos 142 milhões de eleitores em 2014 são mulheres. Além disso, atualmente, já ocupam os maiores cargos políticos, como aponta Rebecca Tavares: No campo político, o Brasil elegeu em 2010 a primeira mulher Presidenta da República, Dilma Rousseff, que nomeou nove mulheres ministras e priorizou o empoderamento econômico das mulheres e o enfrentamento à violência baseada no gênero. O Congresso Nacional está analisando propostas de reforma política que garantam mais mulheres nos corpos legislativos estaduais e federais, e o país tem levado muito a sério seus compromissos com diversas Convenções e Tratados internacionais que garantem os direitos das mulheres, incluindo-se a Convenção sobre Todas as Formas de Discriminação Contra as Mulheres (Cedaw) e a Convenção de Belém do Pará.2 O fato de o Brasil ter como chefe de Estado uma mulher não significa, porém, que existe igualdade de gênero na nossa sociedade. Nota-se, então, que, para fortalecer o gênero feminino, o caminho que vem sendo trilhado pelas mulheres é a educação, como mostrado por Dilvo Ristoff, para quem: A trajetória da mulher brasileira nos últimos séculos é, para dizer pouco, extraordinária: de uma educação no lar e para o lar, no período colonial, para uma participação tímida nas escolas públicas mistas do século 19; depois, uma presença significativa na docência do ensino primário, seguida de uma presença hoje majoritária em todos os níveis de escolaridade, bem como de uma expressiva participação na docência da educação superior.3 3 RISTOFF, 2006 (online). 4 ROSEMBERG e MADSEN, 2011. 5 BRASIL, 2013. Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 1 MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO que aproveitam todas as oportunidades de formação, principalmente a formal. De acordo com o Centro de Gestão e Estudos Estratégicos em Ciência, Tecnologia e Inovação (CGEE), o Brasil é pioneiro entre os países que conseguiram alcançar esse marco histórico da igualdade de gênero no nível mais elevado da formação educacional, o doutorado, o que ocorreu a partir do ano de 2004.7 Apesar desses avanços, a inserção das mulheres no mundo da ciência ainda enfrenta obstáculos. Gilda Olinto8 chama a atenção para o fato de que se mantêm as diferenças entre homens e mulheres no que diz respeito à sua inclusão nos diversos campos profissionais e no campo científico. Exemplo disso é a Academia Brasileira de Ciências, entidade que congrega os mais eminentes cientistas do Brasil nos campos das Ciências Matemáticas, Físicas, Químicas, da Terra, Biológicas, Biomédicas, da Saúde, Agrárias, da Engenharia e Sociais: de seus 896 membros afiliados, apenas 117 são mulheres.9 9 ABC, 2014. 9 ABC, 2014. Diante dessa realidade, decidiu-se desenvolver uma pesquisa que investigasse a presença feminina nas ciências no Brasil, procurando identificar, principalmente, suas áreas de interesse de pesquisa. Para tal, foi feito um mapeamento sobre a participação feminina nas áreas da ciência e tecnologia, a partir da análise dos currículos Lattes de mulheres que defenderam suas teses de doutorado entre os anos de 2000 e 2013. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO No caso dos homens, o aumento das matrículas foi de 67,9%; - No nível de doutorado: o aumento do número de matrículas femininas foi de 104% e as masculinas totalizaram 69,2%. Nos anos seguintes, essa tendência se manteve, como demonstram os dados do censo da educação superior, realizado em 2010 pelo Inep. De acordo com esse censo, as matrículas femininas foram majoritárias no período de 2001 a 2010, atingindo 57,0% das matrículas e, ao considerar os alunos que terminam os cursos, a participação feminina é de 60,9%.6 6 INEP, 2011. Assim, pela educação, as mulheres têm se posicionado na sociedade. Elas já superaram a desvantagem que tinham na área educacional em relação aos homens, mostrando 13 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 14 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL teoria psicanalítica se difundiu no pensamento popular francês. Para esse autor, as representações coletivas de Durkheim, que estão relacionadas a conceitos, resumo e categorias que são produzidas e que, coletivamente, formam toda a cultura de uma sociedade, incluíam excessos de formas intelectuais. Sendo assim, como explica Moscovici,10 “qualquer crença, ideia ou emoção presente na sociedade era considerada uma representação”. 10 MOSCOVICI, 2003. 11 SÁ, 2002. De acordo com Celso Sá,11 na perspectiva de Moscovici, as representações deveriam ser reduzidas às formas de conhecimento da vida cotidiana, com a função de possibilitar a comunicação entre os sujeitos e a orientar o comportamento. Durkheim tinha uma concepção estática das representações, mas, na sociedade atual, o que interessa a Moscovici,12 as representações possuem caráter plástico; logo, elas passam a ser consideradas dinâmicas. 12 MOSCOVICI, 2003. A representação do trabalho docente sempre foi alvo de muitos questionamentos, em especial o da professora, já que ela foi incumbida, historicamente, pela educação dos pequenos, o que, muitas vezes, perpassou o papel de instruir, sendo uma figura associada à educação, zelo, higiene, alimentação – uma figura que se assemelhava ao papel maternal. 13 LOURO, 1997. Conforme relatado por Guacira Louro,13 a primeira representação da professora foi a de figura severa, de poucos sorrisos, ou seja, distante dos seus alunos. Essa representação muda quando o discurso sobre o espaço escolar passa a ser associado a um ambiente prazeroso. Surge, então, a “professorinha”, termo muitas vezes hoje utilizado em tom pejorativo para designar profissionais que possuem poucos conhecimentos em sua área de atuação, já que uma concepção errônea de educação infantil se propagou por muito tempo: a de que profissionais que trabalham na educação infantil precisam apenas gostar de criança para que sejam boas profissionais, o que é uma inverdade, pois o processo educacional vai além do simples fato de se gostar ou não de crianças. Essa representação de “professorinha” aproximou a profissional um pouco mais dos alunos, apesar de ainda existir um distanciamento. À medida que as teorias psicológicas e sociológicas ganham maior representatividade no âmbito pedagógico, a professora passa a ser denominada educadora. No final dos anos 60 e início dos anos 70, com o regime militar e a tecnização do ensino, substitui-se a imagem da professora como mãe, passando ela a ser considerada uma profissional de ensino. O lugar das mulheres na arena educacional O lugar das mulheres na arena educacional O lugar das mulheres na arena educacional O lugar das mulheres na arena educacional O lugar das mulheres na arena educacional na sociedade da informação na sociedade da informação na sociedade da informação na sociedade da informação na sociedade da informação O trabalho consiste em um processo pelo qual o indivíduo transforma a natureza com determinados fins e objetivos que melhor satisfaçam às suas necessidades e, nesse processo, ao transformar a natureza, é reciprocamente transformado por ela. Ao longo da história, verificam-se diversas alterações em todos os segmentos da sociedade, seja, por exemplo, no que se refere às concepções de trabalho, do desenvolvimento das técnicas, dos processos produtivos e das relações entre os homens. As mudanças são igualmente observadas no contexto educacional, em que a representação docente também tem sido afetada, em especial no que diz respeito ao papel das mulheres. Diversos pesquisadores dedicaram-se aos estudos sobre as teorias das representações sociais. O precursor do termo foi Serge Moscovici, com seu trabalho intitulado A psicanálise: sua imagem e seu público, publicado na França, em 1961, onde o autor estudou as formas como a 14 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 12 MOSCOVICI, 2003. 13 LOURO, 1997. 17 Disponível em: http://www.uis. unesco.org. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO torna mais técnica. Esse novo discurso contrapunha-se à concepção do magistério como uma extensão das atividades maternais, de cuidado, apoio emocional, etc. Quase como uma negação à falta de afetividade, os professores e professoras começam a utilizar o termo tia para identificarem-se. Essa denominação, contudo, acaba sendo apropriada pelo discurso tecnológico, pois favorece o anonimato da professora, excluída do processo de produção do saber. A tia não tem mais o papel de pensar ou decidir sobre a educação das crianças, é uma mera executora, anônima no processo de ensino.14 Nesse contexto, surgiram diversos movimentos de lutas por melhores salários e condições de trabalho, os quais fizeram com que despertasse uma nova forma de tratamento para essas profissionais como trabalhadoras da educação. Tudo isso propiciou um novo olhar para os profissionais da educação. A partir daí, as mulheres ampliaram sua atuação em direção às ciências, como pontua Zuleica Oliveira: A busca da promoção de igualdade de oportunidades entre os homens e as mulheres nas carreiras educacio- nais na área de Ciência e Tecnologia se constitui em um dos desafios para o desenvolvimento da sociedade da informação no Brasil. A maior inserção das mulheres nas profissões que compreendem esta área torna-se uma necessidade de natureza econômica e social.15 15 OLIVEIRA, 2005, p. 3. 15 OLIVEIRA, 2005, p. 3. De acordo com essa autora, é preciso que exista uma política de informação na área de Ciência e Tecnologia que leve em conta a dimensão de gênero, para subsidiar a elaboração de políticas públicas que promovam maior participação das mulheres nessa carreira. AS MULHERES PRATICANDO CIÊNCIA NO BRASIL De acordo com Glaucia Santos, as tarefas dos professores e professoras passam a ser mais burocráticas, com atividades de ordem admi- nistrativa e de controle. Sua ação didática também se 15 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 14 SANTOS, 2000, p. 174-175. 16 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 15 OLIVEIRA, 2005, p. 3. Mulheres na ciência e tecnologia Mulheres na ciência e tecnologia Mulheres na ciência e tecnologia Mulheres na ciência e tecnologia Mulheres na ciência e tecnologia Conforme o Instituto de Pesquisas Tecnológicas (IPT), foi a partir das décadas de 60 e 70 que a participação das mulheres na Ciência e Tecnologia tornou-se mais frequente.16 O IPT aponta como uma das razões para esse aumento o movimento feminista ocorrido nessas décadas, que se tornou mais frequente. Observou-se, nesse período que mais espaços foram abertos às mulheres participarem dos processos científicos, uma vez que cresceu, também, o número de mulheres com cursos universitários. 16 IPT, 2014. Pesquisas indicam que cada vez mais aumenta essa participação, como a realizada pela United Nations Educational Scientific and Cultural Organization (UNESCO17), em 2009, a qual revelou que 29% dos pesquisadores no mundo são mulheres. Esse número aumenta quando os dados são analisados apenas na América Latina, onde as 16 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL mulheres representam 46% dos cientistas. Já, no Brasil, a participação das mulheres na ciência é ainda maior, chegando a igualar ao número dos pesquisadores mas- culinos.18 18 CNPq, 2013. De acordo com o Conselho Nacional de Desenvolvi- mento Científico e Tecnológico (CNPq), o número de cientistas do gênero feminino é praticamente o mesmo do gênero masculino.19 Dados do censo de 2010 do CNPq mostram que estavam cadastrados em sua base de dados aproxima- damente 128,6 mil pesquisadores, sendo que metade são mulheres.20 Na tabela 1 tabela 1 tabela 1 tabela 1 tabela 1, tem-se a evolução dessa participação feminina nos últimos 15 anos, a qual, segundo o CNPq, se deve à universalização da educação e ao avanço da ciência e da tecnologia nos últimos vinte anos. Porém, o relatório indica que as posições de liderança nos grupos de pesquisas ainda são ocupadas por pesquisadores masculinos; a participação feminina nesse requisito cai para 45%. Entretanto, se o critério comparativo for apenas por participação em grupos de pesquisas, o percentual de mulheres supera o de homens: 52% contra 48%, respectivamente (tabela 1).21 21 CNPq, 2013. Fonte: Fonte: Fonte: Fonte: Fonte: CNPq (2013). Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 17 25 BRUSCHINI e AMADO, 1998, p. 7. 23 Disponível em: http://www.cnpq. br/web/guest/apresentacao2. Mulheres na ciência e tecnologia Mulheres na ciência e tecnologia Mulheres na ciência e tecnologia Mulheres na ciência e tecnologia Mulheres na ciência e tecnologia TTTTTabela 1 abela 1 abela 1 abela 1 abela 1 – Distribuição percentual dos pesquisadores segundo o sexo - 1993-2010 TTTTTabela 1 abela 1 abela 1 abela 1 abela 1 – Distribuição percentual dos pesquisadores segundo o sexo - 1993-2010 Atualmente, a presidência da Sociedade Brasileira para o Progresso da Ciência (SBPC) é exercida por uma mulher: a professora Helena Nader, da Universidade Federal de São Paulo, que enfatiza o fato de as mulheres estarem cada vez mais se titulando e produzindo ciência, mas que, ao mesmo tempo, há um número pequeno que chega a posições de chefia. Nader22 fala sobre a desigualdade de gênero na academia, onde as mulheres têm evoluído bem, mas não o suficiente. A professora enfatiza que, hoje, as mulheres continuam tendo dois papéis, como profissional e dentro de casa, na criação dos filhos. 22 NADER, 2011. Vale ressaltar um exemplo de incentivo governamental para a participação das mulheres nas pesquisas científicas: é o Programa Mulher e Ciência, lançando em 2005, que teve como objetivo aumentar a participação feminina no campo das ciências e nas carreiras acadêmicas, bem como estimular a produção científica e a reflexão acerca das relações de gênero. O programa foi o resultado de trabalho 17 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 18 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 24 OCDE, 2014. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO Independente das áreas de escolha, a formação acadêmica e o ingresso nas ciências pelas mulheres são fundamentais para a diminuição das diferenças entre homens e mulheres no mercado de trabalho, como pontua Olinto: O distanciamento entre homens e mulheres na ciência é um processo que envolve diversos tipos de ganhos 18 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL que beneficiam os homens: a promoção, a obtenção de bolsas de estudo, a ocupação de cargos de chefia ou liderança, assim como os ganhos salariais.26 26 OLINTO, 2011, p. 71. 27 PASSINATO, 2008. Nesse sentido, Cristina Passinato27 chama a atenção para o fato de que, historicamente, nem sempre as mulheres tiveram as suas participações relatadas na Ciência. Para essa autora, a contribuição feminina nas ciências só pôde vir com muita luta e estudos, provas e debates. Passinato conti- nua afirmando que as mulheres que tentaram transgredir alguma regra sociocultural, econômico-educacional, aspec- to religioso-político em alguma época ou cenário no passado tornaram-se verdadeiras afrontas perante a hegemônica presença masculina nessa área. Reforçando o papel que as mulheres sempre trouxeram para elas, como o de educadoras, Passinato28 considera que a presença feminina sempre foi mais destacada na formação das pessoas, atuando ativamente como professoras, e, prossegue, afirmando que, na Ciência, isso não foi diferente, verificando-se um constante paralelo. Na ótica da OECD, a participação das mulheres na ciência e pesquisa não é apenas uma questão de gênero, mas, também, uma questão econômica: “Deixar as mulheres para trás significa não somente desprezar as importantes contribuições que as mulheres trazem para a economia, mas também desperdiçar anos de investimento em educação de meninas e jovens mulheres”.29 É a persistência desse distanciamento que levou à realização desta pesquisa, cuja metodologia é apresentada na próxima seção. 28 PASSINATO, 2008. 29 2012, p. 2. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO realizado por um grupo interministerial, do qual fazem parte a Secretaria Especial de Políticas para as Mulheres (SPM), o Ministério da Ciência e Tecnologia (MCT), o CNPq e o MEC, dentre outros.23 No que diz respeito à escolha de carreira, um estudo divulgado pela Organização para a Cooperação e Desen- volvimento Econômico (OCDE), em 2014, baseado em dados da edição de 2012 do Programa Internacional de Avaliação de Alunos (Pisa), mostrou que as meninas brasileiras pesqui- sadas mencionaram, em proporções muito maiores do que os meninos, áreas que já são normalmente associadas às mulheres, como a saúde e ciências sociais. Apenas 38% das meninas planejam seguir carreira que envolva matemática, sendo que isso faz parte dos planos de 53% dos meninos.24 24 OCDE, 2014. O afastamento das meninas nas carreiras científicas ditas como duras pode estar associado à edificação social do gênero. O que os homens devem ser e saber fazer social- mente foi construído histórica e socialmente de forma dico- tômica. Às mulheres, na mesma medida, foram associadas características como delicadeza, zelo, afetividade. Características essas associadas quase que exclusivamente às funções do magistério. Um dos discursos que contribui para reforçar a concepção de feminização do magistério é marcado historicamente pela ideia de vocação da mulher para o desenvolvimento da prática. Assim, de acordo com Cristina Bruschini e Tina Amado, historicamente, o conceito de vocação foi aceito e expresso pelos próprios educadores e educadoras, que argumentavam que, como a escolha da carreira devia ser adequada à natureza feminina, atividades requerendo sentimento, dedicação, minúcia e paciência deveriam ser preferidas. Ligado à ideia de que as pessoas têm aptidões e tendências inatas para certas ocupações, o conceito de vocação foi um dos mecanismos mais eficientes para induzir as mulheres a escolher as profissões menos valorizadas socialmente.25 Tratam-se, pois, de características historicamente dadas como opostas às masculinas, marcadas pelo senso de disputa, racionalidade, objetividade e força. Enfim, características essas que excluem e estigmatizam a mulher no cenário científico. Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 19 31 VERGARA, 2009. 28 PASSINATO, 2008. Resultados e análises Resultados e análises Resultados e análises Resultados e análises Resultados e análises Os dados coletados foram analisados apenas por meio da utilização do número de ocorrências. Caminhos da pesquisa Caminhos da pesquisa Caminhos da pesquisa Caminhos da pesquisa Caminhos da pesquisa A pesquisa aqui relatada teve caráter descritivo. Segundo Antônio Gil,30 esse tipo de pesquisa visa descrever as características de determinadas populações ou fenômenos. No caso desta pesquisa, buscou-se descrever as características de um fenômeno contemporâneo, que é a participação feminina no desenvolvimento de pesquisas no Brasil. Adotou-se, nesse trabalho, a pesquisa documental. Segundo Sylvia Vergara,31 a pesquisa documental é um estudo feito em documentos encontrados em órgãos públicos ou privados, que, no caso da pesquisa aqui descrita, tratou- se da base de dados da Plataforma Lattes, do CNPq. De acordo com os dados dessa Plataforma, de julho de 2014, existem, ali cadastrados, 84.502 currículos Lattes de mulheres doutoras que constituíram o universo desta pesquisa. Ressalta-se que esse número é a soma de doutoras que atuam em dois tipos de atividade: Pesquisa e Ensino; e Administrativas, Técnicas e outras. 31 VERGARA, 2009. 19 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO Para definição do tamanho da amostra, utilizou-se a técnica de amostragem aleatória simples, cuja premissa básica é de que cada elemento da população estudada tem a mesma chance de ser escolhido para compor a amostra, de acordo com Naresh Malhotra.32 Então, foi utilizada a margem de erro, também chamada por erro amostral, que é uma estatística que representa a quantidade de erro da amostragem aleatória em um resultado de pesquisa. Quanto menor a margem de erro, maior a confiança nos resultados, ou seja, a amostra trabalhada corresponde aos valares para a população inteira, sendo que o erro amostral pode ser definido para qualquer nível de confiança desejado, como, por exemplo, 90%, 95% ou 99%. Nessa pesquisa foi considerado um erro amostral de 2%, que, conforme Malhotra,33 corresponde a um nível de confiança de 99%. Dessa forma, a necessidade da amostra aqui estudada é de pelo menos 3.953 currículos a serem analisados, o que corresponde a 4,68% do universo. 33 MALHOTRA, 2011. 33 MALHOTRA, 2011. Como critério de delimitação da amostra, definiu-se um corte temporal nos dados, sendo considerados os currículos Lattes de mulheres que defenderam suas teses de doutorado entre os anos de 2000 e 2013. A consulta à Plataforma Lattes iniciou em dezembro de 2013 e se estendeu até julho de 2014..... Foram analisados 4.970 currículos, escolhidos aleato- riamente, o que corresponde a 5,88% do universo, superando, portanto, a amostra mínima necessária. Para cada um dos currículos analisados, foram levantadas as seguintes variáveis: - Ano de defesa da tese; - Ano de defesa da tese; - Realização de pós-doutorado (encerrado ou em curso); - Região geográfica em que a tese foi defendida; Á - Áreas e grandes áreas do conhecimento em que as pesquisas de doutorado foram desenvolvidas; - Dependências administrativas onde essas pesquisas foram desenvolvidas; - Agências financiadoras das pesquisas realizadas; - Tipo de carreira profissional da pesquisadora – variável foi dividida em duas categorias, docentes e não docentes, subdivididas em: servidoras públicas e celetistas. Ano de defesa da tese Ano de defesa da tese Ano de defesa da tese Ano de defesa da tese Ano de defesa da tese Verificou-se que o interesse das mulheres em pesquisas e formação acadêmica tem sido de grande relevância, visto 20 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL que é visível o crescimento dessas publicações, princi- palmente por meio da curva de evolução, como mostra o gráfico 1 gráfico 1 gráfico 1 gráfico 1 gráfico 1. Destaca-se que, no período entre 2009 e 2011, ou seja, em dois anos, houve um aumento significativo no número de teses publicadas. A partir deste levantamento pode-se fazer a inferência de que este número tende a aumentar ainda mais, considerando seu crescimento a cada ano. que é visível o crescimento dessas publicações, princi- palmente por meio da curva de evolução, como mostra o gráfico 1 gráfico 1 gráfico 1 gráfico 1 gráfico 1. Destaca-se que, no período entre 2009 e 2011, ou seja, em dois anos, houve um aumento significativo no número de teses publicadas. A partir deste levantamento pode-se fazer a inferência de que este número tende a aumentar ainda mais, considerando seu crescimento a cada ano. Fonte: Fonte: Fonte: Fonte: Fonte: Dados da pesquisa. Gráfico 1 Gráfico 1 Gráfico 1 Gráfico 1 Gráfico 1 – Número de teses publicadas anualmente por mulheres no período de 200 2013 Gráfico 1 Gráfico 1 Gráfico 1 Gráfico 1 Gráfico 1 – Número de teses publicadas anualmente por mulheres no período de 2000 a 2013 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 Região geográfica em que a tese foi Região geográfica em que a tese foi Região geográfica em que a tese foi Região geográfica em que a tese foi Região geográfica em que a tese foi defendida defendida defendida defendida defendida Das 4.970 teses defendidas por mulheres no período pesquisado, 4.835 foram desenvolvidas em instituições brasileiras e 135 no exterior. Das teses desenvolvidas no Brasil, no período de 2000 a 2013, 64,34% foram defendidas no Sudeste do país, representando 3.111 defesas de tese. A região Sudeste é seguida pelas regiões Sul, com 18,97%; Nordeste, com 9,08%; Centro-Oeste, com 6,64%, e, finalmente, a região Norte, com 0,97% das teses defendidas no período. O expressivo número de teses defendidas na região Sudeste deve-se, principalmente, às universidades que oferecem cursos de doutorado em São Paulo, pois 36,40% do total das teses defendidas no Brasil foram desenvolvidas nesse estado. Na mesma região, encontram-se, ainda, os estados do Rio de Janeiro, com 14,00%, e de Minas Gerais, com 13,65% do total das teses defendidas no Brasil. Considerando as 135 mulheres que fizeram e defen- deram suas teses no exterior, a tabela 2 tabela 2 tabela 2 tabela 2 tabela 2 relaciona os países onde as teses foram desenvolvidas e defendidas, desta- cando-se a Espanha como o país onde mais brasileiras (38) foram fazer pesquisa, seguida pelos Estados Unidos (25) e pela França (15). Os motivos para essa escolha podem ser vários – desde o pessoal, como melhorar a fluência em uma língua estrangeira e ampliar a network profissional, ao interesse acadêmico, como a troca de experiências em fazer pesquisas com métodos e culturas diferentes. Outro dado encontrado na pesquisa foi a opção pela realização do doutorado sanduíche, que é um programa de bolsa de estudo no qual a pesquisadora fez parte do seu curso de doutorado em uma instituição internacional, alternativa, adotado por 84 das doutoras pesquisadas. Estão registrados, também na tabela 2, os países onde essas pesquisadoras fizeram parte de suas pesquisas. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO na pesquisa, pois a maioria das doutoras que estão fazendo o pós-doutorado ainda não tem vínculo de trabalho. Realização de pós-doutorado (encerrado Realização de pós-doutorado (encerrado Realização de pós-doutorado (encerrado Realização de pós-doutorado (encerrado Realização de pós-doutorado (encerrado ou em curso) ou em curso) ou em curso) ou em curso) ou em curso) Dentro da amostra selecionada nessa pesquisa, verificou-se que 20% (994) das doutoras brasileiras, tituladas no intervalo de tempo analisado nessa pesquisa, fizeram o estágio de pós-doutoramento ou estão em curso dessa ativi- dade acadêmica. Percebe-se, portanto, que as mulheres cada vez mais investem em sua educação, o que corrobora os dados da educação superior apresentados em 2010 pelo Inep. Convém salientar, no entanto, que esse resultado pode estar associado ao fato de que nem sempre um pesquisador sai do doutorado com um emprego garantido. Quando isso ocorre, o pós-doutorado é uma opção, garantindo a continui- dade da doutora nas atividades de pesquisa, que, normal- mente, recebe uma bolsa: além de melhorar seu currículo, visa ao ingresso na carreira acadêmica. Isso foi observado 21 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 23 MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO Ciências Humanas, em que foram realizadas 688 pesquisas, Ciências Agrárias (657), Ciências Exatas e da Terra (509), Linguística, Letras e Artes (420) e Ciências Sociais Aplicadas, com 400 pesquisas. Em último lugar, fica a grande área das Engenharias, com apenas 230 pesquisas realizadas, indi- cando ser essa uma área de menor participação feminina, o que confirma os dados apresentados por Olinto,34 Bruschini e Amado35 ao explicar que a participação das mulheres em certas áreas, como, por exemplo, as ciências humanas e sociais, está relacionadas à vocação feminina para carreiras que exigem sentimento, dedicação, minúcia e paciência. Ciências Humanas, em que foram realizadas 688 pesquisas, Ciências Agrárias (657), Ciências Exatas e da Terra (509), Linguística, Letras e Artes (420) e Ciências Sociais Aplicadas, com 400 pesquisas. Em último lugar, fica a grande área das Engenharias, com apenas 230 pesquisas realizadas, indi- cando ser essa uma área de menor participação feminina, o que confirma os dados apresentados por Olinto,34 Bruschini e Amado35 ao explicar que a participação das mulheres em certas áreas, como, por exemplo, as ciências humanas e sociais, está relacionadas à vocação feminina para carreiras que exigem sentimento, dedicação, minúcia e paciência. 34 OLINTO, 2011. 35 BRUSCHINI e AMADO, 1998. Analisando os currículos das doutoras, percebeu-se que as pesquisas são oriundas de diversas áreas do conhecimento. Foram identificadas 87 diferentes áreas de estudo. As dez áreas que mais se sobressaíram foram: Educação (310 teses), Agronomia (293), Medicina (250), Química (247), Linguística (213), Psicologia (160), Medicina Veterinária (147), Odontologia (125), Bioquímica (118) e Microbiologia (115). Ao se analisar as áreas e grandes áreas do conheci- mento à que as doutoras declaram pertencer suas teses, verificou-se o forte caráter interdisciplinar de suas pesquisas. Neste estudo, pode ser comprovado que a interdisciplinari- dade é a representação do diálogo que se faz necessário não apenas no âmbito da teoria, mas, principalmente, no âmbito da prática para a realização de pesquisas científicas. Quanto a essa questão, foi observado que, no que se refere às grandes áreas do conhecimento, 520 teses (10,46%) pertenciam a duas ou três grandes áreas, com 53 combinações diferentes de relação entre as nove grandes áreas do conhecimento, sendo que a área Ciência da Saúde foi a que mais fez interface com as demais. Ela é seguida pelas áreas das Ciências Humanas, Engenharias e Ciências Exatas e da Terra, Ciências Sociais Aplicadas, Ciências Agrárias, Ciências Biológicas e outras. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO A área da Linguística, Letras e Artes foi a que apresentou o menor grau de interdisciplinaridade. Em relação às áreas do conhecimento, aumenta muito o diálogo nas pesquisas. Foram 394 combinações diferentes de relação entre 71 diferentes áreas do conhecimento, sendo que as duas áreas que mais dialogaram com outras foram: Engenharia Agrícola e Bioquímica. Áreas e grandes áreas do conhecimento Áreas e grandes áreas do conhecimento Áreas e grandes áreas do conhecimento Áreas e grandes áreas do conhecimento Áreas e grandes áreas do conhecimento em que as pesquisas de doutorado foram em que as pesquisas de doutorado foram em que as pesquisas de doutorado foram em que as pesquisas de doutorado foram em que as pesquisas de doutorado foram desenvolvidas desenvolvidas desenvolvidas desenvolvidas desenvolvidas Verificando-se as grandes áreas do conhecimento em que as pesquisas de doutorado foram desenvolvidas pelas mulheres tituladas no período considerado (tabela 3 tabela 3 tabela 3 tabela 3 tabela 3), o predomínio é das Ciências Biológicas, com 845 pesquisas, e das Ciências da Saúde, com 838. A seguir, encontram-se as 22 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL TTTTTabela 2 abela 2 abela 2 abela 2 abela 2 – Países onde as pesquisas foram desenvolvidas, no período de 2000 a 2013 Fonte: Fonte: Fonte: Fonte: Fonte: Dados da pesquisa. TTTTTabela 3 abela 3 abela 3 abela 3 abela 3 – Número de pesquisas por grande área do conhecimento Fonte: Fonte: Fonte: Fonte: Fonte: Dados da pesquisa. TTTTTabela 2 abela 2 abela 2 abela 2 abela 2 – Países onde as pesquisas foram desenvolvidas, no período de 2000 a 2013 Fonte: Fonte: Fonte: Fonte: Fonte: Dados da pesquisa. TTTTTabela 2 abela 2 abela 2 abela 2 abela 2 – Países onde as pesquisas foram desenvolvidas, no período de 2000 a 2013 TTTTTabela 3 abela 3 abela 3 abela 3 abela 3 – Número de pesquisas por grande área do conhecimento Fonte: Fonte: Fonte: Fonte: Fonte: Dados da pesquisa. TTTTTabela 3 abela 3 abela 3 abela 3 abela 3 – Número de pesquisas por grande área do conhecimento Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 23 34 OLINTO, 2011. 35 BRUSCHINI e AMADO, 1998. 24 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 Dependências administrativas onde as Dependências administrativas onde as Dependências administrativas onde as Dependências administrativas onde as Dependências administrativas onde as pesquisas foram desenvolvidas pesquisas foram desenvolvidas pesquisas foram desenvolvidas pesquisas foram desenvolvidas pesquisas foram desenvolvidas O levantamento das instituições brasileiras em que as doutoras defenderam suas teses mostrou que mais de 90% dos trabalhos de doutorado se efetivaram em institui- 24 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL ções públicas, sendo que foram 2.618 teses defendidas em instituições federais, 1.814 em instituições estaduais e apenas 403 em instituições particulares. ções públicas, sendo que foram 2.618 teses defendidas em instituições federais, 1.814 em instituições estaduais e apenas 403 em instituições particulares. Agências financiadoras das pesquisas Agências financiadoras das pesquisas Agências financiadoras das pesquisas Agências financiadoras das pesquisas Agências financiadoras das pesquisas realizadas realizadas realizadas realizadas realizadas No que diz respeito ao financiamento das pesquisas no período considerado, a Capes se destaca como a agência que financiou o maior número de pesquisas (1.371), seguida pelo CNPq, com 1.166, e pelas fundações de amparo à pesquisa dos Estados, que financiaram a produção de 698 pesquisas. Um número pequeno (44) foi financiado pelas próprias instituições onde o doutorado foi realizado, e 270 receberam financiamento de outras agências. Nesse quesito, chama a atenção o alto número de pesquisas (1.421) que não tiveram financiamento algum, mostrando que, além de realizar todo o trabalho inerente ao desenvolvimento de uma pesquisa, essas mulheres tiveram, também, que arcar com os custos decorrentes dessa atividade. Esses investimentos em suas carreiras não necessariamente garantem um retorno financeiro e/ou plano de carreira. Fator este que dependerá da instituição na qual estejam trabalhando. Cabe, nesse contexto, uma reflexão para a falta de financiamento aos pesquisadores brasileiros, independente do gênero. De acordo com Nader,36 a falta de financiamento é o principal entrave da ciência brasileira nos dias atuais. Ela destaca que o Brasil investe apenas 1,1% do seu Produto Interno Bruto (PIB) em pesquisas, enquanto a China investe mais de 3%. 36 NADER, 2014. 36 NADER, 2014. Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 36 NADER, 2014. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO número se justifica pela exigência cada vez maior de titulação por parte das instituições de ensino e assinala, ainda, um retorno social muito expressivo, já que a maioria dessas pesquisadoras dedica-se à Educação. Dessa forma, os conhecimentos adquiridos ao longo da formação do doutorado poderão vir a ser disseminados entre os estudantes e, consequentemente, difundidos na sociedade. Fonte: Fonte: Fonte: Fonte: Fonte: Dados da pesquisa. Obs: A categoria das que não informaram inclui: monitoras, bolsistas, voluntárias, apenas estudando, sem nenhuma dessas categorias ou não informou nada. TTTTTabela 4 abela 4 abela 4 abela 4 abela 4 – Tipo de carreira profissional das doutoras pesquisadas TTTTTabela 4 abela 4 abela 4 abela 4 abela 4 – Tipo de carreira profissional das doutoras pesquisadas TTTTTabela 4 abela 4 abela 4 abela 4 abela 4 – Tipo de carreira profissional das doutoras pesquisadas Fonte: Fonte: Fonte: Fonte: Fonte: Dados da pesquisa. Obs: A categoria das que não informaram inclui: monitoras, bolsistas, voluntárias, apenas estudando, sem nenhuma dessas categorias ou não informou nada. Fonte: Fonte: o e o e Fonte: Dados da pesquisa. Obs: A categoria das que não informaram inclui: monitoras, bolsistas, voluntárias, apenas estudando, sem nenhuma dessas categorias ou não informou nada. Dentre as 1.267 doutoras que não exercem a função de docência, a maioria (843) é de servidoras públicas, o que pode ser explicado pelo incentivo à titulação, geralmente oferecido pelos diversos níveis de governo. As 425 doutoras restantes têm atividade profissional desenvolvida em empresas privadas, o que mostra que, mesmo assim, há pouca inserção de doutoras nas empresas. Os resultados apresentados nessa pesquisa indicam que as mulheres têm praticado cada vez mais ciências no Brasil, e nas mais diversas áreas do conhecimento, inclusive nas áreas tradicionalmente ocupadas pelos homens, como: engenharias, agronomia, medicina e medicina veterinária. Isso representa um avanço das mulheres em nossa sociedade. Cabe observar que a maioria das pesquisadoras são professoras. A razão desse fato pode ser a necessidade da formação acadêmica para a profissão docente, pois o doutorado significa manutenção do emprego, aumento salarial e prestígio acadêmico. O número de pesquisadores que se encontram no mercado de trabalho, fora das instituições de ensino, contudo, ainda não é expressivo. Vale ressaltar que os resultados corroboram a afirmação de Passinato,37 que declara que a presença feminina sempre foi mais destacada na formação das pessoas, por meio da Educação, até mesmo nas suas atuações nas Ciências. Tipo de carreira profissional da Tipo de carreira profissional da Tipo de carreira profissional da Tipo de carreira profissional da Tipo de carreira profissional da pesquisadora pesquisadora pesquisadora pesquisadora pesquisadora Essa variável foi dividida em duas categorias, docen- tes e não docentes, sendo essas duas categorias subdivi- didas em outras duas: - Servidoras públicas: nessa pesquisa foram consideradas servidoras públicas as profissionais que são concursadas em instituições públicas federais, estaduais e municipais, bem como as profissionais que atuam em instituições públicas, mas com contrato em regime celetista; ç g - Não servidoras: são as profissionais celetistas pertencentes às empresas privadas e as profissionais liberais. ç g - Não servidoras: são as profissionais celetistas pertencentes às empresas privadas e as profissionais liberais. Os resultados estão consolidados na tabela 4 tabela 4 tabela 4 tabela 4 tabela 4, que mostra que a maioria das doutoras (2.140) é constituída por professoras servidoras públicas, seguidas por professoras não servidoras (1.147), totalizando 3.287 docentes. Esse 25 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 37 PASSINATO, 2008. 26 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 37 PASSINATO, 2008. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO Percebeu-se, também, o incentivo que o governo federal e as fundações de amparo à pesquisa dos diversos 26 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL Estados têm proporcionado às pesquisadas, sob forma de financiamento dos projetos e distribuição de bolsas de pesquisa, destacando-se a Capes e o CNPq. 38 2012. Portanto, confirma-se a visão da OECD,38 de que é preciso incentivar a participação das mulheres na ciência e pesquisa, até mesmo por uma questão econômica e para não desperdiçar os anos de investimento feitos na educação dessas mulheres. Considerações finais Considerações finais Considerações finais Considerações finais Considerações finais Apesar de todas as transformações pelas quais passou a sociedade brasileira e de ações voltadas para o empoderamento das mulheres, elas ainda enfrentam obstáculos para se inserirem no mundo da ciência. A pesquisa relatada neste artigo investigou o perfil da participação de mulheres na realização de pesquisas, a partir de dados coletados nos currículos Lattes de 4.970 mulheres que defenderam suas teses de doutorado entre os anos de 2000 e 2013. Os resultados mostram um crescimento constante, no grupo e no período pesquisado, no número de mulheres que concluem o doutorado por ano, sendo que uma parcela significativa (20%) delas fez ou está fazendo pós-doutorado, o que mostra que as mulheres cada vez mais investem em sua educação. Entretanto, considerando as grandes áreas do conhe- cimento em que as mulheres pesquisadas realizaram seu doutorado, observa-se uma participação feminina maior nas áreas das Ciências Biológicas, das Ciências da Saúde e das Ciências Humanas, sendo que a menor participação se dá nas Engenharias. A expressiva maioria das doutoras pes- quisadas atua na docência, carreira tradicionalmente ligada às mulheres. Isso comprova que, apesar dos avanços alcan- çados pelas mulheres, ainda persiste a desigualdade de papéis entre mulheres e homens dentro da ciência. Por isso, é um dos desafios da sociedade atual a busca da promoção de igualdade de oportunidade entre os gêneros nas carreiras educacionais na área de Ciências e Tecnologia, como apontado por Oliveira.39 Espera-se que esta pesquisa contribua para uma melhor compreensão da participação das mulheres no mundo da ciência, pois é essa compreensão que pode levar à adoção de medidas para aumentar tal participação. 39 OLIVEIRA, 2005. 39 OLIVEIRA, 2005. 39 OLIVEIRA, 2005. Referências Referências Referências Referências Referências ACADEMIA BRASILEIRA DE CIÊNCIAS – ABC. Sítio institucional. Disponível em: <http://www.abc.org.br>. Acesso em: 08 set. 2014. 27 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO BRASIL. Presidência da República. Secretaria de Políticas para as Mulheres. Plano Nacional de Políticas para as Mulheres. Brasília: Secretaria de Políticas para as Mulheres, 2013. 114p. BRUSCHINI, Cristina; AMADO, Tina. “Estudos sobre mulher e educação”. Cadernos de Pesquisa. São Paulo, n. 64, p. 4-13, fev., 1988. CENTRO DE GESTÃO E ESTUDOS ESTRATÉGICOS EM CIÊNCIA - CGEE. Doutores 2010: estudos da demografia da base técnico-científica brasileira. Brasília-DF: CGEE, 2010. Disponível em: <http://www.cgee.org.br/busca/ ConsultaProdutoNcomTopo.php?f=1&idProduto=6401>. Acesso em: 29 jan. 2014. CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPq. Número de mulheres cientistas já iguala o de homens, 06 mar. 2013. Disponível em: <http://www.cnpq.br/web/guest/noticiasviews/-/ journal_content/56_INSTANCE_a6MO/10157/905361>. Acesso em: 02 jul. 2014. GIL, Antônio Carlos. Como elaborar projetos de pesquisa. São Paulo: Atlas, 2009. 184p. GROSSI, Márcia Gorett Ribeiro. Estudo das características de Software e implementação de um software livre para o sistema de gerenciamento de bibliotecas universitárias federais brasileiras. Tese (Doutorado em Ciência da Informação) - Escola Ciência da Informação. Universidade Federal de Minas Gerais, Belo Horizonte, 2008. 253f. INSTITUTO DE PESQUISAS TECNOLÓGICAS - IPT. A mulher na ciência e na Tecnologia. Disponível em: <http://www.ipt. br/institucional/campanhas/8-a_mulher_na_ciencia_e_ tecnologia.htm>. Acesso em: 14 jul. 2014. g INSTITUTO NACIONAL DE ESTUDOS E PESQUISAS EDUCACIONAIS ANÍSIO TEIXEIRA – INEP, 2011. Censo da Educação Superior 2010. 2011. Disponível em: <http://download.inep.gov.br/ educacao_superior/censo_superior/documentos/2010/ divulgacao_censo_2010.pdf>. Acesso em: 31 jan. 2014. LOURO, Guacira Lopes. Mulheres na sala de aula. In: DEL PRIORE, Mary. História das mulheres no Brasil. São Paulo: Contexto/UNESP, 1997, p. 443-481. MALHOTRA, Naresh K. Pesquisa de Marketing: foco na decisão. São Paulo: Pearson Prentice Hall, 2011. 492 p. MOSCOVICI, Serge. A representação social da psicanálise::::: investigações em psicologia social. Petrópolis: Vozes, 2003. NADER, Helena. Helena Nader é reeleita presidente da SBPC. 2011. Disponível em: <http://www.sbpcnet.org.br/site/ noticias/materias/detalhe.php?id=1866>. Acesso em: 20 jul. 2015. NADER, Helena. Helena Nader é reeleita presidente da SBPC. 2011. Disponível em: <http://www.sbpcnet.org.br/site/ noticias/materias/detalhe.php?id=1866>. Acesso em: 20 jul. 2015. 28 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 AS MULHERES PRATICANDO CIÊNCIA NO BRASIL AS MULHERES PRATICANDO CIÊNCIA NO BRASIL ______. Financiamento ainda é o principal desafio da ciência no Brasil, 2014. Disponível em: <http://agenciabrasil.ebc. com.br/pesquisa-e-inovacao/noticia/2014-07/ financiamento-ainda-e-o-principal-desafio-da-ciencia- no-brasil>. Acesso em: 20 jul. 2015. ______. Financiamento ainda é o principal desafio da ciência no Brasil, 2014. Disponível em: <http://agenciabrasil.ebc. com.br/pesquisa-e-inovacao/noticia/2014-07/ financiamento-ainda-e-o-principal-desafio-da-ciencia- no-brasil>. Acesso em: 20 jul. 2015. OLINTO, Gilda. “A inclusão das mulheres nas carreiras de ciência e tecnologia no Brasil”. Inclusão Social. Brasília, DF, v. 5, n. 1, p. 68-77, jul./dez., 2011. MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO Disponível em: <revista.ibict.br/inclusao/index.php/inclusao/article/ view/240/208>. Acesso em: 07 set. 2014. OLIVEIRA, Zuleica Lopes Cavalcanti de Oliveira. Pensando as estatísticas públicas sobre carreiras educacionais na área de ciência e tecnologia, por gênero. In: ENCONTRO NACIONAL DE PESQUISA EM CIÊNCIA DA INFORMAÇÃO, 6, 2005, Florianópolis. Anais... Florianópolis: Escola de Ciência da Informação, 2005. Disponível em: <http:// www.uff.br/ppgci/editais/zuleicaleituras.pdf>. Acesso em: 10 mar. 2014. ORGANIZAÇÃO PARA COOPERAÇÃO E DESENVOLVIMENTO ECONÔMICO – OCDE. Gender equality in education, employment and entrepreneurship: final report to the MCM, 2012. Disponível em: <http://www.ocde.org/ education/48111145.pdf>. Acesso em: 29 set.2014. Ã Ã ORGANIZAÇÃO PARA COOPERAÇÃO E DESENVOLVIMENTO ECONÔMICO – OCDE. Are boys and girls equally prepared for life?, 2012. Disponível em: <http:// www.oecd.org/pisa/pisaproducts/PIF-2014-gender- international-version.pdf>. Acesso em: 29 set. 2014. PASSINATO, Cristiana de Barcellos. Atuação da mulher na ciên- cia, 2008. Disponível em: <http://http://www.ebah.com.br/ content/ABAAAAjX0AE/atuacao-mulher-na-ciencia#>. Acesso em: 20 jul. 2015. RISTOFF, Dilvo. “A trajetória da mulher na educação brasilei- ra”. Folha de São Paulo. São Paulo, 08 mar. 2006. Caderno Opinião. Disponível em: <http://www1.folha.uol.com.br/ fsp/opiniao/fz0803200610.htm>. Acesso em: 09 fev. 2014. ROSEMBERG, Fulvia; MADSEN, Nina. “Educação formal, mulheres e gênero no Brasil contemporâneo”. In: BARSTED, Leila Linhares e PITANGUY, Jacqueline (Orgs.). O Progresso das Mulheres no Brasil 2003-2010. Rio de Janeiro: CEPIA; Brasília: ONU Mulheres, 2011, p. 390-434. Disponível em: <http://www.cepia.org.br/progresso.pdf>. Acesso em: 09 fev. 2014. SÁ, Celso Pereira de. Núcleo central das representações sociais. Petrópolis: Vozes, 2002. SANTOS, Glaucia Romualdo dos. “Mulher negra e magistério primário: a construção da identidade racial pela repre- sentação do outro”. Educação em Revista. Belo Horizonte, n. esp., p. 169-185, set. 2000. Disponível em: <http://educa. SANTOS, Glaucia Romualdo dos. “Mulher negra e magistério primário: a construção da identidade racial pela repre- sentação do outro”. Educação em Revista. Belo Horizonte, n. esp., p. 169-185, set. 2000. Disponível em: <http://educa. 29 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 30 Estudos Feministas, Florianópolis, 24(1): 11-30, janeiro-abril/2016 MÁRCIA G. R. GROSSI, SHIRLEY D. BERNARDES, ALINE M. LOPES E ALEIXINA MARIA L. ANDALÉCIO fcc.org.br/pdf/edur/nnumeroespecial/nnumeroespeciala 09.pdf>. Acesso em: 10 mar. 2014. fcc.org.br/pdf/edur/nnumeroespecial/nnumeroespeciala 09.pdf>. Acesso em: 10 mar. 2014. TAVARES, Rebecca Reichmann. “Igualdade de gênero e empoderamento das mulheres”. In: BARSTED, Leila Linhares; PITANGUY, Jacqueline (Orgs.). O Progresso das Mulheres no Brasil 2003-2010. Rio de Janeiro: CEPIA; Brasília: ONU Mulheres, 2011. Disponível em: <http://www.cepia.org.br/ progresso.pdf> Acesso em: 09 fev. 2014. p g p TRIBUNAL SUPERIOR ELEITORAL. Eleições 2014: eleitorado feminino cresce 5,81% em 4 anos. Disponível em: <http:/ /www.tse.jus.br/noticias-tse/2014/Julho/eleicoes-2014- eleitorado-feminino-cresce-5-81-em-quatro-anos>. Acesso em: 04 nov. 2014. g TRIBUNAL SUPERIOR ELEITORAL. Eleições 2014: eleitorado feminino cresce 5,81% em 4 anos. Disponível em: <http:/ /www.tse.jus.br/noticias-tse/2014/Julho/eleicoes-2014- eleitorado-feminino-cresce-5-81-em-quatro-anos>. Acesso em: 04 nov. 2014. g TRIBUNAL SUPERIOR ELEITORAL. Eleições 2014: eleitorado feminino cresce 5,81% em 4 anos. Disponível em: <http:/ /www.tse.jus.br/noticias-tse/2014/Julho/eleicoes-2014- eleitorado-feminino-cresce-5-81-em-quatro-anos>. Acesso em: 04 nov. 2014. VERGARA, Sylvia Constant. Projetos e relatórios de pesquisa em administração. 9.ed. São Paulo: Atlas, 2009. 92p. VERGARA, Sylvia Constant. Projetos e relatórios de pesquisa em administração. 9.ed. São Paulo: Atlas, 2009. 92p. [Recebido em 7 de novembro de 2014 e aceito para publicação em 14 de setembro de 2015] [Recebido em 7 de novembro de 2014 e aceito para publicação em 14 de setembro de 2015] [Recebido em 7 de novembro de 2014 e aceito para publicação em 14 de setembro de 2015] The Women Practicing Science in Brazil The Women Practicing Science in Brazil The Women Practicing Science in Brazil The Women Practicing Science in Brazil The Women Practicing Science in Brazil The Women Practicing Science in Brazil The Women Practicing Science in Brazil The Women Practicing Science in Brazil The Women Practicing Science in Brazil The Women Practicing Science in Brazil Abstract Abstract Abstract Abstract Abstract: The woman participation in different areas of society has been increasing, but she still faces obstacles, including her integration in the world of science. Seeking a better understanding of this phenomenon, it was carried out a survey that mapped the female participation in research development in Brazil, from the analysis of the Lattes curricula of 4,970 women who presented their doctoral dissertations between 2000 and 2013. In regarding to technical procedures, it was used the documentary research, which data collection took place from December 2013 to July 2014. The results show that women have made many advances in this area, but that inequality of roles between women and men persists within the science. K d K d K d K d K d W Ed ti S i R h D t t
https://openalex.org/W2096982239	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/1205DBFA100E03288141D3F80F192CD6/S0021875814001844a.pdf/div-class-title-a-jewish-american-monster-stanley-kubrick-anti-semitism-and-span-class-italic-lolita-span-1962-div.pdf	English	null	A Jewish American Monster: Stanley Kubrick, Anti-Semitism and<i>Lolita</i>(1962)	Journal of American studies	2,014	cc-by	9,078	Journal of American Studies, (), , – © C b d P d B h A Journal of American Studies, (), , – © C b d P d B h A Journal of American Studies, (), , – © Cambridge University Press and British Association for American Studies . This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/./), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:./S First published online November  Journal of American Studies, (), ,  © Cambridge University Press and British Association for American Studies . This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/./), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:./S First published online November  SCSM, Bangor University. Email: n.abrams@bangor.ac.uk. Frederic Raphael, Eyes Wide Open: A Memoir of Stanley Kubrick (London: Orion, ), ; see also Geoﬀrey Cocks, “Indirected by Stanley Kubrick,” Post Script, , (), –. A Jewish American Monster: Stanley Kubrick, Anti-Semitism and Lolita () NATHAN ABRAMS This article presents a case study of the ﬁlmmaker Stanley Kubrick, considering how his ﬁlms can be considered an emotional response to the Holocaust, the legacy of European anti- Semitism, and stereotypes of the Jewish American woman. It will argue that there are various clues in Kubrick’s ﬁlms which produce Jewish moments; that is, where, through a com- plementary directing and acting strategy, in particular one of misdirection, the viewer is given the possibility of “reading Jewish,” albeit not with certainty, for Jewishness is “textually sub- merged.” Its focus is Kubrick’s adaptation of Vladimir Nabokov’s Lolita (), in par- ticular the character of Charlotte Haze, played by Shelley Winters, especially in light of Kubrick’s choice of casting for the role, and Winters’s subsequent performance of it. It will conclude that Holocaust and anti-Semitic stereotypes/reverse stereotypes haunt Kubrick’s version of Lolita as an emotional, yet sub-epidermis, presence. INTRODUCTION Filmmaker Stanley Kubrick was rarely thought of as a Jewish director who made Jewish ﬁlms (however that may be deﬁned). Yet, born in , and growing up as the Holocaust was taking place in Europe, the awareness of the inescapability of his Central European Jewish heritage arguably had a sig- niﬁcant emotional impact upon him. Although Kubrick said very little about the Holocaust, its presence is felt in his ﬁlms, but it is approached obliquely, often via analogies and metaphors, sometimes by overt, albeit brief, moments which explore the very same issues raised by the Shoah. Frederic Raphael, who collaborated with Kubrick on the screenplay for his ﬁnal ﬁlm, Eyes Wide Shut (), suggested, “S. K. proceeds by indirection . . . [his] work could be viewed, as responding, in various ways, to the unspeakable (what lies beyond spoken explanation).”And John Orr and Elżbieta Ostrowska have pointed out, “Kubrick, who never realised his Holocaust ﬁlm project, nonetheless had a https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press Nathan Abrams  post-Holocaust vision of the contemporary world.”This may well have been ampliﬁed by his third marriage, in , to Christiane Harlan, the niece of Veit Harlan, who had directed the notoriously anti-Semitic propaganda ﬁlm, Jud Süss in . Kubrick had met Harlan in and wanted to make a ﬁlm about him, and Kubrick therefore was surely sensitive to the impact on the Harlan family of Harlan’s decision to work so closely with the Nazi leadership. How this post-Holocaust sensibility operated in Kubrick’s ﬁlms will be explored via a detailed case study of a key character in one of his ﬁlms, namely Charlotte Haze, played by Shelley Winters, in his adaptation of Vladimir Nabokov’s Lolita ().She has been chosen because in casting and performance Winters’s real-life Jewishness and her performance of Haze’s onscreen persona provide a key prism through which to consider Kubrick’s own ethnicity and attitudes towards it, as well as his post-Holocaust sensibility, at a crucial stage in his career and in postwar Hollywood. It will be argued here that, if, as Daniel Anderson has suggested, “The language and the visible world of Lolita are so deeply conditioned by their post-Holocaust circumstances,” then they must have also inﬂuenced Kubrick.Consequently, the Holocaust haunts his version of Lolita as an emotional, yet submerged, presence, producing an intriguing representation of the Jewish American Mother. Jerold J. Abrams, “The Logic of Lolita: Kubrick, Nabokov, and Poe,” in Abrams (ed.), The Philosophy of Stanley Kubrick (Lexington: University of Kentucky Press, ), –; Susan L. Mizruchi, “Lolita in History,” American Literature, , (), –; Andrea Pitzer, The Secret History of Vladimir Nabokov (New York: Pegasus, ). Mizruchi, . https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press John Orr and Elżbieta Ostrowska, The Cinema of Roman Polanski: Dark Spaces of the World (London: Wallﬂower, ), . Geoﬀrey Cocks, The Wolf at the Door: Stanley Kubrick, History, and the Holocaust (New York: Peter Lang, ), . Reasons of length preclude a consideration of other characters and elements in the ﬁlm, especially those that are also integral to a post-Holocaust sensibility. This may include a reckoning with the relationship between sexuality and perversity and “Jewishness,” so central to anti-Semitism as played out through Humbert in particular. The casting of Peter Sellers was yet another interesting and signiﬁcant casting choice; Seller’s maternal Jewishness was also, I would argue, important to his selection. Furthermore, there are many possible ways and “coded clues” to reading his character/performance as Jewish. In both cases, then, there is certainly the implication that Humbert and Quilty might also be coded as Jewish, connecting their “inappropriate” sexuality to anti-Semitism. Douglas Anderson, “Nabokov’s Genocidal and Nuclear Holocausts in ‘Lolita’,” Mosaic, ,  (), –, . Jerold J. Abrams, “The Logic of Lolita: Kubrick, Nabokov, and Poe,” in Abrams (ed.), The Philosophy of Stanley Kubrick (Lexington: University of Kentucky Press, ), –; Susan L. Mizruchi, “Lolita in History,” American Literature, , (), –; Andrea Pitzer, The Secret History of Vladimir Nabokov (New York: Pegasus, ). Mizruchi, . Douglas Anderson, “Nabokov’s Genocidal and Nuclear Holocausts in ‘Lolita’,” Mosaic, ,  (), –, .  John Orr and Elżbieta Ostrowska, The Cinema of Roman Polanski: Dark Spaces of the World (London: Wallﬂower, ), . Geoﬀrey Cocks, The Wolf at the Door: Stanley Kubrick, History, and the Holocaust (New York: Peter Lang, ), . Reasons of length preclude a consideration of other characters and elements in the ﬁlm, especially those that are also integral to a post-Holocaust sensibility. This may include a reckoning with the relationship between sexuality and perversity and “Jewishness,” so central to anti-Semitism as played out through Humbert in particular. The casting of Peter Sellers was yet another interesting and signiﬁcant casting choice; Seller’s maternal Jewishness was also, I would argue, important to his selection. Furthermore, there are many possible ways and “coded clues” to reading his character/performance as Jewish. In both cases, then, there is certainly the implication that Humbert and Quilty might also be coded as Jewish, connecting their “inappropriate” sexuality to anti-Semitism. Reasons of length preclude a consideration of other characters and elements in the ﬁlm, especially those that are also integral to a post-Holocaust sensibility. This may include a reckoning with the relationship between sexuality and perversity and “Jewishness,” so central to anti-Semitism as played out through Humbert in particular. The casting of Peter Sellers was yet another interesting and signiﬁcant casting choice; Seller’s maternal Jewishness was also, I would argue, important to his selection. Furthermore, there are many possible ways and “coded clues” to reading his character/performance as Jewish. In both cases, then, there is certainly the implication that Humbert and Quilty might also be coded as Jewish, connecting their “inappropriate” sexuality to anti-Semitism. INTRODUCTION Scholars have already detected the novel’s underlying concerns with the Holocaust.Susan L. Mizruchi, for example, has elucidated the novel’s “holocaust subtext”; that is, “a consistent pattern of references to Nazi persec- ution and genocide in Europe.”Many of the metaphors and descriptions in the novel evoke the trains, camps, and other details of the Holocaust, both John Orr and Elżbieta Ostrowska, The Cinema of Roman Polanski: Dark Spaces of the World (London: Wallﬂower, ), . Geoﬀrey Cocks, The Wolf at the Door: Stanley Kubrick, History, and the Holocaust (New York: Peter Lang, ), . Reasons of length preclude a consideration of other characters and elements in the ﬁlm, especially those that are also integral to a post-Holocaust sensibility. This may include a reckoning with the relationship between sexuality and perversity and “Jewishness,” so central to anti-Semitism as played out through Humbert in particular. The casting of Peter Sellers was yet another interesting and signiﬁcant casting choice; Seller’s maternal Jewishness was also, I would argue, important to his selection. Furthermore, there are many possible ways and “coded clues” to reading his character/performance as Jewish. In both cases, then, there is certainly the implication that Humbert and Quilty might also be coded as Jewish, connecting their “inappropriate” sexuality to anti-Semitism. Jerold J. Abrams, “The Logic of Lolita: Kubrick, Nabokov, and Poe,” in Abrams (ed.), The Philosophy of Stanley Kubrick (Lexington: University of Kentucky Press, ), –; Susan L. Mizruchi, “Lolita in History,” American Literature, , (), –; Andrea Pitzer, The Secret History of Vladimir Nabokov (New York: Pegasus, ). Mizruchi, . https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press A Jewish American Monster  directly and subtextually. Vladimir Nabokov, Lolita (London: Transworld, ; ﬁrst published ), , . Anderson, –; Mizruchi, . See also Cocks, “Indirected,” –, on Kubrick’s use in The Shining of a painting by Paul Peel, After the Bath (), which depicts two naked little girls in front of a ﬁreplace. Mizruchi, . Nabokov, . Ibid., –. Ibid., . Alfred Appel Jr. (ed.), The Annotated “Lolita” (London: Penguin, ), . See Cocks, The Wolf at the Door, for a full list. INTRODUCTION Nabokov refers to “the brown wigs of tragic old women who had just been gassed” or “the ashes of our predecessors.”In , the year of Lolita’s ﬁctive birth, Hitler passed the Nuremberg Laws and Anderson reads an imaginative equation between the Nazis’ obsession with race and therefore sexual reproduction and Humbert’s paedophilia, while Mizruchi posits that Humbert’s “case” parallels the ongoing trials of Nazi war criminals in Nuremberg from to .The repetition of twins and twinning in the novel – the twin beds and the picture of twins in the motel, the twin girls in blue bathing suits who almost discover Lolita and Humbert Humbert (itself a twinned name), the four pairs of twins in Lolita’s class list (at least one of whom, “Cowan”, may be read as Jewish as it was common to alter the name “Cohen” to that) – evokes the notorious pseudoscientiﬁc medical experiments of Dr. Josef Mengele at Auschwitz. Kubrick would later go on to make use of twins in The Shining (), which like the famous Diane Arbus photograph Identical Twins () that inspired him, also prompts audience reference to Mengele and Auschwitz. Mizruchi also observes Lolita’s “attention to American anti-Semitism.” Humbert is often mistaken for being Jewish. Before marrying him, Charlotte ﬁrst wants to ﬁnd out precisely how “foreign” Humbert is: “Looking down at her ﬁngernails, she also asked me had I not in my family a certain strange strain.” She can tolerate a “Turk” as one of his ancestors, as long as he himself is truly Christian; however, “if she ever found out I did not believe in Our Christian God, she would commit suicide.”Likewise, her friends, John and Jean Farlow, also have a vague suspicion he may be Jewish because of his dark looks and exotic name. So when John is about to make disparaging remarks about Jews in Humbert’s presence, “Of course, too many of the tradespeople here are Italians . . . but on the other hand we are still spared,” she cuts him oﬀ.Humbert attempts to check in to the Enchanted Hunters Hotel but is initially refused entry because it is restricted, advertising itself as being “Near Churches,”a coded expression used in adverts to indicate its discriminatory, restrictive practices.Nabokov also makes continuous use in the novel of the number , as the workings of what Humbert regarded as “McFate” stalking him to his doom. Ibid., . Cocks, “Indirected,” . Anderson, . Calder Willingham, Lolita screenplay, SK///, the Stanley Kubrick Archives, University of the Arts, London (hereafter SKA); Kubrick, telegram to Vladimir Nabokov, Dec. , MSS Nabokov, the Berg Collection, New York Public Library (hereafter Berg). Kubrick, telegram to Nabokov, Dec. , Berg. Nabokov later published his version, enabling comparisons to Kubrick’s ﬁlm. James B. Harris, interview (Fall ), at www.hollywoodﬁveo.com/archive/issue/exclusive/ harris/harris.htm, accessed July . “The Diary of Anne Frank,” Commentary, , (), –; “The Diary of Anne Frank – II,” Commentary, , (), –. INTRODUCTION The number also recurs in his Lolita screenplay. Cocks suggests that was “conscious and unconscious cultural shorthand Ibid., . https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press Nathan Abrams  for the Holocaust.”Consequently, Anderson argues that “the novel’s rich amalgamation of post-war America with pre-war Europe” evokes the “unbear- able memory of genocidal holocaust.”Yet Kubrick omitted many of these details, consistent with his practice of writing Jews out of his ﬁlms, although he did reference them indirectly by various means. For example, he did use as the number of the room at the Enchanted Hunters Hotel in which Lolita and Humbert ﬁrst have sex (and as a reference to the Holocaust throughout The Shining, which is set in the haunted Overlook Hotel). g These concerns may have been one of the motivating factors behind Kubrick’s desire to ﬁlm the novel in the ﬁrst place, although, given his refusal to be explicit on the subject, we will never know for certain. Kubrick solicited writer Calder Willingham to produce a screenplay, but Kubrick rejected it on the grounds that it was “not worthy” of the book, its “most serious fault not realizing characters.”Kubrick subsequently approached Nabokov himself, telling him, “you are only one for screen play. If ﬁnancial details can be agreed would you be available quick start for May Production appreciate cable.” Nabokov then began the laborious task of adapting his own novel, producing various draft screenplays, little of which Kubrick ultimately used.Instead, what became the ﬁnal screenplay was written by Kubrick and his producer, James B. Harris, using the book, Nabokov’s various drafts, and their own ideas, as well as those generated from the rehearsals and the process of shooting itself. Nonetheless, they decided to give the screenwriting credit to Nabokov. These concerns may have been one of the motivating factors behind Kubrick’s desire to ﬁlm the novel in the ﬁrst place, although, given his refusal to be explicit on the subject, we will never know for certain. Kubrick solicited writer Calder Willingham to produce a screenplay, but Kubrick rejected it on the grounds that it was “not worthy” of the book, its “most serious fault not realizing characters.”Kubrick subsequently approached Nabokov himself, telling him, “you are only one for screen play. Anderson, . https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press INTRODUCTION If ﬁnancial details can be agreed would you be available quick start for May Production appreciate cable.” The Holocaust was much in the news and in popular culture at precisely the same time as the ﬁlm was in preproduction. The Diary of Anne Frank had been published and serialized in the leading New York intellectual magazine Commentary in .It was subsequently adapted for the stage, and then made into the ﬁlm (directed by George Stevens) for which Winters won the Oscar for Best Supporting Actress. The following year, in , high- ranking Nazi bureaucrat Adolf Eichmann – one the chief architects of the Nazi genocide – was captured in Argentina, kidnapped and transported to Israel, where he was imprisoned while awaiting trial. Incidentally, at some point during his incarceration, one of Eichmann’s guards gave him a copy of Anderson, . https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press A Jewish American Monster  the recently published German translation of Lolita (), as German Jewish émigré philosopher Hannah Arendt puts it, “for relaxation.”After two days Eichmann returned it, visibly indignant, telling his guard, “Quite an unwhole- some book.”(Is it possible that Eichmann rejected Lolita not only because of its sexual content but also because he detected it as being somehow “Jewish”?) In , Judgment at Nuremberg (Stanley Kramer), with camp footage, was released and Raul Hilberg published his magisterial and ground- breaking Holocaust study, The Destruction of European Jews, which Kubrick subsequently read.That same year, with much publicity and international attention, Eichmann’s trial for war crimes began in Jerusalem. As a result, secular Jewish intellectuals, particularly in the United States, became much more conscious of the devastation of the Holocaust. Furthermore, they were vocal about it, using the Shoah to mould public opinion, increasingly making explicit comparisons between the Nazi genocide and nuclear mass death in the s and early s. Even Kubrick suggested it in his next ﬁlm, Dr. Hannah Arendt, Eichmann in Jerusalem: A Report on the Banality of Evil, revised and enlarged edn (New York: Penguin, ), . See also Leland de la Durantaye, “Eichmann, Empathy, and Lolita,” Philosophy and Literature, , (), –. Arendt  g g g In Kubrick asked his brother-in-law Jan Harlan to read Hilberg’s book; in he sent a copy to Michael Herr, describing it as “monumental.” Herr recalled how Kubrick was “absorbed” by it. Michael Herr, Kubrick (London: Pan, ), , original emphasis; Geoﬀrey Cocks, “Death by Typewriter: Stanley Kubrick, the Holocaust, and The Shining,” in Geoﬀrey Cocks, James Diedrick and Glenn Perusek (eds.), Depth of Field: Stanley Kubrick, Film, and the Uses of History (Madison: Wisconsin University Press, ), –, . However, de la Durantaye, , notes, “given Eichmann’s radical conventionality one could hardly imagine him liking – or even very well understanding – much of the book.” https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press f y y See Jon Petrie, “The Secular Word HOLOCAUST: Scholarly Myths, History, and t Century Meanings,” Journal of Genocide Research, , (), –. enlarged edn (New York: Penguin, ), . See also Leland de la Durantaye, Eichmann, Empathy, and Lolita,” Philosophy and Literature, , (), –. Arendt, . However, de la Durantaye, , notes, “given Eichmann’s radical conventionality one could hardly imagine him liking – or even very well understanding – much of the book.” In Kubrick asked his brother-in-law Jan Harlan to read Hilberg’s book; in he sent a copy to Michael Herr, describing it as “monumental.” Herr recalled how Kubrick was “absorbed” by it. Michael Herr, Kubrick (London: Pan, ), , original emphasis; Geoﬀrey Cocks, “Death by Typewriter: Stanley Kubrick, the Holocaust, and The Shining,” in Geoﬀrey Cocks, James Diedrick and Glenn Perusek (eds.), Depth of Field: Stanley Kubrick, Film, and the Uses of History (Madison: Wisconsin University Press, ), –, . See Jon Petrie, “The Secular Word HOLOCAUST: Scholarly Myths, History, and th Century Meanings,” Journal of Genocide Research, , (), –. See Patricia Erens’s The Jew in American Cinema (Bloomington: Indiana University Pres ); and Lester D. Friedman, The Jewish Image in American Film (Secaucus, NJ: Citade ). Shelley Winters, Shelley II: Best of Times, Worst of Times (London: Muller, ), . Ibid. These were A Double Life (), A Place in the Sun () and Night of the Hunter (). Gene D. Philips and Rodney Hill, The Encyclopedia of Stanley Kubrick (New York: Hill & Wang, ), . J. Hoberman, “Shelley Winters,” in J. Hoberman and Jeﬀrey Shandler (eds.), Entertaining America: Jews Movies, and Broadcasting (Princeton, NJ: Princeton University Press, ), . Winters, . Ibid., . Ibid., . Shelley Winters, Shelley: Also Known as Shirley (London: Granada, ), . Anderson, . INTRODUCTION Strangelove or: How I Learned to Stop Worrying and Love the Bomb (), for example.The s were also a time when American Jewish ﬁlmmakers began to introduce a wider range of Jewish themes and characters, including the Holocaust, into their ﬁlms in a fashion not seen since the s. Kubrick’s decision to cast Shelley Winters as the pseudo-intellectual suburban housefrau, Charlotte Haze, is perhaps the most signiﬁcant clue to reading this ﬁlm as an emotional response to his own Jewishness, as well as to the Holocaust, and goes some way to recovering Nabokov’s underlying con- cerns in the novel. It is certainly hard to ignore Winters’s own ethnicity and previous roles as a consideration in Kubrick’s casting of her as Charlotte. Winters was born Jewish, as Shirley Schrift, in , but took her mother’s f y ( y , ),  ,  See Jon Petrie, “The Secular Word HOLOCAUST: Scholarly Myths, History, and th Century Meanings,” Journal of Genocide Research, , (), –. y g f See Patricia Erens’s The Jew in American Cinema (Bloomington: Indiana University Press, ); and Lester D. Friedman, The Jewish Image in American Film (Secaucus, NJ: Citadel, ). https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Pre Nathan Abrams  maiden name. She had already played Natalia Landauer, a German Jewish girl, in I Am a Camera (), based on Christopher Isherwood’s Berlin Stories (), about the doomed intelligentsia in prewar Berlin. Winters had lost an aunt and cousins in the Holocaust: “our family had missing relatives who, we found out later, died in the concentration camps.”As a result, she refused to ﬁlm exterior shots in Germany “because I could not reconcile the thought of doing so with the image of my Holocaust-survivor uncle Yaekel.” Winters then played a variety of roles, in which she specialized as lower-class blondes murdered halfway through the ﬁlm.Thereafter, she progressed to “more matronly roles.”As mentioned above, she eventually won an Oscar for her portrayal of the Jewish refugee Mrs. Petronella Van Daan in The Diary (a part for which she gained twenty-ﬁve pounds). From then on, comments J. Hoberman, “Winters would never return to glamour roles.” Arguably, Winters’s role as Van Daan inﬂuenced all of her subsequent per- formances. She recalled, When we started shooting the ﬁlm, Stevens had all the adult actors come to a projection room. https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press INTRODUCTION He showed us the ﬁlms his unit in the Special Services had taken of the concentration camps. His Army unit had been the ﬁrst into Dachau. Watching those horrendous ﬁlms possibly made me play that role so that I won the Oscar, but I believe that shooting that ﬁlm scarred me for life. I can never read or watch anything about the Holocaust. Winters spent almost six months on the set of The Diary.“I learned something about acting that I was to use for the rest of my life.”She also stated that it was “Anne Frank whose memory and words have inspired me all of my adult life.”Winters, then, brought what she had learned on The Diary to her performance in Lolita. Given the prominence and success of this role, only three years before Lolita, it seems impossible to ignore that this was a consideration in her casting. Furthermore, Anderson argues that the circumstances surrounding Lolita “so perfectly reverse” those of the manuscript of Anne Frank that “Nabokov envisioned Lolita as a ﬁctional mirror image, an opposite twin, of her celebrated nonﬁctional contemporary.”He points out that Lolita’s https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press A Jewish American Monster forerunner, Annabel Leigh, “died of typhus – the endemic disease of the concentration camps in the closing months of the war, and perhaps not so coincidentally the cause of Anne Frank’s death in Bergen–Belsen early in .”He further notes how The Diary appeared in and Lolita was published in .In a further twist of uncanny symmetry, The Diary was made into a ﬁlm in and Lolita in – again three years apart – and both featuring Winters as a supporting actress. Winters also recalled that her Jewishness came through on the set of Lolita. Ibid. Ibid. Winters, Shelley II, . Ibid., . “Lolita Daily Production Progress Reports,” Nov. –March , SK///, SKA. Vincent LoBrutto, Stanley Kubrick: A Biography (New York: Donald I. Fine, ), . Ibid., . “Lolita Daily Production Progress Reports.” INTRODUCTION She wrote how, refusing to drop a silk robe with her back to the camera and instead hitting the microphone with her head, mixing up her lines and breaking James Mason’s glasses (Mason was playing Humbert), she was named “the klutz”, as a speciﬁcally Jewish put-down of herself.Furthermore, she remembered, “At one point, when I was squirming with embarrassment under the covers with just panties on, Mason whispered to me: ‘Would it make you feel more comfortable if I tell you that a long time ago my name was Moskowitz, and not Mason?’” Winters was certainly essential to Kubrick’s thinking; so much so that he was willing to cast her despite the obstacles to doing so. The ﬁrst potential hurdle was the Eady Levy that came into eﬀect on September , which provided indirect funding to ﬁlm producers but only if a ﬁlm qualiﬁed as “British.” In order to qualify as a British ﬁlm no less than eighty-ﬁve per cent of the ﬁlm had to be shot in the United Kingdom or the Commonwealth, and only three non-British individual salaries could be excluded from the costs of the ﬁlm, ensuring the employment of British actors, technicians and ﬁlm crew. The other two principal actors – Mason as Humbert and Peter Sellers as Clare Quilty – were British where Winters was not, and she had to be ﬂown over and put up at considerable expense. Second, the daily production reports indicate that she proved to be a pain on the set.“Winters tried Kubrick’s patience,” wrote Kubrick’s biographer, Vincent Lobrutto.“Winters was very diﬃcult,” recalled Oswald Morris, Lolita’s cinematographer, wanting to do everything her own way. She was very nearly ﬁred oﬀthe ﬁlm. At one point Kubrick said to me, “I think the lady’s gonna have to go” – which would have been very serious halfway through production. But he’d have got rid of her, he really didn’t care about the consequences. She was also ill with stomach problems and diarrhoea, delaying the shoot. Nonetheless, Kubrick persevered with her either because the cost of replacing her so late into the shoot was prohibitive, or because of what Kubrick valued https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press Nathan Abrams  that she speciﬁcally brought to the role. ( ) Incidentally, Winters would play an explicitly Jewish version of this stereotype in Next Stop, Greenwich Village (), directed by Paul Mazursky, who acted in Kubrick’s very ﬁrst ﬁlm, Fear and Desire (). Hoberman, . Ibid. https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press Quoted in Patrick Webster, Love and Death in Kubrick: A Critical Study of the Films from Lolita through Eyes Wide Shut (Jeﬀerson, NC: McFarland, ), . See Joyce Antler, You Never Call! You Never Write! A History of the Jewish Mother (New York: Oxford University Press, ). Quoted in Patrick Webster, Love and Death in Kubrick: A Critical Study of the Films from Lolita through Eyes Wide Shut (Jeﬀerson, NC: McFarland, ), . Richard Corliss, Lolita (London: BFI, ), . Incidentally, Winters would play an explicitly Jewish version of this stereotype in Next Stop, Greenwich Village (), directed by Paul Mazursky, who acted in Kubrick’s very ﬁrst ﬁlm, Fear and Desire (). Hoberman, . Ibid. g y (J Richard Corliss, Lolita (London: BFI, ), . See Joyce Antler, You Never Call! You Never Write! A History of the Jewish Mother (New York: Oxford University Press, ). Q d P k W b L d D h K b k A C l S d f h F l f INTRODUCTION Frustratingly, however, Kubrick’s archives contain no explicit reference to his reasons for casting her, or to her ethnicity, so we will never know for certain what it was that Kubrick speciﬁcally valued about what Winters brought to the role. Although there is no indication in the novel that Charlotte is Jewish, nor is there any other explicit evidence in the ﬁlm beyond the fact of Winters’s own ethnicity and previous roles, a series of clues combine to allow us to read her as Jewish. First, Charlotte is the embodiment of the stereotype of the Jewish American Mother (JAM) that began to emerge in postwar American Jewish literature at exactly the same time as Lolita was published. In Herman Wouk’s best-selling novel Marjorie Morningstar produced a stereotype that would be much copied over the coming years. Unlike her pre-Second World War counterpart, the yiddische Mama, who was viewed with aﬀection, the Jewish Mother was not. She was presented as meddlesome, domineering and controlling. Toward the end of the decade, the Jewish mother and her spoiled suburban daughter became the objects of literary ridicule, as evidenced by Philip Roth’s Goodbye Columbus (), a template which, in many ways, ﬁtted Charlotte and Lolita Haze.According to Susan Bordo, Charlotte is “the monster of the story.”Like the JAM, Charlotte is pretentious, irritating, bossy, “a behemoth mom.”Charlotte is a baalebusteh who cooks and kibitzes, nagging her daughter incessantly, and henpecking Humbert, as her husband, into desperation and longing for a means of escape.In return, Humbert describes her as a “brainless ba-ba,” a designation attributed to his ﬁrst wife in Nabokov’s novel but attached to Charlotte in the ﬁlm. It is surely no coincidence that, following Lolita, Winters was thereafter typecast. She played Jewish women/mothers in A House Is Not a Home (), Enter Laughing (), Wild in the Streets (), Buona Sera, Mrs. Campbell (), The Poseidon Adventure (), Blume in Love () and Next Stop, Greenwich Village (). As J. Hoberman put it, “No actress since Gertrude Berg has been more associated with the Jewish mother than Shelley Winters.”Signiﬁcantly, he continues, she “was a Jewish mother for the s: blowzy, strident, and generally overwhelming.”Although Hoberman does not list Lolita in his discussion of Winters, his description neatly ﬁts the role of Charlotte. A Jewish American Monster  Charlotte manifests other stereotypical Jewish tics. She is zaftig (Yiddish: plump). Norman Podhoretz, Making It (New York: Random House, ), . Is this a sly reference to the role she played as a Dutch Jewish refugee in Amsterdam in The Diary? Homi Bhabha, The Location of Culture (London: Routledge, ), ; emphasis in the original. INTRODUCTION Her taste in clothing and interior decoration is vulgar. She wears fur wraps and leopard-print dresses and belts. Her kitchen is hideously decorated with very loud wallpaper covered in food motifs. Similarly, her taste in art and artefacts – a porcelain cat sits on a dresser beneath a painting at which Humbert contemptuously stares – reveals the levels of her vulgarity. Indeed, her house is littered with so many tshatshkes (Yiddish: ornaments, trinkets, knickknacks) that it might well have come straight out of a Mad magazine caricature. She displays a lack of civility and decorum and her body language lacks the required reserve. She stuﬀs her mouth with a hotdog at the summer dance. She encroaches upon the personal space of others and is unaware of their discomfort. She talks too much and fails to read the cues, particularly when alone with Humbert, who does all he can to reject her sexual advances, which, however, she fails to notice. At one point he simply walks out of the frame and Winters keeps yakking. As Norman Podhoretz wrote, the “associ- ation of Jewishness with vulgarity and lack of cultivation” is fairly widespread, “not least among Jews.” Furthermore, Charlotte is desperate to hide her origins. Consequently, she is determined to mimic her idea of a cultivated and sophisticated suburbanite. She aﬀects a French accent, referring to Humbert as “Oh M’sieur.” She smokes through a cigarette holder. She belongs to a book club, is “Chairman of the great Books Committee,” and decorates her house with her idea of high art and artefacts. She name-drops at every opportunity, citing Dufy, Van Gogh, Monet, Schweitzer and Zhivago as evidence of her insistence on just how cultured, progressive and advanced she really is. She informs Humbert, “We’re really very fortunate here in West Ramsdale. Culturally, we’re a very advanced group with lots of good Anglo-Dutchand Anglo-Scotch stock and we’re very progressive intellectually.” At the same time, Charlotte’s choice of words, which were taken verbatim from the novel and retained by Kubrick, suggest an implicit postwar racism of the genteel Gentleman’s Agreement type in which covenanted neighbourhoods prevented Jews from buying or renting property there. Max Horkheimer and Theodor Adorno, Dialectic of Enlightenment, trans. John Cumming (London: Allen Lane, ), . John Murray Cuddihy, The Ordeal of Civility: Freud, Marx, Lévi-Strauss, and the Jewish Struggle with Modernity (Boston: Beacon, ), –. “Lolita” Dialogue Continuity, Sept. , labelled “S. Kubrick corrected copy,” containing notes to translators and dubbing directors, SK///, SKA. Martha A. Ravits, “The Jewish Mother: Comedy and Controversy in American Popular Culture,” MELUS, , (), –, . Winters, Shelley, . INTRODUCTION Charlotte’s multiple references to culture reveal her attempt to pass – and to make a pass at Humbert – but in reality they suggest an excess, a trying too hard to be the same but failing, becoming, in Homi Bhabha’s famous formulation, “almost the same, but not quite.”It is revealed, for example, by https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press Nathan Abrams  the fact that her artworks are merely reproductions or simulacra, as well as by her mispronunciation of the name Van Gogh as “Van Gock.” Charlotte’s mimicry, which surely can be described as “undisciplined,” has long been felt to mark the Jewish condition. For Max Horkheimer and Theodor Adorno, “undisciplined mimicry” was “engraved in the living substance of the dominated and passed down by a process of unconscious imitation in infancy from generation to generation, from the down-at-heel Jew to the rich banker.”This was because “Jewish Emancipation involved Jews in collisions with the diﬀerentiations of Western society [and] Jews were being asked, in eﬀect, to become bourgeois, and to become bourgeois quickly.”Charlotte here is desperately trying, but failing, to pass by masking her Jewish roots through her failing mimicry (a faux posh accent, use of words, intellectual/ cultural airs and graces), but it is the very excess of her mimicry that gives her away, revealing her failure to pass, and echoing the Jewish saying that “Jews are like everybody else, only more so.” As if to stress the point, and to make sure that the translators and dubbing directors understood his intentions, Kubrick annotated the Dialogue Continuity script with instructions. For example, when Humbert is shown around Charlotte’s house, Kubrick has written, “Note to translators and dubbing directors: Charlotte Haze’s choice of words in English are pretentious and awkwardly pseudo-intellectual. Try to retain that feeling because it is the basis of much of the comedy.” When she says, “Oh Paris . . . France . . . Madame,” Kubrick noted, “a good example of her pretentious and awkward choice of words.”These mannerisms precisely ﬁt the emerging JAM stereotype of the s and s as described by Martha A. Ravits: “she personiﬁes garish ethnic manners and materialistic, middle- class pretensions.”In this respect, it certainly seems very illuminating that Winters drew upon someone she knew (although whom she does not actually reveal) in playing the part of Charlotte. https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press INTRODUCTION “I had known a pseudointellectual suburbanite like Charlotte, the character I played, during my childhood days in Jamaica, Queens, and Stanley Kubrick knew what acting buttons to press in my acting computer to bring her back.” Charlotte also aﬀects a Christian/Catholic religious identity. In her letter of love and confession to Humbert she writes, “Last Sunday in church, my dear one, when I asked the Lord what do about it . . .”. She keeps her late husband’s https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press A Jewish American Monster  ashes in an urn on a sideboard, in a bedroom shrine, complete with a cruciﬁx and ﬂanked by Catholic icons, as if copying Tennessee Williams in Rose Tattoo, noted the Brooklyn Tablet.This display again reveals the excess of her mimicry, for arguably only a Jew could conceive of such a Christian/Catholic shrine and indeed the shrine is the product of the Jewish imagination: Kubrick’s. Furthermore, as the Brooklyn Tablet further noted, while Charlotte “prattles about God” she “gives daughter Lolita neither religious training nor good example.”As if recognizing this fact, John Baxter has written that Kubrick replaced a cruciﬁx with a triptych of Our Lady of Perpetual Succour, following complaints about the juxtaposition of Mr. Haze’s ashes with a cruciﬁx in Charlotte’s bedroom, resulting in “a Byzantine image that probably looked suﬃciently exotic to count as Jewish or Middle European.”Making Charlotte Catholic/Christian and have her attend church is Kubrick’s misdirection. Where the novel, as mentioned above, was full of allusions, both direct and indirect, to the Second World War and the Holocaust, the ﬁlm removes these, but their traces remain. The one explicit remaining reference is when Charlotte tells Lolita (Sue Lyon) oﬀand orders her not to disturb “Professor Humbert.” In reply Lolita mimics a Hitler salute, albeit with her left hand, and says “Sieg heil.” As if responding to the gesture, in the scene that immediately follows, Charlotte informs Humbert that she has been “too liberal” and is send- ing Lolita oﬀ“long-distance” to a “camp” for “isolation.” The phraseology here, through its close juxtaposition with the direct invocation of Hitler, uncannily echoes the Nazis’ euphemistic language (“ﬁnal solution,” “solution possibilities,” “special treatment,” “cleansing operation,” “deportation,” “dis- placement,” “resettlement,” and “evacuation”),as well as anticipating Betty Friedan’s striking comparison of Nazi concentration camps to American suburban homes one year later. y John Baxter, Stanley Kubrick: A Biography (New York: HarperCollins, ), . That Mr. Haze was, in Charlotte’s words “in insurance,” “left [her] well-provided for,” and “was a lovely human being” (i.e. a mensch), could also be read as further Jewish clues. https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press The Tablet (Brooklyn), June , SK///i, SKA. Ibid.  The Tablet (Brooklyn), June , SK///i, SKA.  g ( ) J Hilberg, , , . In this respect, it is signiﬁcant that the daily continuity report for this shot, dated Jan. , reported, “The dialogue oﬀscreen is not the dialogue used in the shot – that is only very approximately – Humbert speaking with a German accent, and calling himself Rommel etc. simply to give reaction to Lolita.” Daily continuity reports, SK///, SKA. As if reinforcing this underlying German subtext, prior to Lolita, Mason had starred as Field Marshal Erwin von Rommel in The Desert Fox () and The Desert Rats (). The Tablet (Brooklyn), June , SK///i, SKA. Ibid. John Baxter, Stanley Kubrick: A Biography (New York: HarperCollins, ), . That Mr. Haze was, in Charlotte’s words “in insurance,” “left [her] well-provided for,” and “was a lovely human being” (i.e. a mensch), could also be read as further Jewish clues. Hilberg, , , . In this respect, it is signiﬁcant that the daily continuity report for this shot, dated Jan. , reported, “The dialogue oﬀscreen is not the dialogue used in the shot – that is only very approximately – Humbert speaking with a German accent, and calling himself Rommel etc. simply to give reaction to Lolita.” Daily continuity reports, SK///, SKA. As if reinforcing this underlying German subtext, prior to Lolita, Mason had starred as Field Marshal Erwin von Rommel in The Desert Fox () and The Desert Rats (). INTRODUCTION In one of the most potentially shocking passages of her The Feminine Mystique (), Friedan claimed that “the women who ‘adjust’ as housewives, who grow up wanting to be ‘just a house- wife,’ are in as much danger as the millions who walked to their own death in Hilberg, , , . In this respect, it is signiﬁcant that the daily continuity report for this shot, dated Jan. , reported, “The dialogue oﬀscreen is not the dialogue used in the shot – that is only very approximately – Humbert speaking with a German accent, and calling himself Rommel etc. simply to give reaction to Lolita.” Daily continuity reports, SK///, SKA. As if reinforcing this underlying German subtext, prior to Lolita, Mason had starred as Field Marshal Erwin von Rommel in The Desert Fox () and The Desert Rats (). https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press Nathan Abrams  the concentration camps.”Friedan went on to explore this analogy for several pages, and then continued to use the phrase “comfortable concen- tration camps” to refer to suburban homes throughout the rest of the book. Furthermore, in another shift from the novel, in the ﬁlm Humbert lies on the marital bed contemplating murdering Charlotte. In the foreground, a gun is on the bedside table. The following conversation, transposed almost verbatim from the novel, takes place: Charlotte: Darling, you’ve gone away. Humbert: Just a minute, darling, I’m following a train of thought. . . . Charlotte: Am I on that train? Humbert: Yes. Humbert’s thoughts, in the form of a voice-over narrative, conﬁrm the suspicion: “No man can bring about the perfect murder. Chance, however, can do it. Just minutes ago she had said it wasn’t loaded. What if I had playfully pulled the trigger then? She said it wasn’t loaded. It belonged to the late Mr. Haze.” The proximity of the gun, and Humbert’s assumed thoughts, suggest a connection between trains and killing, what Cocks refers to as the “association with Nazi mechanics of murder that would show up in The Shining.”Again, to repeat a key point, in light of Winters’s starring in The Diary only three years earlier, both of these conversations are particularly poignant and suggestive. Signiﬁcantly, Kubrick made various other changes which deviated from Nabokov’s novel. Betty Friedan, The Feminine Mystique (New York: W. W. Norton, ), . Cocks, The Wolf at the Door, . Greg Jenkins, Stanley Kubrick and the Art of Adaptation: Three Novels, Three Films, (Jeﬀerson: McFarland, ), –. Ibid., –. INTRODUCTION In his close textual comparison of the novel and ﬁlm, Greg Jenkins registered that Kubrick’s few changes work to the detriment of Charlotte, magnifying her undesirable qualities . . . the Charlotte of the ﬁlm is more brazen than the original, practically launching herself at Humbert. She is more noxious, rambling angrily in Winters’ diva voice; the ﬁctional Charlotte condemns her daughter in nothing but indirect quotations, a device that distances the reader from her fury. Again, the ﬁlm craftily maneuvers us away from Charlotte; it asks us to take sides, to view her unsym- pathetically. These alterations served to emphasize the negative aspects of Charlotte’s character. As Jenkins put it, “All these adjustments undercut the image, not sterling to begin with, of Charlotte . . . rendering her less sympathetic, more vulgar.”Richard Corliss adds, “Winters does appear to be twenty pounds https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press A Jewish American Monster A Jewish American Monster  heavier, ﬁfteen decibels higher and ten I. Q. points lower than Charlotte deserves.” Contemporary reviewers, especially those who were part of the intelligent- sia, certainly picked up on this characterization. Writing in Partisan Review, Pauline Kael described Charlotte as “the culture-vulture rampant . . . Shelley Winters’ Charlotte is a triumphant caricature, so overdone it recalls Blake’s ‘You never know what is enough until you know what is enough.’”Arthur Schlesinger Jr. felt, meanwhile, that “Winters, as Lolita’s mother, gives the performance of her life, laying bare with delicate exactitude the genteel pretension, the tremulous hope and prurient passion of what Nabokov ap- parently regards as the typical American middle-class woman.”Finally, Dwight Macdonald opined, “Miss Winters plays her so fortissimo that she becomes a brawling Bronx ﬁshwife whom one cannot imagine having poor Charlotte’s cultural pretensions.”Indeed, he labelled her a “Monsterette.” Macdonald misses the point that her casting intentionally transformed Charlotte from genteel to brawling, but he does inadvertently pick up on the implicit Jewishness of her character in locating her in the Bronx, where Kubrick grew up. Again, a brief glimpse of Kubrick’s intentions is seen in his notes, where he refers to Charlotte’s “ugliness.”At the same time, Kubrick coaxed a performance out of Winters that emphasized Charlotte’s worst qualities. This led her to reﬂect, I think the role of Charlotte in Lolita is one of the best performances I ever gave in any medium. Kubrick, “Last Scene Notes,” n.d., SK///, SKA. Winters, Shelley II, . Pauline Kael, “Movie Chronicle: Little Men,” Partisan Review, , (), –, . ( ) Arthur Schlesinger Jr., “Little Women,” Show Magazine, July , . g J g J y   Dwight Macdonald, “Of Nymphets and Monsterettes,” Esquire, Sept. , Press Binder, SK //iii, SKA. Ibid.  Corliss, Lolita, . Corliss, Lolita, . Pauline Kael, “Movie Chronicle: Little Men,” Partisan Review, , (), –, . Arthur Schlesinger Jr., “Little Women,” Show Magazine, July , . Dwight Macdonald, “Of Nymphets and Monsterettes,” Esquire, Sept. , Press Binder, SK/ //iii, SKA. Ibid. Kubrick, “Last Scene Notes,” n.d., SK///, SKA. Winters, Shelley II, . Arthur Schlesinger Jr., Little Women, Show Magazine, July , . Dwight Macdonald, “Of Nymphets and Monsterettes,” Esquire, Sept. , Press Binder, SK/ //iii, SKA. Ibid. Kubrick, “Last Scene Notes,” n.d., SK///, SKA. Winters, Shelley II, . INTRODUCTION She is dumb and cunning, silly, sad, sexy, and bizarre, and totally American and human. Until I saw the whole ﬁlm cut together, I did not realize the gift that Kubrick had given me. I was enchanted with Charlotte and very proud of her. Kubrick had the insight to ﬁnd the areas of me that were pseudointellectual and pretentious. We all have those things in us. A later, and similar, Kubrick casting decision supports this reading. Winters’s role anticipates a comparable choice he took concerning the casting of Miriam Karlin in his A Clockwork Orange (). Before attacking Miss Weathers, the “Cat Lady” (played by Karlin), Georgie (James Marcus) justiﬁes robbing her because her house “is full up with like gold, and silver, and like jewels.” Cocks comments, That this might be an echo of a common stereotype of Jews is suggested by the fact the woman is played by Miriam Karlin. Karlin is a British actress active in Jewish causes https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press Nathan Abrams  and a prominent member of the Anti-Nazi League, which was one of the responses to the stirrings of neo-fascism in Britain at the time. Her mother came to England from Holland, and had lost her entire family at Auschwitz. Furthermore, as Cocks has argued, since the Cat Lady is “by conventional Hollywood standards a less than physically and personally attractive person,” the audience is not encouraged to sympathize with her – just like it is not with Charlotte Haze.Thus she ﬁts into a pattern of “Kubrick’s indirect insinuation of the issue of Jews and anti-Semitism.”In this respect, it is certainly signiﬁcant that when Winters saw the ﬁlm, she wrote to Kubrick praising it and jokingly asking why she had not been asked to play “the very British woman who gets raped in this ﬁlm.”Winters recalled, “He did not get the joke. He sent me back a very stern reply and informed me that he would cast me in any role I was suited for in any one of his ﬁlms. And that was ﬁnal.” The question remains, then, why did Kubrick create such a negative caricature of a Jewish woman and mother? Ibid., –. Cocks, The Wolf at the Door, . Ibid., . Winters, Shelley II, . Ibid. LoBrutto, Stanley Kubrick, . Cocks, The Wolf at the Door, . LoBrutto, . INTRODUCTION One answer would be to suggest an emotional and psychological impulse of misogyny and self-hatred; that is, that after two marriages to two diﬀerent Jewish women – he divorced Toba Metz in and Ruth Sobotka in – Kubrick took a dim view of Jewish femininity, buying into the caricature of the JAM that had begun to emerge in the mid-s. Evidence that might ﬁt this last assertion may lie in the fact that Kubrick married Christiane Harlan, a non-Jewish German woman, who grew up during the Third Reich, for his third (and last) wife. Christiane recalled how “I was the little girl who moved in where Anne Frank was pushed out.” However, this answer seems far too easy. Kubrick was close to his (Jewish American) mother Gertrude (and she to him). According to Kubrick’s third wife, Christiane, his mother was still buying him clothes as late as and was “more up on his ﬁlms” than his father.According to LoBrutto, she was an “intelligent” and “well-spoken woman” from whom “Kubrick had inherited his looks.”Indeed, in the sole ﬁlm in which Kubrick allowed someone to make the only formal record of him at work – Making The Shining (), directed by his daughter Vivian, Gertrude appears, paying an on-set visit to her son. She is seen discussing with Jack Nicholson the daily script changes and the meaning of the colour of the script pages. Kubrick played a heavy hand in its editing, so the ﬁnal cut had his approval, indicating a certain warmth towards his mother. https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press A Jewish American Monster  Rather, an alternative suggestion will be posited here, that it may well be that Kubrick was deliberately playful with that very JAM stereotype, using an underlying and Jewish-inﬂected humour to make emotional, cogent and deeply serious points about anti-Semitism. Much has been written about the function of stereotypes in general and Jewish ones in particular, especially how they perform cultural work in demonizing minority groups from the outside, and emotionally perpetuating group solidarity and continuity from the inside. As Bhabha suggests, the stereotype oﬀers “a secure point of identiﬁcation”; that is, emotional reassurance. p gy y A. A. Berger, Jewish Jesters: A Study in American Popular Comedy (Cresskill, NJ: Hampton Press, ), –, . Michel Foucault, The History of Sexuality, Volume I, An Introduction, trans. Robert Hurley (London: Penguin, ), . Bhabha, . Bhabha, , original emphasis. Daniel Boyarin, Unheroic Conduct: The Rise of Heterosexuality and the Invention of the Jewish Man (Berkeley: University of California Press, ), xxiii. Ibid. Slavoj Žižek, The Sublime Object of Ideology (London: Verso, ), xi. Bhabha, . Vasiliki P. Neofotistos, “The Muslim, the Jew and the African American: America and the Production of Alterity in Borat,” Anthropology Today, , (), –, –. A. A. Berger, Jewish Jesters: A Study in American Popular Comedy (Cresskill, NJ: Hampton Press, ), –, . Michel Foucault, The History of Sexuality, Volume I, An Introduction, trans. Robert Hurley (London: Penguin, ), . Bhabha, . https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press  Vasiliki P. Neofotistos, “The Muslim, the Jew and the African American: America and the Production of Alterity in Borat,” Anthropology Today, , (), –, –. ( y y ) Slavoj Žižek, The Sublime Object of Ideology (London: Verso, ), xi. h bh Bhabha, , original emphasis.  Daniel Boyarin, Unheroic Conduct: The Rise of Heterosexuality and the Invention of the Jewish Man (Berkeley: University of California Press, ), xxiii. Ibid. INTRODUCTION Daniel Boyarin called this form of comfort “Jewisssance.”Itself a play on the French term jouissance – literally translating as “orgasm,” but also referring to physical or intellectual pleasure, delight, or ecstasy – Boyarin deﬁned Jewissance as “a pleasure” that “brings to many men and women an extraordinary richness of experience and a powerful sense of being rooted somewhere in the world, in a world of memory, intimacy, and connectedness.” Yet, on a deeper level, stereotypes contain a “surplus value,” which provides “enjoyment or jouissance [and] enables us to understand the logic of exclusion.”Bhabha similarly suggested that the stereotype is characterized by a “productive ambivalence” between “pleasure and desire” and “power and domination.”In other words, stereotypes are enjoyed because they allow us to see contested images at work and understand their ideological implications. They entertain us, as well as serve to ridicule the logic of exclusion.This use of Jewish stereotypes by Kubrick, then, reveals a deeper strategy beyond Jewissance and pleasure. The reversal of insult, or “victim humour,” is a tech- nique against anti-Semitism, to “disguise the aggression and hostility by turning it on oneself.”This is comparable to what Michel Foucault labelled a “reverse discourse,” which seeks to “demand that its legitimacy . . . be acknowl- edged, often in the same vocabulary, using the same categories by which it was . . . disqualiﬁed.”Bhabha pointed out how “the same stereotype maybe read in a contradictory way or, indeed, be misread.”This “reverse stereotype,” then, achieves the status of what Foucault called “a hindrance, a stumbling https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press Nathan Abrams  block, a point of resistance and a starting point for an opposing strategy.”Or what Sigmund Freud described as “a rebellion against authority, a liberation from its pressures,”glossed by Bhabha as “a strategy of cultural resistance and agency committed to a community’s survival.” As we have seen with the character of Charlotte, reverse stereotypes may take the form of “mimicry,”which “is never a simple reproduction of those traits. Rather, the result is a ‘blurred copy’ . . . that can be quite threatening. https://doi.org/10.1017/S0021875814001844 Published online by Cambridge University Press INTRODUCTION This is because mimicry is never very far from mockery, since it can appear to parody whatever it mimics.”The reverse stereotype and mimicry, therefore, was a means for Kubrick to draw upon his Jewish background and Yiddishkeit (Yiddish: “Jewishness” or “Jewish culture”) as a means to mimic, mock and critique the representation of the Jewish woman, particularly at a time when explicit Jews, played by Jews, were not much in evidence in Hollywood cinema and when representations of the Holocaust and anti-Semitism were only really beginning to emerge into mass media in the United States. Furthermore, if, as has been argued, the way that Kubrick adapted Nabokov’s novel retained its concerns with the Holocaust and anti-Semitism, it ﬁt into a period from the early to mid-s when various Jewish American intellectuals, who had grown up while the Holocaust was happening, used Nazism to forge emotional and deeply personal expressions of identity. Foucault, –. Foucault, . Sigmund Freud, Jokes and Their Relation to the Unconscious (Harmondsworth: Penguin, ), . Bhabha, xvii. Bhabha, . Bill Ashcroft, Gareth Griﬃths and Helen Tiﬃn, Post-colonial Studies: The Key Concepts (London: Routledge, ), . Bill Ashcroft, Gareth Griﬃths and Helen Tiﬃn, Post-colonial Studies: The Key Concepts (London: Routledge, ), . g See Kirsten Fermaglich, American Dreams and Nazi Nightmares (Waltham, MA: University Press of New England, ). See Kirsten Fermaglich, American Dreams and Nazi Nightmares (Waltham, MA: University Press of New England, ). Press of New England, ).
https://openalex.org/W2159230150	https://europepmc.org/articles/pmc4272773?pdf=render	English	null	mARC vs. IMRT radiotherapy of the prostate with flat and flattening-filter-free beam energies	Radiation oncology	2,014	cc-by	6,027	© 2014 Dzierma et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH Open Access Abstract Background: There as yet exists no systematic planning study investigating the novel mARC rotational radiotherapy technique, which is conceptually different from VMAT. We therefore present a planning study for prostate cancer, comparing mARC with IMRT treatment at the same linear accelerator equipped with flat and flattening-filter-free (FFF) photon energies. Methods: We retrospectively re-contoured and re-planned treatment plans for 10 consecutive prostate cancer patients. Plans were created for a Siemens Artiste linear accelerator with flat 6 MV and FFF 7 MV photons, using the Prowess Panther treatment planning system. mARC and IMRT plans were compared with each other considering indices for plan quality and dose to organs at risk. All plans were exported to the machine and irradiated while measuring scattered dose by thermoluminescent dosimeters placed on an anthropomorphic phantom. Treatment times were also measured and compared. Results: All plans were found acceptable for treatment. There was no marked preference for either technique or energy from the point of view of target coverage and dose to organs at risk. Scattered dose was significantly decreased by the use of FFF energies. While mARC and IMRT plans were of very similar overall quality, treatment time could be markedly decreased both by the use of mARC and FFF energy. Conclusions: Highly conformal treatment plans could be created both by the use of flat 6 MV and FFF 7 MV energy, using IMRT or mARC. For all practical purposes, the FFF 7 MV energy and mARC plans are acceptable for treatment, a combination of both allowing a drastic reduction in treatment time from over 5 minutes to about half this value. Keywords: mARC rotational treatment, Flattening-filter-free photons, Prostate cancer, Planning Dzierma et al. Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 Dzierma et al. Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 mARC vs. IMRT radiotherapy of the prostate with flat and flattening-filter-free beam energies Yvonne Dzierma, Katharina Bell, Jan Palm, Frank Nuesken, Norbert Licht and Christian Rübe Correspondence: yvonne.dzierma@uks.eu Department of Radiotherapy, Saarland University Medical Center, Kirrberger Str. Geb. 6.5, 66421 Homburg, Germany Patients and methods Table 2 DVH criteria for plan acceptability PTV V(95%) >95% V(105%) <5% Bladder V75Gy <15% V70Gy <20% V50Gy <50% Rectum V70Gy <10% V60Gy <30% V50Gy <50% V40Gy <70% V30Gy <80% Posterior rectal wall D(max) <60 Gy V50Gy <15% V40Gy <30% Femoral heads V50Gy <5% p p y PTV V(95%) >95% V(105%) <5% Bladder V75Gy <15% V70Gy <20% V50Gy <50% Rectum V70Gy <10% V60Gy <30% V50Gy <50% V40Gy <70% V30Gy <80% Posterior rectal wall D(max) <60 Gy V50Gy <15% V40Gy <30% Femoral heads V50Gy <5% For the present study we chose 10 consecutive patients di- agnosed with intermediate and high-grade prostate cancer that had previously been treated in our department. Due to the retrospective nature of this study, no ethics board approval was required. Patient characteristics are shown in Table 1. Computed tomography (CT) datasets had been acquired on a dedicated scanner (Brilliance CT - Big Bore Oncology, Philips, Koninklijke) with 3 mm spacing be- tween slides. For all patients additional MRI data of the small pelvis was present for coregistration. For reasons of standardization contouring was completely redone by one radiation oncologist according to the Radiation Therapy Oncology Group (RTOG) Trial 0126 [7] con- touring guidelines. Gross tumor volume (GTV), clinical target volume (CTV), planning target volume (PTV) and normal tissues were outlined on all CT slices in which the structures existed. PTV The GTV encompassed the prostate gland, the CTV encompassed the GTV plus the proximal bilateral sem- inal vesicles (only the first 1.0 cm of the seminal vesicle tissue adjacent to the prostate). The PTV was generated by adding a surrounding margin of 7 mm to the CTV. Organs at risk were outlined according to the Male RTOG Normal Pelvis Atlas [8]. A dose of 76 Gy was prescribed to the PTV, for plan evaluation we used our in-house DVH (dose-volume histogram) criteria as shown in Table 2 that are mainly based on the data pub- lished by Quantitative Analysis Of Normal Tissue Effects In The Clinic (QUANTEC) [9,10]. Therefore, any differences in plan quality will reflect mainly the difference in beam profiles. For mARC plans, one complete (360°) gantry rotation was used with optimization points spaced 10° and arclet length 4°. IMRT plans consisted of 11 beams (gantry an- gles 205°, 235°, 265°, 295°, 330°, 0°, 30°, 65°, 95°, 125°, 155°), with 3 segments per beam. Background of organs at risk (OAR), yet only requires one gantry ro- tation to achieve a highly conformal dose distribution (compare, e.g., [6]). We therefore present a planning study for prostate cancer with mARC for a Siemens Artiste machine equipped with flat 6 MV and flattening- filter-free (FFF) 7 MV energies. The combination of FFF beams with mARC treatment is of particular interest since this offers the greatest potential for a reduction in treatment time. The mARC (“modulated arc”) technique has recently been introduced as a rotational intensity-modulated ra- diation therapy (IMRT) technique for Siemens linear ac- celerators [1,2]. Although the dosimetric accuracy has been assessed by various methods and first patient treat- ment has been reported [3,4], no systematic planning studies have yet been carried out to assess the quality of mARC treatment as compared with IMRT delivered at the same linear accelerator. Although the mARC technique is a Siemens Artiste specific modality and primarily interesting for Siemens customers, we hope that the comparison of the flat and FFF beam lines, both for mARC and IMRT treatment, will be useful for a wide range of readers. First applications have centered on prostate treatment for mARC [4,5], which appears to be an ideal indication as it benefits from inverse planning due to the proximity * Correspondence: yvonne.dzierma@uks.eu Department of Radiotherapy, Saarland University Medical Center, Kirrberger Str. Geb. 6.5, 66421 Homburg, Germany Dzierma et al. Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 Page 2 of 8 Dzierma et al. Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 Table 2 DVH criteria for plan acceptability Patients and methods In a prior test, it was checked if plans were improved by allowing 5 segments per beam (the “gold standard” for prostate IMRT at our institution). Since no significant difference was observed, we here limit our analysis to IMRT plans with a total of 33 segments or less, which is nearly the same number of de- grees of freedom as for the mARC plans (36 optimization points). The collimator angle was 90° for all plans, also based on previous tests. At our institution, the mARC technique is available at one Siemens Artiste linear accelerator with flat 6 MV and FFF 7 MV energy, equipped with 160 multi-leaf collimator (MLC, leaf width 5 mm). The two energies are particularly well suited for comparative planning as the slightly in- creased nominal energy of the FFF 7 MV beam compen- sates for the spectral softening caused by the removal of the flattening filter, so that the percent depth dose of the FFF 7 MV beam matches the flat 6 MV beam closely [11]. Planning is performed in the Prowess Panther V5.10r2 treatment planning system (TPS) on a 3 mm dose grid using the collapsed cone dose algorithm. IMRT and mARC inversion are closely similar, both using a simu- lated annealing approach for direct aperture optimization. Planning is performed in the Prowess Panther V5.10r2 treatment planning system (TPS) on a 3 mm dose grid using the collapsed cone dose algorithm. IMRT and mARC inversion are closely similar, both using a simu- lated annealing approach for direct aperture optimization. Based on a set of inversion objectives, the optimization can be carried out interactively by adjusting the DVH con- straints and weights until the desired shape is reached. Criteria for optimization are listed in Table 2. Table 1 Patient characteristics Mean Range Age (years) 69 59-74 T-stage 2a 1 - 2b 1 - 2c 7 - 3a 1 - Gleason grading at diagnosis 7 6-9 PSA at diagnosis (ng/ml) 26.56 3.8-94 Anti androgen deprivation (yes/no) 10/0 Prostate volume (ccm) 61.6 39.55-91.22 Table 1 Patient characteristics Table 1 Patient characteristics Plan quality was compared for the four scenarios (IMRT vs. mARC, 6 MV vs. FFF 7 MV) based on the conformity index (CI), the homogeneity index (HI), V(50Gy) for blad- der and rectum, and V(40Gy) of the posterior rectal wall. Results TVPIV is the volume of the PTV surrounded by the pre- scribed isodose (95%). All plans (DVH and dose distributions) were reviewed by at least one senior radiotherapist and were all deemed acceptable for treatment. All plans satisfied the criteria that at least 95% of the planning target volume received 95% of the prescribed dose of 76 Gy, and all organs at risk remained below the imposed limits (Table 2). The homogeneity index is calculated as HI ¼ DPTV 2% ð Þ−DPTV 98% ð Þ DPTV 50% ð Þ A visual comparison of the four plan scenarios for each patient did not show a marked preference for either tech- nique or energy (example dose distributions and DVH shown in Figures 1 and 2). DVHs of the four scenarios are closely similar for each patient. Relying on the quality mea- sures (Table 3), the comparison of IMRT with mARC plans for the same energy never yielded a significant difference. For comparison of 6 MV with FFF 7 MV, homogeneity index and conformity index were significantly better for 6 MV than 7 MV both for mARC and IMRT plans, separ- ately. Considering dose to OAR, there was no significance for comparison of IMRT plans (6 MV vs. 7 MV), but 6 MV mARC plans performed better than 7 MV mARC. However, even in those cases where a statistical signifi- cance was shown, the differences were very small and hardly of clinical significance: For the bladder, a median of 20.8% of the volume received a dose of 50 Gy in the 6 MV mARC plans, whereas this was 22.4% for 7 MV mARC. The rectum V50 increases from 16.8% for 6 MV mARC to 17.5% for 7 MV mARC. As all these values remain far below the allowed limits, they would not have caused rejec- tion of the plans for clinical treatment. For all these values, the variation between different patients was considerably larger than the variation from one plan scenario to the next, which can be seen by the overlapping ranges of values (Table 3) even in cases where a small statistical significance was found. where DPTV(x %) means the dose received by x % of the PTV volume. All plans were exported to the machine for treatment and irradiated on an Alderson anthropomorphic phan- tom positioned with the prostate at the approximate lo- cation of the isocenter. Patients and methods The conformity index is defined by [12]: CI ¼ TV PIV 2 TV⋅PIV where TV denotes the volume of the PTV, PIV is the volume enclosed in the prescribed isodose (95%), and Page 3 of 8 Page 3 of 8 Page 3 of 8 Dzierma et al. Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 Dzierma et al. Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 TVPIV is the volume of the PTV surrounded by the pre- scribed isodose (95%). Treatment times Treatment times depend on the time for gantry and MLC movement on the one hand and on the time re- quired to irradiate the monitor units on the other hand. As it is more time-consuming to stop the gantry at pre- cise angles rather than just move it through an angular range, mARC saves treatment time in comparison with step-and-shoot plans that would use the same number of gantry angles (i.e., 36 with one segment per beam). The use of FFF beam energies saves time as the higher dose rate allows faster irradiation of the ca. 400 MU. We therefore assess the dependence of treatment time on the number of MU for the four scenarios (Figure 3). In all cases, a linear fit can be made, with parameters given in Table 4. Based on the above considerations, the y-axis intercept should be the same for both IMRT plans and for both mARC plans, respectively, since it is mainly determined by the irradiation geometry. The slope of the curves should be similar for the 6 MV plans and the 7 MV plans, respectively, since it depends on the available dose rate. Scattered dose is significantly decreased by the use of FFF energies, which is physically reasonable since head scatter is reduced in the absence of a flattening filter. For identical plan scenarios, the FFF 7 MV energy pro- duces only about 58–85% of the scattered dose mea- sured for flat 6 MV, with strongest reduction at larger distance from the treatment field (lens). A difference be- tween IMRT and mARC plans cannot be observed for 6 MV; for 7 MV, the out-of-field dose is slightly higher for mARC (up to 108% of the 7 MV IMRT plan, but still much lower than for the 6 MV plans). Indeed, the y-axis intercepts of the IMRT plans for dif- ferent energies differ less than one standard error; the same applies to the mARC plans. The slope of the curves for 6 MV plans also agree within less than one standard error, with an approximate value of 0.27 s/MU – this cor- responds to an average dose rate of 219 MU/min. For the 7 MV plans, the slope (again within less than one standard error) of ca. 0.122 s/MU corresponds to an average dose rate of 492 MU/min. These values are reasonable – the maximum available dose rate for 6 MV is 300 MU/min. Results Thermo-luminescent dosimeters (Harshaw TLD 100H) were placed at three positions on the surface of the phantom (navel, manubrium sterni, right eye lens) to measure the scattered dose outside the treatment field. At each position, three TLDs were placed in close proximity and the measurements averaged. The average standard deviation of the three measurements was below 5% for the measurements at the navel, and between 5 and 10% for the lower dose values measured at the ster- num and lens. During irradiation, treatment times were measured for comparison. Plans were compared pair-wise for mARC vs. IMRT and for 6 MV vs. 7 MV energy, considering the mea- sures of quality defined above, monitor units, treatment time and scattered dose. The Shapiro-Wilk test was per- formed to check for normality. In cases where this could not be refuted, the t-test for paired data was applied, otherwise the Wilcoxon signed-rank test was used. A value of p = 0.05 or below was considered to be statisti- cally significant. Figure 1 Example dose distributions (transverse slices). The isodose lines are given relative to the reference point, which receives 76 Gy. The PTV is displayed as filled cyan contour. Figure 1 Example dose distributions (transverse slices). The isodose lines are given relative to the reference point, which receives 76 Gy. The PTV is displayed as filled cyan contour. ndard p (7 IMR mAR n.s. n.s. n.s. n.s. n.s. n.s. 0.00 n.s. n.s. 0.02 andard V p (7 IMR mA n.s. n.s. n.s. n.s. n.s. n.s. 0.00 n.s. n.s. 0.02 standard MV T – RC) p (7 IMR mA n.s. n.s. n.s. n.s. n.s. 7 n.s. 2 0.00 n.s. n.s. 0.02 mA n.s. n.s. n.s. n.s. n.s. n.s. 0.00 n.s. n.s. 0.02 or the four plan scenarios (median values ± standard C 7 MV mARC p (6 MV IMRT – mARC) p (7 IMR mA 7 (0.878-0.925) 0.883 ± 0.024 (0.850-0.926) n.s. n.s. 1 (0.085-0.121) 0.102 ± 0.010 (0.088-0.126) n.s. n.s. (6.0-49.3) 22.4 ± 14.6 (6.7-49.8) n.s. n.s. 5.3-22.3) 17.5 ± 5.5 (5.7-23.4) n.s. n.s. 0-8.3) 4.8 ± 4.2 (0.0-11.6) n.s. n.s. 65–452) 411 ± 43 (366–507) 0.047 n.s. (3:20–3:55) 2:27 ± 0:09 (2:13–2:39) 0.002 0.00 2.7-19.0) 12.4 ± 2.2 (10.3-16.7) n.s. n.s. .33-1.87) 1.29 ± 0.2 (0.97-1.58) n.s. n.s. 50 (0.563-0.705) 0.378 ± 0.037 (0.322-0.449) n.s. 0.02 Page 5 of 8 Dzierma et al. Results Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 beam: this was indeed observed for large PTV [13,14]. Therefore, the position of the isocenter is more critical for the FFF energies. The mARC plans required slightly more monitor units than the IMRT plans, but the difference was negligible (not significant except for 6 MV IMRT vs. mARC, with 402 and 414 MU, respectively, p = 0.047). However, treatment time could be markedly decreased both by the use of mARC and FFF energy. In moving from 6 MV to FFF 7 MV, about one minute treatment time was saved both in IMRT and mARC, respectively. In moving from IMRT (11 beams) to mARC, treatment times were re- duced by about two minutes both for 6 MV and FFF 7 MV, respectively. By combining mARC treatment with FFF 7 MV energy, the treatment time could effectively be reduced by half (median 2:27 min for FFF mARC ver- sus 5:21 min for 6 MV IMRT). Treatment times For FFF 7 MV, 2000 MU/min are theoretically available. However, for small segments/arclets with low MU, the linac firmware automatically reduces the dose rate for bet- ter linearity (for about 400 MU distributed over 33 seg- ments or 36 arclets, the linac will nearly always operate at a reduced dose rate since most segments receive only about 10 MU). We therefore find that both the FFF IMRT and mARC plans operate at an average dose rate consider- ably below the maximum available; but still about twice as fast as the flat energy. Monitor units In this study, monitor units were not observed to differ significantly between mARC and IMRT, and for 6 MV vs. FFF 7 MV, respectively. This changes markedly if the isocenter is displaced from the centre of the PTV. In this case, the monitor units required by the 6 MV plans do not change systematically – sometimes increasing, some- times decreasing, but remaining within 30 MU of the original value. For the FFF 7 MV plans, however, the value increases strongly, sometimes exceeding 500 MU. This is plausible, because if the isocenter moves to the side or even outside of the PTV, the dose intensity de- creases with distance from the central axis, creating a constant dose gradient in the target. Additional monitor units are hence required to add dose at greater distance from the axis. This effect does not occur for the flat in- tensity profile of the 6 MV beam. If the isocenter is placed in the centre of the PTV, the dose profile of the FFF 7 MV beam peaks inside the PTV and only deviates from a flat profile at distances of several centimetres from the axis. It appears that the prostate PTV is suffi- ciently small to exhibit no notable difference in monitor units between 6 MV and 7 MV plans if the isocenter is centrally placed. If the PTV extended farther in the cra- niocaudal direction, it might be expected that the dose fall-off to the sides of the central axis – although symmet- rical – would also require more monitor units for the FFF Considering the treatment time, the question arises what the technical limit for the mARC operation may be. Given the results above, it might be imagined that treatment times could be further improved if the plans were irradiated with the maximum available dose rates (300 MU/min for 6 MV and 2000 MU/min for FFF 7 MV). In addition, the spacing of optimization points and distance of MLC leaf travel between successive arclets will influence treatment time. Current work at our institution Dzierma et al. Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 Page 6 of 8 Figure 2 Example dose-volume histogram (same patient as in Figure 1). Figure 2 Example dose-volume histogram (same patient as in Figure 1). (for an overview, see [15]). Monitor units While details differ between these studies – due on the one hand to variations in the considered prostate PTV contours, and on the other hand to different planning approaches – it was generally found that VMAT treatment offers at least as good quality plans as IMRT treatment, sometimes even with better spar- ing of organs at risk. Depending on whether one or two arcs were chosen for VMAT treatment and whether con- stant or variable dose rate irradiation was allowed, plan quality measures and OAR DVH values sometimes favoured IMRT, sometimes VMAT treatment [16-24]; how- ever, all studies observed a marked reduction in treatment time by VMAT treatment, and generally a drastic reduction in monitor units. is aiming to find the optimum mARC scenarios for fast irradiation, and the technical constraints imposed on treatment time (Dzierma et al., in prep.). From the technical/legal point of view, gantry rotation is re- strained to be no faster than 360° per minute, but only plan scenarios with hardly any MLC movement and few monitor units per arclet appear to be capable of achieving this speed. Future work will show where the practical limits for treatment times are, and treatment planning systems will then be evaluated by how closely they can approach this technical limit. Conclusion used, the smaller would be the improvement by any add- itional beam, with an optimum number of ca. 10–20 beams depending on the target size [26]. In fact, our in-house standards have evolved over the past few years from IMRT plans with 5–7 beams to 11–13 beams for prostate cancer. More beams are never used since they have not shown to yield any further benefit, which may explain why the mARC plan quality is not notably improved over the IMRT plans. Although small differences in plan quality exist, none of these were found to be clinically significant. Highly con- formal treatment plans could be created both by the use of flat 6 MV and FFF 7 MV energy, using IMRT or mARC. For all practical purposes, the FFF 7 MV energy and mARC plans are acceptable for treatment, allowing a drastic reduc- tion in treatment time from over 5 minutes to about half this value. As expected on physical grounds and based on past studies, scattered dose is reduced by the FFF 7 MV energy. Considering the monitor units, we do not observe a marked decrease for mARC vs. IMRT plans, whereas most studies considering VMAT vs. IMRT treatment do. The monitor units for the mARC plans are comparable to those found by, e.g., [6,16,19-21,23,24,27] all of whom except for Ost et al. [27] had considerably higher IMRT monitor units. It cannot be decided whether the low amount of MU also for IMRT is indebted to the plan- ning scenario (more degrees of freedom offered by 11 beam angles) or the planning system (the direct aper- ture optimization algorithm used in Prowess Panther has been observed to require fewer MUs for IMRT in comparison with other planning systems [28]). The low number of monitor units for our IMRT plans also entails relatively fast IMRT treatment (5:21 minutes for flat and 4:31 minutes for FFF beams), which is at the lower limit of what is observed in other studies (4–6 min [6,17,19,20,22,23,27,29,30]; 8 min [21,31]). mARC treatment times of 3:35 min for flat and 2:27 min for FFF treatment plans are slower than times reported for VMAT treatment with single arcs (1–2 minutes [6,17,19-21,27]; FFF: 60–90 sec [14,30,32]), but within the range of times found for two arcs (3–5 min [6,20,21,23]). Comparison with other studies Figure 3 Treatment time as a function of monitor units for the ten patients, with linear fits. Fit parameters are given Competing interests d Our department is Siemens reference center and customer user test site (CUT-Site). The present study was carried out outside the scope of the CUT-Site and after any contract about the mARC installation was terminated. No Siemens member had any participation or insight in this study. Authors’ contributions YD and FN designed the concept of the study. JP selected the patient collective and contoured the target volume and organs at risk. KB created the treatment plans and calculated the quality indices. JP assessed and approved the treatment plans. KB, YD and FN performed the statistical analysis. All authors participated in the discussion of the results. YD and JP drafted the manuscript. All authors read and approved the final manuscript. Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images. Comparison with other studies Only few studies have investigated the plan properties and treatment times associated with mARC planning [1,4,5]. Their observed treatment times for prostate can- cer are of the same order of magnitude as those reported here, again for comparable plan qualities between mARC and IMRT treatments. Our study explicitly created the scenarios in such a way to offer approximately the same number of degrees of freedom to both IMRT and mARC optimization pro- cesses, which may explain the similar quality outcome. Besides, it should be pointed out that the studies com- paring VMAT with IMRT all relied on IMRT plans with fewer fields – most chose 5 or 7 gantry angles for the IMRT, so it can be expected that our IMRT plans with 11 beams might yield better plans, hence closing the gap to rotational modulated treatment. Still, it has been ob- served that the plan quality can be improved by including more gantry angles even for the same number of segments [25], which is not surprising since it offers more freedom to the optimization from a geometrical point of view and might allow for better mARC plan quality even with a simi- lar number of free parameters for the optimization. How- ever, it also appears plausible that this effect should saturate for plans with many gantry angles – the more beams are Several past studies have evaluated plan quality for VMAT/RapidArc treatment as compared with IMRT Table 4 Linear fit parameters for treatment time vs. monitor units (Figure 2) Table 4 Linear fit parameters for treatment time Table 4 Linear fit parameters for treatment time vs. monitor units (Figure 2) Plan y-axis intercept (s) Slope (s/MU) Value Standard error Value Standard error 6 MV IMRT 216.7 30.5 0.263 0.076 7 MV IMRT 233.5 18.3 0.119 0.045 6 MV mARC 98.7 41.1 0.286 0.099 7 MV mARC 94.1 23.9 0.125 0.057 Dzierma et al. Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 Page 7 of 8 Figure 3 Treatment time as a function of monitor units for the ten patients, with linear fits. Fit parameters are given in Table 4. Figure 3 Treatment time as a function of monitor units for the ten patients, with linear fits. Fit parameters are given in Table 4. Figure 3 Treatment time as a function of monitor units for the ten patients, with linear fits. Fit parameters are given in Table 4. References Bortfeld T: The number of beams in IMRT – theoretical investigations and implications for single-arc IMRT. Phys Med Biol 2010, 55(1):83–97. 6. Guckenberger M, Richter A, Krieger T, Wilbert J, Baier K, Flentje M: Is a single arc sufficient in volumetric-modulated arc therapy (VMAT) for complex-shaped target volumes? Radiother Oncol 2009, 93(2):259–265. 6. Guckenberger M, Richter A, Krieger T, Wilbert J, Baier K, Flentje M: Is a single arc sufficient in volumetric-modulated arc therapy (VMAT) for complex-shaped target volumes? Radiother Oncol 2009, 93(2):259–265. 27. Ost P, Speleeers B, De Meerleer G, De Neve W, Fonteyne V, Villeirs G, De Gersem W: Volumetric arc therapy and intensity-modulated radiotherapy for primary prostate radiotherapy with simultaneous integrated boost to intraprostatic lesion with 6 and 18 MV: a planning comparison study. Int J Radiat Oncol Biol Phys 2011, 79:920–926. 7. Michalski J, Purdy J, Watkins-Bruner D: RTOG P-0126: A Phase III Randomized Study of High-Dose 3D-CRT/IMRT Versus Standard Dose 3D-CRT/IMRT in Patients Treated for Localized Prostate Cancer. Available online at: http://www. rtog.org/ClinicalTrials/ProtocolTable/StudyDetails.aspx?study=0126. 28. Sabatino M, Kretschmer M, Zink K, Würschmidt F: The impact of direct aperture optimization on plan quality and efficiency in complex head and neck IMRT. Radiat Oncol 2012, 7:7. 8. Gay HA, Barthold HJ, O’Meara E, Bosch WR, El Naga I, Al-Lozi R, Rosenthal SA, Lawton C, Lee WR, Sandler H, Zietman A, Myerson R, Dawson LA, Willett C, Kachnic LA, Jhingran A, Portelance L, Ryu J, Small W Jr, Gaffney D, Viswanathan AN, Michalski JM: Pelvic normal tissue contouring guidelines for radiation therapy: a radiation therapy Oncology Group Consensus Panel Atlas. Int J Radiat Oncol Biol Phys 2012, 83(3):e353–e362. 29. Tsai CL, Wu JK, Chao HL, Tsai YC, Cheng JC: Treatment and dosimetric advantages between VMAT, IMRT, and helical tomotherapy in prostate cancer. Med Dosim 2011, 36:264–271. 30. Fogarty GB, Ng D, Liu G, Haydu LE, Bhandari N: Volumetric modulated arc therapy is superior to conventional intensity modulated radiotherapy – a comparison among prostate cancer patients treated in an Australian centre. Radiat Oncol 2011, 6:108. 9. Michalski JM, Gay H, Jackson A, Tucker SL, Deasy JO: Radiation dose-volume effects in radiation-induced rectal injury. Int J Radiat Oncol Biol Phys 2010, 76:S123–S129. 10. Viswanathan AN, Yorke ED, Marks LB, Eifel PJ, Shipley WU: Radiation 10. Viswanathan AN, Yorke ED, Marks LB, Eifel PJ, Shipley WU: Radiation dose-volume effects of the urinary bladder. Int J Radiat Oncol Biol Phys 2010, 76:S116–S122. 31. Acknowledgements h k We thank two anonymous reviewers for their very constructive comments, which have greatly improved this manuscript. Received: 18 August 2014 Accepted: 4 November 2014 Page 8 of 8 Page 8 of 8 Dzierma et al. Radiation Oncology 2014, 9:250 http://www.ro-journal.com/content/9/1/250 21. Yoo S, Wu QJ, Lee WR, Yin FF: Radiotherapy treatment plans with RapidArc for prostate cancer involving seminal vesicles and lymph nodes. Int J Radiat Oncol Biol Phys 2010, 76:935–942. References 1. Kainz K, Chen GP, Chang YW, Prah D, Qi XS, Shukla HP, Stahl J, Li XA: A planning and delivery study of a rotational IMRT technique with burst delivery. Med Phys 2011, 38:5104–5118. 22. Davidson MTM, Blake SJ, Batchelar DL, Cheung P, Mah K: Assessing the role of volumetric modulated arc therapy (VMAT) relative to IMRT and helical tomotherapy in the management of localized, locally advanced, and post-operative prostate cancer. Int J Radiat Oncol Biol Phys 2011, 80(5):1550–1558. 2. Salter BJ, Sarkar V, Wang B, Shukla H, Szegedi M, Rassiah-Szegedi P: Rotational IMRT delivery using a digital linear accelerator in a very high dose rate ‘burst mode’. Phys Med Biol 2011, 56:1931–1946. 2. Salter BJ, Sarkar V, Wang B, Shukla H, Szegedi M, Rassiah-Szegedi P: Rotational IMRT delivery using a digital linear accelerator in a very high dose rate ‘burst mode’. Phys Med Biol 2011, 56:1931–1946. 3. Dzierma Y, Nuesken F, Licht N, Rübe C: A novel implementation of mARC treatment for non-dedicated planning systems using converted IMRT plans. Radiat Oncol 2013, 8:193. 3. Dzierma Y, Nuesken F, Licht N, Rübe C: A novel implementation of mARC treatment for non-dedicated planning systems using converted IMRT plans. Radiat Oncol 2013, 8:193. 23. Pasler M, Wirtz H, Lutterbach J: Impact of gantry rotation time on plan quality and dosimetric verification – Volumetric Modulated Arc Therapy (VMAT) vs. Intensity Modulated Radiotherapy (IMRT). Strahlenther Onkol 2011, 187:812–819. 4. Dzierma Y, Nuesken FG, Kremp S, Palm J, Licht NP, Rübe C: Commissioning and first clinical application of mARC treatment. Strahlenther Onkol 2014, 190:1046–1052. 24. Peters S, Schiefer H, Plasswilm L: A treatment planning study comparing Elekta VMAT aand fixed field IMRT using the varian treatment planning system eclipse. Radiat Oncol 2014, 9:153. 5. Spahn U, Prott FJ: Erste Erfahrungen mit der modulierten Rotationsbestrahlung mARC am Linearbeschleuniger ARTISTE (First experiences with the modulated rotational mARC treatment at the Artiste linear accelerator). Strahlenther Onkol Suppl 2013, 1:62–63. 25. Bratengeier K, Gainey MB, Flentje M: Fast IMRT by increasing the beam number and reducing the number of segments. Radiat Oncol 2011, 6:170. 25. Bratengeier K, Gainey MB, Flentje M: Fast IMRT by increasing the beam number and reducing the number of segments. Radiat Oncol 2011, 6:170. 26. Bortfeld T: The number of beams in IMRT – theoretical investigations and implications for single-arc IMRT. Phys Med Biol 2010, 55(1):83–97. 26. References Rao M, Yang W, Chen F, Sheng K, Ye J, Mehta V, Shepart D, Cao D: Comparison of Elekta VMAT with helical toomotherapy and fixed field IMRT: plan quality, delivery efficiency and accuracy. Med Phys 2010, 37:1350–1359. p y dose-volume effects of the urinary bladder. Int J Radiat Oncol Biol Phys 2010, 76:S116–S122. 11. Dzierma Y, Licht N, Nuesken F, Ruebe C: Beam properties and stability of a flattening-filter-free 7 MV beam–an overview. Med Phys 2012, 39(5):2595–2602. 32. Zwahlen DR, Lang S, Hrbacek J, Glanzmann C, Kloeck S, Najafi Y, Streller T, Studer G, Zaugg K, Luetolf UM: The Use of photon beams of a flattening filter-free linear accelerator for hypofractionated volumetric modulated Arc therapy in localized prostate cancer. Int J Radiat Oncol Biol Phys 2012, 83(5):1655–1660. 12. Paddick I: A simple scoring ratio to index the conformity of radiosurgical treatment plans. Technical note. J Neurosurg 2000, 93(Supp 3):219–222. 13. Dzierma Y, Nuesken FG, Fleckenstein J, Melchior P, Licht NP, Rübe C: Comparative planning of flattening-filter-free and flat beam IMRT for hypopharynx cancer as a function of beam and segment number. PLoS One 2014, 9(4):e94371. doi:10.1186/s13014-014-0250-2 Cite this article as: Dzierma et al.: mARC vs. IMRT radiotherapy of the prostate with flat and flattening-filter-free beam energies. Radiation Oncology 2014 9:250. 14. Gasic D, Ohlhues L, Brodin NP, Fog LS, Pommer T, Bangsgaard JP, Af Rosenschöld PM: A treatment planning and delivery comparison of volumetric modulated arc therapy with or without flattening filter for gliomas, brain metastases, prostate, head/neck and early stage lung cancer. Acta Oncol 2014, 53:1005–1011. 15. Teoh M, Clark CH, Wood K, Whitaker S, Nisbet A: Volumetric modulated arc therapy: a review of current literature and clinical use in practice. Br J Radiol 2011, 84:967–996. 16. Palma D, Vollans E, James K, Nakano S, Moiseenko V, Shaffer R, McKenzie M, Morris J, Otto K: Volumetric modulated arc therapy for delivery of prostate radiotherapy: comparison with intensity-modulated radiotherapy and three-dimensional conformal radiotherapy. Int J Radiat Oncol Biol Phys 2008, 72:996–1001. References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 17. Zhang P, Happersett L, Hunt M, Jackson A, Zelefsky M, Mageras G: Volumetric modulated arc therapy: planning and evaluation for prostate cancer cases. Int J Radiat Oncol Biol Phys 2010, 76:1456–1462. • Convenient online submission 18. Kjaer-Kristoffersen F, Ohlhues L, Medin J, Korreman S: RapidArc volumetric modulated therapy planning for prostate cancer patients. Acta Oncol 2009, 48:227–232. 19. Hardcastle N, Tome WA, Foo K, Miller A, Carolan M, Metcalfe P: Comparison of prostate IMRT and VMAT biologically optimized treatment plans. Med Dosim 2011, 36:292–298. 20. Wolff D, Stieler F, Wlzel G, Lorenz F, Abo-Madyan Y, Mai S, Herskind C, Polednik M, Steil V, Wenz F, Lohr F: Volumetric modulated arc therapy (VMAT) vs. serial tomotherapy, step-and-shoot IMRT and 3D-conformal RT for treatment of prostate cancer. Radiother Oncol 2009, 93:226–233.
https://openalex.org/W2803210097	https://link.springer.com/content/pdf/10.1007/s10661-018-6713-4.pdf	English	null	Evaluating organochlorine pesticide residues in the aquatic environment of the Lake Naivasha River basin using passive sampling techniques	Environmental monitoring and assessment	2,018	cc-by	7,387	Environ Monit Assess (2018) 190: 349 https://doi.org/10.1007/s10661-018-6713-4 Evaluating organochlorine pesticide residues in the aquatic environment of the Lake Naivasha River basin using passive sampling techniques Yasser Abbasi & Chris M. Mannaerts Received: 3 November 2017 /Accepted: 2 May 2018 /Published online: 18 May 2018 # The Author(s) 2018 Abstract Passive sampling techniques can improve the discovery of low concentrations by continuous collecting the contaminants, which usually go undetect- ed with classic and once-off time-point grab sampling. The aim of this study was to evaluate organochlorine pesticide (OCP) residues in the aquatic environment of the Lake Naivasha river basin (Kenya) using passive sampling techniques. Silicone rubber sheet and Speedisk samplers were used to detect residues of α- HCH, β-HCH, γ-HCH, δ-HCH, heptachlor, aldrin, heptachlor epoxide, pp-DDE, endrin, dieldrin, α-endo- sulfan, β-endosulfan, pp-DDD, endrin aldehyde, pp- DDT, endosulfan sulfate, and methoxychlor in the Malewa River and Lake Naivasha. After solvent extrac- tion from the sampling media, the residues were ana- lyzed using gas chromatography electron capture detec- tion (GC-ECD) for the OCPs and gas chromatography- mass spectrometry (GC-MS) for the PCB reference compounds. Measuring the OCP residues using the silicone rubber samplers revealed the highest concen- tration of residues (∑OCPs of 81 (± 18.9 SD) μg/L) to be at the Lake site, being the ultimate accumulation environment for surficial hydrological, chemical, and sediment transport through the river basin. The total OCP residue sums changed to 71.5 (± 11.3 SD) μg/L for the Middle Malewa and 59 (± 12.5 SD) μg/L for the Upper Malewa River sampling sites. The concentration sums of OCPs detected using the Speedisk samplers at the Upper Malewa, Middle Malewa, and the Lake Naivasha sites were 28.2 (± 4.2 SD), 31.3 (± 1.8 SD), and 34.2 (± 6.4 SD) μg/L, respectively. An evaluation of the different pesticide compound variations identi- fied at the three sites revealed that endosulfan sulfate, α-HCH, methoxychlor, and endrin aldehyde residues were still found at all sampling sites. However, the statistical analysis of one-way ANOVA for testing the differences of ∑OCPs between the sampling sites for both the silicone rubber sheet and Speedisk samplers showed that there was no significant difference from the Upper Malewa to the Lake site (P < 0.05). Finally, the finding of this study indicated that continued mon- itoring of pesticides residues in the catchment remains highly recommended. Keywords Pesticide residues . Passive sampling . Silicone rubber sheet . Speedisk . Lake Naivasha Y. Abbasi (*): C. M. Mannaerts Department of Water Resources (WRS), Faculty of Geo-information Sciences and Earth Observation (ITC), University of Twente (UT), P.O. Box 217, 7500AE, Enschede, The Netherlands e-mail: y.abbasi@utwente.nl C. M. Mannaerts e-mail: c.m.m.mannaerts@utwente.nl Introduction The first application of organochlorine pesticides such as DDT and dieldrin dates back to 1956 and 1961, respectively, but due to the long half-life and their bioaccumulation in animal body, they were banned C. M. Mannaerts e-mail: c.m.m.mannaerts@utwente.nl Environ Monit Assess (2018) 190: 349 349 Page 2 of 12 349 globally in 1976 (Keating 1983), except for regulated use of DDT for the control of malaria. After application of the chemicals, their residues can reach non-targets such as plants, soil, water, and sediment, by which these environmental compartments could be contaminated. Kaoga et al. (2013) explained that over 95% of applied insecticides and herbicides end up in non-target areas. This could potentially endanger the environment and also contribute to public health problems (Mutuku et al. 2014). Although most of the pesticides have a short half-life and are easily degradable, there are still persistent pesticides such as first-generation organo- chlorine pesticides (OCPs), which have long time half- life and are persistent in environment. Consequently, they can be washed off to water bodies and cause considerable environmental risk (Gitahi et al. 2002). Lakes and reservoirs are typical accumulation sites for runoff, sediment, and chemicals in catchments, and therefore, these aquatic environments are at risk of being contaminated. Bearing in mind that due to the threat of pesticides, residues pose to aquatic life and ecosystems, careful evaluation is needed. contaminants. Passive sampling provides means for continuous water quality monitoring from short term to long term (a week to some months) and allows determining time-weighted average (TWA) of contam- inant concentrations (Ahrens et al. 2015). The chemi- cal potential discrepancy between passive sampler me- dia and the dissolved pollutants in the aquatic phase causes a partitioning of contaminants between water and the sampler (Allan et al. 2009). Moreover, in comparison with organisms, which undergo biotrans- formation and changing physiological conditions, the uptake of pollutants using passive samplers is more feasible (Smedes and Booij 2012). These features of passive sampling facilitate chemical examination of surface and other water bodies and provide an alterna- tive approach to biomonitoring (Fox et al. 2010; Meyn et al. 2007; Munoz et al. 2010; Wille et al. 2011). There are various kinds of non-polar passive sam- plers, which have been used for evaluating organic contaminants in aquatic environments (Brockmeyer et al. 2015). Introduction Passive samplers trap the pollutants in a kinetic or equilibrium diffusion, in which the whole process including selective analyte, isolation, and pre- concentration occurs simultaneously (Vrana et al. 2005). The mass transfer of an analyte—an organic or inorgan- ic compound—proceeds until the equilibrium phase occurs or the sampling is finished (Górecki and Namieśnik 2002). Silicone rubber (SR) sheets and Speedisk samplers were selected in this study for the determination of organochlorine pesticides because sil- icone rubber samplers for more hydrophobic com- pounds and Speedisk samplers for more hydrophilic compounds are ideal samplers that have some advan- tages such as simple construction, robust for installing in the rivers or the Lake, cheap, and commonly available (Smedes et al. 2010). Aquatic monitoring programs are usually based on grab samples collected within a short time span. Grab sampling can only provide a snapshot of pollution levels (Hernando et al. 2007; Vrana et al. 2005) and also is associated with logistical and practical difficul- ties including transportation, filtration, extraction, and storage. Moreover, grab sampling as a way of moni- toring pesticide pollution in aquatic environments can- not encompass all of the changes in pollutant concen- trations (Ahrens et al. 2015). In other words, determin- ing the dynamic status of pollution accurately during low and high flows is not entirely feasible with grab sampling. Consequently, the outcomes do not directly relate to the average load of pollutants (Jordan et al. 2013). In spite of these facts, grab sampling is useful for finding information quickly. However, extending measurements to cover fluctuations in flow and pollut- ant concentrations requires increasing the sampling frequency and sample numbers, which is expensive and time consuming while the results remain uncertain (Rozemeijer et al. 2010). The Lake Naivasha catchment is a major agricultural areas in Kenya, and because of dense agricultural activ- ity and population, there is a high demand for pesticides. Although there was a decline in organochlorine pesti- cide imports into the country, there is still risk of these pesticides’ application in agricultural areas. Therefore, a monitoring plan for pesticide residue pollution in aquat- ic environments is necessary to evaluate their potential risk on ecosystems and humans in general. The aim of this research was to gain understanding in the organo- chlorine pesticide residue pollution and their spatial variation in the Lake Naivasha catchment using passive sampling techniques. Sampler preparation and installation Sampler preparation and installation Materials and methods Large AlteSil SR sheets were cut into pieces with 55 × 90 × 0.5-mm dimensions and about 100 cm2— both sides—surface area. The SR samplers were pre- cleaned in Soxhlet apparatus with ethyl acetate for at least 100 h to remove all chains of oligomers. Then, they were air dried and spiked with performance reference compounds (PRCs). Based on Smedes and Booij (2012), the SR samplers need to be spiked with at least six PRCs that have a sampler-water partition coefficient (logKpw) between 3.5 and 5.5 as well as a PRC that is rarely depleted (logKpw > 6) and a completely depleted PRC (logKpw < 3.3) for modeling water sampling rate and the concentration of the pollutants. Therefore, the applied PRCs in the SR samplers were BIP-D10, PCB001, PCB002, PCB003, PCB010, PCB014, PCB030, PCB050, PCB021, PCB104, PCB055, PCB078, PCB145, and PCB204. Then, the prepared SR samplers were Introduction Due to the challenges related to grab sampling, the passive sampling technique is considered as a promis- ing alternative method for measuring pollutants in aquatic environments. This method allows the accu- mulation of contaminants in the samplers, making it possible to determine very low concentrations of Environ Monit Assess (2018) 190: 349 Page 3 of 12 349 Study area Lake Naivasha catchment is located in the eastern part of the Rift Valley region in Kenya with an area of about 3400 km2. The eastern rift has a tropical climate with two dry and two rainy seasons. The upper and middle parts of the catchment are mostly subjected to small- holder mixed farming for producing various crops. Moreover, there are various local dwellings in villages and towns all around the catchment that can influence the rivers and the Lake water quality located in the lower catchment. Input from upstream into the Lake includes water from the Malewa, Karati, and Gilgil Rivers plus surface runoff that drains from the catchment and reaches the Lake. But as the Malewa River accounts for approximately 80% of the inflow into Lake Naivasha, the samplers were installed in the Upper Malewa, Middle Malewa River, and the Lake (Fig. 1). Fig. 1 The study area and locations of passive samplers (P1, P2, and P3 are Upper Malewa, Middle Malewa, and the Lake sites, respectively) Fig. 1 The study area and locations of passive samplers (P1, P2, and P3 are Upper Malewa, Middle Malewa, and the Lake sites, respectively) Environ Monit Assess (2018) 190: 349 349 Page 4 of 12 349 kept in air-tightened amber glass bottles in the freez- er (−20 °C) until installation. with methanol and water) and the water of the same sampling site. Then, the samplers were kept in a cool box (about 5 °C) during transfer and at −20 °C in the laboratory till treatment and analysis (Monteyne et al. 2013; Smedes and Booij 2012). Speedisk extraction samplers, H2O-philic DVB low capacity (0.6 g) produced by Avantor, were also used for more hydrophilic substances. The Speedisks were con- ditioned by eluting them slowly with 15 mL dichloro- methane (HPLC grade, 99.9%), 10 mL acetone (HPLC grade, 99.5%), and 20 mL distilled water, sequentially. They were then stored in a bottle of purified water and stored at + 4 °C until deployment. Fig. 2 Mounting Speedisk (SD; left) and silicone rubber sheet (SR; right) passive samplers for deployment Calculations The amounts of PRC fraction (fexp) indicates sampling rate and was estimated as f exp ¼ Nt N0 ð1Þ f exp ¼ Nt N0 ð1Þ where Nt and N0 are the PRC amounts (ng) in the exposed and the reference samplers, respectively. Booij and Smedes (2010) showed that f is a continuous function of Kpw and the sampling rate (Rs): Extraction and analysis 2 Mounting Speedisk (SD; left) and silicone rubber sheet (SR; right) passive samplers for deployment Environ Monit Assess (2018) 190: 349 Page 5 of 12 349 time of 15 min), and 200 to 275 °C (at 30 °C/min and hold time of 5 min). The carrier gas was helium and nitrogen was used as make-up gas with a continuous stream of 2 mL/min. The injection mode was pulsed- splitless with a volume of 1 μL. The column was a DB-5 (Agilent, USA) with length of 30 m, internal diameter of 0.32 mm, and film thickness of 0.25 μm. The calibration of the machine was done using the standards of organo- chlorine pesticides (purity of more than 99%) in 10 concentration levels of 1, 5, 10, 50, 100, 200, 400, 600, 800, and 1000 μg/L. Finally, the quality control of the results was done by triplication for all the samples, and determination of recovery rates from blank treatments. The column of the GC-MS was also DB-5 (length 30 m, ID 0.25 mm, film 0.25 μm). The temperature program was set as initially 70 °C for 1 min, then ramp 1 increase 20 °C/min to 120 min, hold time 0 min; ramp 2 increase 6 °C/min to 250 °C, hold time 0 min; and ramp 3 17.5 °C/ min to 300 °C, hold time 2.48 min. The low detection limit was also 1 μg/L for all the PRCs. time of 15 min), and 200 to 275 °C (at 30 °C/min and hold time of 5 min). The carrier gas was helium and nitrogen was used as make-up gas with a continuous stream of 2 mL/min. The injection mode was pulsed- splitless with a volume of 1 μL. The column was a DB-5 (Agilent, USA) with length of 30 m, internal diameter of 0.32 mm, and film thickness of 0.25 μm. The calibration of the machine was done using the standards of organo- chlorine pesticides (purity of more than 99%) in 10 concentration levels of 1, 5, 10, 50, 100, 200, 400, 600, 800, and 1000 μg/L. Finally, the quality control of the results was done by triplication for all the samples, and determination of recovery rates from blank treatments. The column of the GC-MS was also DB-5 (length 30 m, ID 0.25 mm, film 0.25 μm). Extraction and analysis The temperature program was set as initially 70 °C for 1 min, then ramp 1 increase 20 °C/min to 120 min, hold time 0 min; ramp 2 increase 6 °C/min to 250 °C, hold time 0 min; and ramp 3 17.5 °C/ min to 300 °C, hold time 2.48 min. The low detection limit was also 1 μg/L for all the PRCs. The sampling rate was estimated by combining Eqs. (2) and (3) and fitting the retained fraction and KpwM0.47 using a solver package. The non-linear least squares (NLSs) method, which takes all of the PRCs into account, was applied for this aim (Booij and Smedes 2010). Booij et al. (2007) showed that the amount of a target compound in the sampler can be presented as Nt ¼ Cw:Kpwm 1−exp −Rst Kpwm ð4Þ ð4Þ Therefore, by adjusting Eqs. (3) and (4), the concen- tration of compounds was determined as Cw ¼ N t Kpwm 1−exp − FAt M 0:47Kpwm ð5Þ ð5Þ Finally, the standard deviations (SDs) of the sam- pling rates as well as the pesticide concentration were calculated and included to the results. The statistical analysis of one-way ANOVA was also applied to ex- plore the differences of total OCPs between the sam- pling sites at 95% confidence. This examination deter- mined the spatial variation from the upper catchment to the Lake for the results of both kinds of passive samplers. Extraction and analysis Various solvents such as acetonitrile, hexane, acetone, dichloromethane, and methanol were used to extract the non-polar contaminants from the samplers. The solvents were all of HPLC grade (> 99% purity) in order to extract the studied OCPs. All of the procedures for both the exposed passive samplers and the blanks (control sam- plers) such as sampler extraction by Soxhlet apparatus, cleanup, concentration, and instrumental analysis were done according to the guidelines by Smedes and Booij (2012) and standard laboratory methods. After solvent extraction from the sampling media, the residues were determined using a gas chromatograph (Agilent 6890N) in combination with an electron capture detector (Agilent μECD) and an auto sampler (Agilent 7683 Series injec- tor) for the OCPs (in Department of Chemistry at Uni- versity of Nairobi, Kenya), and a gas chromatography (Agilent 7890A) coupled with a mass spectrometer (Agilent 7000 Series Triple Quadrupole MS detector) that had a possibility to measure with an MSMS method for the PRCs in Deltares (TNO laboratory, Netherlands). The temperature program of GC-μECD was set as initially 90 °C (3 min), then 90 to 200 °C (at 30 °C/min and hold At the sampling sites, including two sites in the Malewa River and one site in the Lake Naivasha as represented in Fig. 1, three sets of silicone rubber sheets and three sets of Speedisk passive samplers were installed for monitoring the concentrations of α-HCH, β-HCH, γ- HCH, δ-HCH, heptachlor, aldrin, heptachlor epoxide, α- endosulfan, pp-DDE, endrin, dieldrin, β-endosulfan, pp- DDD, endrin aldehyde, pp-DDT, endosulfan sulfate, and methoxychlor in the Malewa River and the Lake Naivasha. Both the Speedisk and silicone rubber sheet samplers were mounted on metal wire mesh (Fig. 2) and immediately deployed in water. Additionally, one sam- pler was exposed to the air while installing the samplers, as reference sampler. Passive samplers were deployed in the water for 1 month from 20 June to 20 July 2016, during the long rainy season when most of the agricul- tural activity and use of pesticides occur. After 1 month, the samples were collected from the sampling sites. As they were covered by some fouling or algae, they were cleaned using a pre-treated scourer (washed and rinsed Fig. Results and discussion The amounts of remaining PRCs with a logKpw of less than 4.2, such as PCB001 and BIP-D10, occurred on less than 20% of the sam- plers. The PRCs of PCB014 and PCB104 with a logKpw of more than 5.1 showed a variation of 73% (± 16.7 SD) to 102% (± 1.1 SD), which was in agree- ment with the results of the study by Monteyne et al. (2013). They indicated that the dissipation of more than 80% and less than 20% of the PRCs leads to difficulties in determining the initial and the final ratio of the PRCs on the samplers. Therefore, it could be concluded that the PRCs with a logKpw of 4.2 (PCB002) to 5.2 (PCB030) would be the most appropriate ones for cal- culating sampling rate. Moreover, as was concluded in other literature (Allan et al. 2009; Monteyne et al. 2013), the transition between linear and equilibrium phases occurred for the PRCs with logKpw between 4.2 and 5.2. Therefore, PCB010 and other compounds with this range of logKpw were still releasing and the sampling was continued. site was 6.2 (± 0.7 SD) L/day, an intermediate result compared to other sites. Silicone sheets have been used mostly for monitoring the PAHs and PCBs in marine aquatic environments. However, comparing the results of this study with other literature showed that these results were comparable with the study by Harman et al. (2009), who reported sampling rates between 4.1 and 14.8 L/day for a duration of 6 weeks. The data of sampling rates were used to determine the pesticide concentrations in the water (Cw). This approach allowed the use of passive sampling technique to monitor non-polar compounds. Calculating the concentration of OCPs with SR samplers showed that the total amount of α-HCH, β-HCH, γ-HCH, δ-HCH, heptachlor, aldrin, isodrin, heptachlor epoxide, α-endosulfan, pp-DDE, en- drin, dieldrin, β-endosulfan, pp-DDD, endrin aldehyde, pp-DDT, endosulfan sulfate, and methoxychlor at the Lake site was the highest with a total amount of organo- chlorine pesticide residue (∑OCPs) of 81 (± 18.9 SD) μg/L. This total amount was 71.5 (± 11.3 SD) and 59 (± 12.6 SD) μg/L for the Middle Malewa and the Upper Malewa river sites, respectively. The reason of reporting the results as a summation of OCPs is that the individual concentrations of the OCPs were mostly low ranging from below detection limit to 56 μg/L. Results and discussion Frequent measurements of different parameters at the sampling sites during sampler exposure time are pre- sented in Table 1. It was found that the average acidity of water in the Malewa River and the Lake was between 7 and 7.8, and no remarkable difference was found. Moreover, the effect of water temperature on sampling rate has been studied by Booij et al. (2003), and they showed that sampling rate at 30 °C was three times more than 20 °C. This issue demonstrates the relations be- tween water temperature and up taking the contami- nants. However, by calculating the sampling rates, the effect of different factors (e.g., oxygen saturation, salin- ity, conductivity, temperature, pH) on the sampler per- formance is taken to account. f cal ¼ e −Rst Kpwm ð2Þ f cal ¼ e −Rst Kpwm ð2Þ where Kpw is the sampler-water partition coefficient (L/kg), Rs is the sampling rate (L/day), m is the sampler weight (kg), and t is the exposure time (days). Rusina et al. (2010) demonstrated that sampling rate was a function of the hydrodynamic situation and the sampler surface area as well as the PRC molar mass (M). There- fore, their proposed Eq. (3) was used to demonstrate the relationship between these factors: The results of analysis showed that after 30 days of passive sampler deployment, the average of minimum remaining PRCs on the silicone samplers was 4.7% Rs ¼ FA M 0:47 ð3Þ Rs ¼ FA M 0:47 Rs ¼ FA M 0:47 ð3Þ Environ Monit Assess (2018) 190: 349 349 Page 6 of 12 Table 1 Physicochemical properties of water at the sampling sites Location/parameter pH T (∘C) EC (μS/cm) Sal. (‰) Sat. O2 (%) The Lake Average 7.8 20.1 352.2 0.11 87.1 Minimum 6.8 18.6 309.0 0.10 53.7 Maximum 8.6 21.0 366.0 0.12 104.8 Middle Malewa Average 7.4 16.3 147.6 0.05 103.6 Minimum 6.7 15.0 111.0 0.04 100.3 Maximum 8.6 18.0 170.0 0.06 106.4 Upper Malewa Average 7.7 16.3 150.4 0.05 103.2 Minimum 7.0 15.6 120.0 0.04 101.7 Maximum 8.9 17.2 174.2 0.06 105.0 EC electrical conductivity, Sal. salinity, Sat.O2 oxygen saturation Table 1 Physicochemical properties of water at the sampling sites EC electrical conductivity, Sal. salinity, Sat.O2 oxygen saturation (± 4.1 SD) for BCP-d10 and the maximum average was 97% (± 7.4 SD) for PCB204. Results and discussion The results of NLS model showed that there was a good fit between the measured and calculated PRC frac- tions (Fig. 3). Inclusion of a PRC with a low logKpw such as BIP-D10, which has a logKpw of 3.6, and PCB204, which has a high logKpw of 7.6, as well as other PRCs within this range led to a sigmoid trend among the retained fractions and log(Kpw.M0.47). With this approach, the results showed a minimum sampling rate occurring in the samplers that deployed in Lake Naivasha with 1.9 (± 0.4 SD) L/day and a maximum rate at the Middle Malewa river site with 13.1 (± 1.7 SD) L/ day. The average sampling rate at the Upper Malewa river The variation in pesticides found at the three sam- pling sites using SR samplers was also investigated to evaluate possible differences in pesticide residue occur- rence in different parts of the catchment (Fig. 4). Al- though there was an increasing trend from the Upper Malewa to the Lake, the results of one-way ANOVA for the means of the three sampling sites using SR samplers showed that there was not a significant spatial variations Environ Monit Assess (2018) 190: 349 Page 7 of 12 349 Page 7 of 12 349 Fig. 3 Example diagram of logKpw versus retained PRC frac- tions (left) and difference of calculated and measured (calc.) (right). The drawn line represents the best non-linear square for two example sites. RSA1 and RSA7 are example samples in the Lake and in Malewa River, respectively Fig. 3 Example diagram of logKpw versus retained PRC frac- tions (left) and difference of calculated and measured (calc.) (right). The drawn line represents the best non-linear square for two example sites. RSA1 and RSA7 are example samples in the Lake and in Malewa River, respectively respectively, that accounted, respectively, for 69, 55, and 58% of ∑OCPs in these sites. The second major pesti- cide residue found on the SR samplers at all of the sites was α-HCH. The concentration of this pollutant varied from 19.3 (± 6.7 SD) μg/L at the Middle Malewa river site (27% of the ∑OCPs) to the amount of 11.3 (± 4.8 SD) μg/L at the Lake site. α-HCH is an isomer of hexachlorocyclohexane (HCH) that has different iso- mers, and the main ones are α-HCH, β-HCH, γ-HCH, and δ-HCH. Results and discussion All of these isomers are insecticides that are mostly used on fruit, vegetables, and animals. α-HCH is by-product of lindane, but due to the persistence in environment and bioaccumulation, it has been classified as persistent organic pollutant (POP) by Stockholm Convention on Persistent Organic Pollutants in 2009 (ATSDR 2005). Methoxychlor was also found in the SR samplers at all sampling sites. It is an insecticide that (P > 0.05). Apart from application of pesticides in the down part of the catchment, as the hydrological stream flow and suspended sediment transport processes is the main reason for agrochemical movement from the upper part of the catchment to the down part, the increasing concentration gradient to the Lake could be due to the effect of downstream transport and accumulation of pesticides. The results showed that α-HCH, endosulfan sulfate, and methoxychlor formed the most prevalent pesticide’s residues. They were detected in almost all of the samplers, and their concentrations generally in- creased from the Upper Malewa to the Lake site. Based on the results of SR samplers, endosulfan sulfate formed the largest component of pesticide residue in the study area with concentrations of 56 (± 18 SD), 39.3 (± 29.3 SD), and 34.2 (± 11.8 SD) μg/L in the Lake Naivasha, Middle Malewa river, and Upper Malewa river sites, Environ Monit Assess (2018) 190: 349 349 Page 8 of 12 Fig. 4 Distribution of organochlorine pesticide residues on the silicone rubber (SR) and Speedisk (SD) passive sampling media at the three sampling sites (based on June–July 2016 sampling campaign data) Fig. 4 Distribution of organochlorine pesticide residues on the silicone rubber (SR) and Speedisk (SD) passive sampling media at the three sampling sites (based on June–July 2016 sampling campaign data) Fig. 4 Distribution of organochlorine pesticide residues on the silicone rubber (SR) and Speedisk (SD) passive sampling media at the three sampling sites (based on June–July 2016 sampling campaign data) pesticide that was found in the SR samplers at all of the sites. Comparing the results from the SR samplers at different sites showed that the Middle Malewa river site had most different kinds of applied pesticides. In addi- tion to the mentioned pesticides that were found at the Lake and Upper Malewa river sites, β-HCH, endrin, β- endosulfan, pp-DDD, and pp-DDE were found at the Middle Malewa river site. Results and discussion pp-DDD and pp-DDE, oc- curring with a concentration of less than 1 μg/L (almost 1% ratio of ∑OCPs) at the Middle Malewa river site, are the metabolite of DDT, which may originate from pes- ticide application in the past still present in the environ- ment. It is noticeable that DDT/(DDD + DDE) ratio is has a wide range of application for controlling the insects on crops, livestock, and homes. It dissolves in the water or evaporates into air very rarely, and once it reaches the ground, it sticks to the soil particles that can be transported to water bodies by runoff. The process of degradation in the environment is slow and may take several months (ATSDR 2002a). The ratio of other pesticides occurred in very low percentages. DDT, for instance, accounts for a very low percentage (1–2%) of residue, and this finding is in agreement with previous studies, indicating that the use of this pesticide has significantly decreased (Gitahi et al. 2002). Although in very low concentration, endrin aldehyde was another has a wide range of application for controlling the insects on crops, livestock, and homes. It dissolves in the water or evaporates into air very rarely, and once it reaches the ground, it sticks to the soil particles that can be transported to water bodies by runoff. The process of degradation in the environment is slow and may take several months (ATSDR 2002a). The ratio of other pesticides occurred in very low percentages. DDT, for instance, accounts for a very low percentage (1–2%) of residue, and this finding is in agreement with previous studies, indicating that the use of this pesticide has significantly decreased (Gitahi et al. 2002). Although in very low concentration, endrin aldehyde was another Environ Monit Assess (2018) 190: 349 Page 9 of 12 349 an indication of DDTapplication history that the amount of less than 1 means that there might not be current input of the parent DDT into the study area and vice versa (Gbeddy et al. 2012). The results showed that this ratio was less than 1 for the sampling sites. produced which are similar to their parents. The residue of these chemicals that are not dissolved easily in water can stick to soil particles and remain in environment up to 15 years (ATSDR 2002b). Results and discussion Passive samplers accumulate pesticide residues from the water during deployment; therefore, they may be more useful than grab sampling for finding OCPs. Moreover, as the behavior of the passive sam- plers for accumulating the contaminants mimics the bioaccumulation by organisms (e.g., uptake of pesti- cides by aquatic biota like fish), the outcomes of passive sampling studies are more comparable with biomonitoring ones (Smedes and Booij 2012). There- fore, the results of this study can be compared with the study by Gitahi et al. (2002) that explored pesti- cide contamination in water resources of Naivasha using fish samples. Their study about organochlorine pesticide pollution in various species of fish, water, and sediment samples in Lake Naivasha demonstrat- ed different levels of lindane, dieldrin, β-endosulfan, and aldrin in the fat of fish. Their results showed a technical use of these pesticides in the studied area, which would agree with the findings of this study. Considering the sampling rates of Speedisk samplers and the total of pesticides taken up by these samplers, the maximum amount of pesticides was ∑OCPs = 34.2 (± 6.5 SD) μg/L that was found at the Lake Naivasha site. The amounts of ∑OCPs from the Speedisk sam- plers at Middle Malewa and Upper Malewa sites were 31.3 (± 1.8 SD) and 28.2 (± 4.2 SD) μg/L, respectively. Based on the one-way ANOVA results of Speedisks for exploring the spatial variations of the OCPs at the sam- pling sites, these amounts were not significantly differ- ent (P > 0.05). Evaluating pesticide variation in the studied area by Speedisk samplers also demonstrated that α-HCH oc- curred at all of the sampling sites (Fig. 4). The Lake site, with a concentration of 16.1 (± 5.1 SD) μg/L, which is equivalent to 47% of ∑OCPs, revealed the highest mea- sured amount. The concentration of this pesticide’s res- idue was 13 (± 1.9 SD) μg/L in the Upper Malewa river and decreased to the 5.9 (± 1.8 SD) μg/L in the Middle Malewa river(19% of ∑OCPs). Although the Middle Malewa and Upper Malewa river sites were situated in the same river, the sites were placed far apart to discover the effect of the surrounding agricultural areas of the sampling sites on the pollution situation of the Malewa River. Results and discussion The selected site location in the Lake Naivasha was also at the opposite side of the Lake to the Malewa river estuary, in order to minimize the effect of Malewa River on the Lake Naivasha sampling site. Therefore, the results of each of the sites can be said to be mostly related to the pollution in adjacent areas. Moreover, although the interview with the farmers about the pesti- cides use for controlling any kind of diseases in their products (e.g., cabbage, tomato, potato, maize) did not show any OCP application, the results of sampler anal- ysis demonstrated the OCP residues in the sampling sites. Nearly all of the explored HCH isomers were found in the Middle and Upper Malewa Rivers. It is noticeable that some of the OCPs such as α-HCH, β- HCH, γ-HCH, δ-HCH, pp-DDT, pp-DDE, and pp- DDD that are metabolites of HCH and DDT can remain in the environment for an extremely long time by accu- mulating in different environmental compartments. For instance, when DDT is broken down by microorganisms or under environmental condition, DDE and DDD are Based on the report of the Pest Control Products Board of Kenya PCPB (2008), import and use of many of the studied pesticides have been discontinued. How- ever, different studies (Gitahi et al. 2002; Onyango et al. 2014) indicated application of these pesticides in the Lake basin. Lindane was a commonly used pesticide in Kenya. It was used as insecticide and for seed dress- ing. HCH and its isomers are the main pesticide residues found in the Speedisk samplers. These results seem to indicate the use of HCH and its isomers in the Lake Naivasha catchment. Endosulfan sulfate is an oxidation product of endosulfan, which has a high acute toxicity and can be potentially bioaccumulated. Because of the threats of endosulfan and its isomers (α-endosulfan, β- endosulfan, and endosulfan sulfate) to human health and the environment, there was a global ban on its applica- tion under the Stockholm Convention. Based on Camacho-Morales and Sánchez (2015), the estimated half-life time of these chemicals (endosulfan and endo- sulfan sulfate) can vary from 9 months to 6 years. How- ever, endosulfan sulfate was found in the SR samplers at all of the sampling sites. 349 Page 10 of 12 Fig. 5 Comparison of measured pesticide residue concentrations at the three sites studied to WHO drinking water standards 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 Aldrin gamma-HCH pp-DDE pp-DDT pp-DDD Dieldrin Endosulfan Sulfate Endrin Methoxychlor Heptachlor Pescides Con. (μg/L) Standard limit The Lake Middle Malewa Upper Malewa 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 Aldrin gamma-HCH pp-DDE pp-DDT pp-DDD Dieldrin Endosulfan Sulfate Endrin Methoxychlor Heptachlor Pescides Con. (μg/L) Standard limit The Lake Middle Malewa Upper Malewa the WHO drinking water standard and limits (Fig. 5). Results of this study are in agreement with another recent pesticide residue study in the Lake basin by Onyango et al. (2015). They reported on 4,4-DDT, 2,4-DDE, 4,4-DDD, γ-HCH, α-HCH, and aldrin con- tamination in the aquatic environment of the Lake Naivasha catchment and concluded that there was no potential effect of these pesticides on human health in drinking water. However, because of bioaccumulation of pesticides in aquatic organisms and the risk of enter- ing the higher food web and chain, the concentration of pesticide residues, even in low concentrations, needs to be monitored continuously. useful technique that could contribute to environmental monitoring and pollution assessment of water resources in the studied area. Acknowledgments This study commenced within the framework of the Integrated Water Resources Assessment Project—Naivasha or IWRAP project—and was completed in cooperation with the BWaterschap Noorderzijlvest^ (the Netherlands) with support from the WWF (World Wildlife Fund for nature, Nairobi office) and the Water Resources Management Authority (WRMA) of Naivasha (Kenya). Passive samplers were prepared and dedicated by Deltares (the Netherlands), and chemical pesticide residue analysis was carried out in Department of Chemistry at University of Nairobi and in Deltares (TNO) laboratories (Utrecht, Netherlands). Authors would like to express thanks to Dr. Jasperien de Weert (Deltares Company, Utrecht, Netherlands), Prof. Onyari John Mmari, Dr. Madadi Vincent Odongo, and Mr. Enock Osoro (University of Nairobi) and the people in the WWF and WRMA, who all facilitated this study. Conclusions This study investigated organochlorine pesticide resi- dues in surface water resources on Naivasha, Kenya using passive sampling techniques. Analysis of OCP concentrations showed that the total amounts at the Lake site was highest. This could be due to the fact that the Lake was the final accumulation site for runoff and suspended sediments from the basin. The ∑OCPs for the Middle Malewa and the Upper Malewa river sites represented the second and third levels, respectively. The results showed that endosulfan sulfate was the main pesticide residue found at all of the sampling locations. The results from the SR samplers showed that there was also contamination by α-HCH, endrin aldehyde, and methoxychlor at all of the sites. Finally, it was concluded that continuous monitoring of OCPs and other pesticides using passive sampling was a very Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestrict- ed use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Results and discussion Comparing the pollution levels of pesticides in the water for different sampling sites with the drinking water standards criteria (WHO 2011) showed that the concentrations of all the studied pesticides were below 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 Aldrin gamma-HCH pp-DDE pp-DDT pp-DDD Dieldrin Endosulfan Sulfate Endrin Methoxychlor Heptachlor Pescides Con. (μg/L) Standard limit The Lake Middle Malewa Upper Malewa Fig. 5 Comparison of measured pesticide residue concentrations at the three sites studied to WHO drinking water standards 349 Page 10 of 12 Environ Monit Assess (2018) 190: 349 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 Aldrin gamma-HCH pp-DDE pp-DDT pp-DDD Dieldrin Endosulfan Sulfate Endrin Methoxychlor Heptachlor Pescides Con. (μg/L) Standard limit The Lake Middle Malewa Upper Malewa Fig. 5 Comparison of measured pesticide residue concentrations at the three sites studied to WHO drinking water standards 349 Page 10 of 12 Environ Monit Assess (2018) 190: 349 Environ Monit Assess (2018) 190: 349 References Temperature-dependent uptake rates of nonpolar or- ganic compounds by semipermeable membrane devices and low-density polyethylene membranes. Environmental Science and Technology, 37(2), 361–366. https://doi. org/10.1021/es025739i. Monteyne, E., Roose, P., & Janssen, C. R. (2013). Application of a silicone rubber passive sampling technique for monitoring PAHs and PCBs at three Belgian coastal harbours. Chemosphere, 91(3), 390–398. https://doi.org/10.1016/j. chemosphere.2012.11.074. g Booij, K., Vrana, B., & Huckins, J. N. (2007). Chapter 7 theory, modelling and calibration of passive samplers used in water monitoring. Comprehensive Analytical Chemistry, 48(6), 141–169. https://doi.org/10.1016/S0166-526X(06)48007-7. Munoz, I., Martinez Bueno, M. J., Aguera, A., & Fernandez-Alba, A. R. (2010). Environmental and human health risk assess- ment of organic micro-pollutants occurring in a Spanish marine fish farm. Environmental Pollution, 158(5), 1809– 1816. https://doi.org/10.1016/j.envpol.2009.11.006. Brockmeyer, B., Kraus, U. R., & Theobald, N. (2015). Accelerated solvent extraction (ASE) for purification and extraction of silicone passive samplers used for the monitor- ing of organic pollutants. Environmental Science and Pollution Research., 22, 19887–19895. https://doi. org/10.1007/s11356-015-5192-1. Mutuku, M., Njogu, P., & Nyagah, G. (2014). Assessment of pesticide use and application practices in tomato based Agrosystems in Kaliluni sub location, Kathiani District, Kenya. JAGST, 16(2), 34–44. Fox, J. T., Adams, G., Sharum, M., & Steelman, K. L. (2010). Passive sampling of bioavailable organic chemicals in Perry County, Missouri cave streams. Environmental Science and Technology, 44(23), 8835–8841. https://doi.org/10.1021 /es1019367. Onyango, J., Kreuzinger, N., Yillia, P., & Kitaka, N. (2014). Potential risks of pesticide application in Kenya : Case of Lake Naivasha catchment. In 7th International Young Water Professional Conference. Taipei, Chinese Taiwan. Gbeddy, G., Yeboah, P., Carboo, D., Doamekpor, L., Afful, S., Nartey, V., et al. (2012). Organochlorine pesticide residues in African catfish muscle , Nile tilapia muscle and gills from the middle Volta basin , Kpando Torkor , Ghana and their poten- tial health risks to humans. Elixir Agriculture, 49(July), 9724–9730. Onyango, J., Kreuzinger, N., & Kitaka, N. (2015). Pesticides residues contamination in Lake Naivasha catchment. Kenya: AVAkademikerverlag. PCPB, (Pest Control Products Board). (2008). List of Pest control products provisionally registered by the Pest control products board for use in Kenya. Nairobi, Kenya. Gitahi, S. M., Harper, D. M., Muchiri, S. M., Tole, M. P., & Ng’ang’a, R. N. (2002). Organochlorine and organophospho- rus pesticide concentrations in water, sediment, and selected organisms in Lake Naivasha (Kenya). Hydrobiologia, 488, 123–128. https://doi.org/10.1023/A:1023386732731. Rozemeijer, J., Van Der Velde, Y., De Jonge, H., Van Geer, F., Broers, H. P., & Bierkens, M. References Ahrens, L., Daneshvar, A., Lau, A. E., & Kreuger, J. (2015). Characterization of five passive sampling devices for moni- toring of pesticides in water. Journal of Chromatography A, 1405, 1–11. https://doi.org/10.1016/j.chroma.2015.05.044. Allan, I. J., Booij, K., Paschke, A., Vrana, B., Mills, G. A., & Greenwood, R. (2009). Field performance of seven passive sampling devices for monitoring of hydrophobic substances. Environ Monit Assess (2018) 190: 349 of 12 349 Page 11 of 12 349 349 Environmental Science & Technology, 43(14), 5383–5390. https://doi.org/10.1021/es900608w. Environmental Science & Technology, 43(14), 5383–5390. https://doi.org/10.1021/es900608w. Albanis, T. (2007). Passive sampling techniques for monitor- ing organic contaminants in aquaculture environment. In Proceedings of the 10th International Conference on Environmental Science and Technology (pp. B276–B283). Albanis, T. (2007). Passive sampling techniques for monitor- ing organic contaminants in aquaculture environment. In Proceedings of the 10th International Conference on Environmental Science and Technology (pp. B276–B283). ATSDR, (Agency for Toxic Substances and Disease Registry). (2002a). Toxicological profile for Methoxychlor. U.S. Department of Health and Human Services, Public Health Service. Jordan, P., Cassidy, R., Macintosh, K. a., & Arnscheidt, J. (2013). Field and laboratory tests of flow-proportional passive sam- plers for determining average phosphorus and nitrogen con- centration in rivers. Environmental Science & Technology, 47(5), 2331–2338. https://doi.org/10.1021/es304108e. ATSDR, (Agency for Toxic Substances and Disease Registry). (2002b). Toxicological profile for DDT, DDE, and DDD. In U.S. Public Health Service: Department of Health and Human Services. Kaoga, J., Ouma, G., & Abuom, P. (2013). Effect of farm pesti- cides on water quality in Lake Naivasha. Kneya. American Journal of Plant Physiology, 8, 105–113. https://doi. org/10.3923/ajpp.2013.105.113. ATSDR, (Agency for Toxic Substances and Disease Registry). (2005). Toxicological profile for alpha-, beta-, gamma-, and Delta-Hexachlorocyclohexane. U.S. Department of Health and Human Services, Public Health Service. Keating, M. I. (1983). Tick control by chemical ixodicides in Kenya: A review 1912 to 1981. Tropical Animal Health and Production, 15(1), 1–6. https://doi.org/10.1007 /BF02250752. Booij, K., & Smedes, F. (2010). An improved method for estimat- ing in situ sampling rates of nonpolar passive samplers. Environmental Science and Technology, 44(17), 6789– 6794. https://doi.org/10.1021/es101321v. Meyn, A., White, P. S., Buhk, C., & Jentsch, A. (2007). Environmental drivers of large, infrequent wildfires: The emerging conceptual model. Progress in Physical Geography, 31(3), 287–312. https://doi.org/10.1177 /0309133307079365. p g Booij, K., Hofmans, H. E., Fischer, C. V., & Van Weerlee, E. M. (2003). WHO. (2011). WHO guidelines for drinking-water quality. WHO chronicle, (Fourth Edition). https://doi.org/10.1016/S1462- Vrana, B., Allan, I. J., Greenwood, R., Mills, G. a., Dominiak, E., Svensson, K., et al. (2005). Passive sampling techniques for monitoring pollutants in water. TrAC - Trends in Analytical Chemistry, 24(10), 845–868. https://doi.org/10.1016/j. trac.2005.06.006. References (2010). Application and eval- uation of a new passive sampler for measuring average solute concentrations in a catchment scale water quality monitoring study. Environmental Science and Technology, 44(3), 1353– 1359. https://doi.org/10.1021/es903068h. Górecki, T., & Namieśnik, J. (2002). Passive sampling. TrAC Trends in Analytical Chemistry, 21(4), 276–291. https://doi. org/10.1016/S0165-9936(02)00407-7. Rusina, T. P., Smedes, F., Koblizkova, M., & Klanova, J. (2010). Calibration of silicone rubber passive samplers: Experimental and modeled relations between sampling rate and compound properties. Environmental Science and Technology, 44(1), 362–367. https://doi.org/10.1021 /es900938r. Harman, C., Thomas, K. V., Tollefsen, K. E., Meier, S., Bøyum, O., & Grung, M. (2009). Monitoring the freely dissolved concen- trations of polycyclic aromatic hydrocarbons (PAH) and alkylphenols (AP) around a Norwegian oil platform by holistic passive sampling. Marine Pollution Bulletin, 58(11), 1671– 1679. https://doi.org/10.1016/j.marpolbul.2009.06.022. Smedes, F., & Booij, K. (2012). Guidelines for passive sampling of hydrophobic contaminants in water using silicone rubber Hernando, M. D., Lambropoulou, D., Konstantinou, I., Martinez Bueno, M. J., Gabrielides, D., Fernández-Alba, A. R., & 349 Page 12 of 12 Environ Monit Assess (2018) 190: 349 349 0758(00)00006-6, Guidelines for Drinking-Water Quality, 2nd edition, Addendum to Volume 1 – Recommendations, World Health Organisation, Geneva, 1998, 36 pages. samplers. ICES Techniques in Marine Environmental Sciences No. 52. Smedes, F., Bakker, D., & Weer, J. de. (2010). The use of passive sampling in WFD monitoring. Delft: Rijkswaterstaat Centre for Water Management, Deltares Project No. 1202337-004. Wille, K., Claessens, M., Rappé, K., Monteyne, E., Janssen, C. R., De Brabander, H. F., & Vanhaecke, L. (2011). Rapid quanti- fication of pharmaceuticals and pesticides in passive sam- plers using ultra high performance liquid chromatography coupled to high resolution mass spectrometry. Journal of Chromatography A, 1218(51), 9162–9173. https://doi. org/10.1016/j.chroma.2011.10.039. Vrana, B., Allan, I. J., Greenwood, R., Mills, G. a., Dominiak, E., Svensson, K., et al. (2005). Passive sampling techniques for monitoring pollutants in water. TrAC - Trends in Analytical Chemistry, 24(10), 845–868. https://doi.org/10.1016/j. trac.2005.06.006. WHO. (2011). WHO guidelines for drinking-water quality. WHO chronicle, (Fourth Edition). https://doi.org/10.1016/S1462-
https://openalex.org/W2108589525	https://europepmc.org/articles/pmc4213457?pdf=render	English	null	When study site contributes to outcomes in a multi-center randomized trial: a secondary analysis of decisional conflict in men with localized prostate cancer	Health and quality of life outcomes	2,014	cc-by	6,883	© 2014 Underhill et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 * Correspondence: meghanl_underhill@dfci.harvard.edu 1The Phyllis F. Cantor Center for Research in Nursing & Patient Care Services, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02115, USA Full list of author information is available at the end of the article Open Access Open Access When study site contributes to outcomes in a multi-center randomized trial: a secondary analysis of decisional conflict in men with localized prostate cancer When study site contributes to outcomes in a multi-center randomized trial: a secondary analysis of decisional conflict in men with localized prostate cancer Meghan L Underhill1, Fangxin Hong2 and Donna L Berry1 Meghan L Underhill1, Fangxin Hong2 and Donna L Berry1 Abstract Purpose: Evaluate baseline factors that may explain the influence of study site on decisional conflict (DC) in men from the Personal Patient Profile: Prostate (P3P) randomized trial. Materials and methods: 476 cases from 5 P3P sites were included. Participants completed baseline demographic assessments, 4 subscales of the DC scale at baseline (uncertainty, informed, values clarity, and support), the Expanded Prostate Cancer Index Composite (short form) and the State-Trait Anxiety Inventory. Site data regarding typical practices were collected. Linear regressions were used to model the relation between baseline DC scores and study site adjusting for the list of variables. Results: Baseline decisional uncertainly (p = 0.001) and informed (p = 0.03) subscales were significantly different across sites. Participant demographic and baseline measures were significantly different (p < 0.05) between sites except for trait anxiety. We identified participant level factors that explained study site differences at baseline for the decisional uncertainty and values clarity subscales: a preferred treatment choice at study entry, whether the study program was accessed at home vs. in clinic, number of doctors consulted pre-study, working status, state anxiety, information from the media or a health care provider, and perceived knowledge level. State anxiety was associated with higher DC across all subscales. Conclusions: Individual characteristics of men seeking consultation for LPC were associated with DC at baseline, not the site alone; anxiety contributed to higher conflict. These findings will inform future development and implementation of the P3P and other decision support interventions. Trial registration: NCT00692653. Keywords: Localized prostate cancer, Decisional conflict, Decision-making option for most men diagnosed with LPC and therefore men often are asked to contribute to the care or treatment decision. These decisions are complex and are made based on a variety of personal and social economic factors, as well as the side effect profile of each approach [2-6]. Background In 2014, prostate cancer will account for 27% of new cancer cases and 10% of cancer related deaths in men [1]. Over 90% will be diagnosed with localized prostate cancer (LPC) [1]. There are a variety of care and treatment “options” for men diagnosed with LPC including surgical treatment, radiation treatment, or active surveillance. There is little medical evidence to support the “best” The Personal Patient Profile: Prostate (P3P) is a Web- based intervention providing tailored, values-based education and communication coaching to men making decisions about management of LPC [7]. The P3P was tested in a multi-site randomized control trial (RCT) from 2007–2009 comparing standard patient education plus P3P to standard patient education alone [8]. The 6 Page 2 of 9 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 study sites in 4 U.S. cities were the Seattle Prostate Institute (SPI) in Seattle, Washington; the University of Washington (UW)/Seattle Cancer Care Alliance (SCCA) in Seattle, Washington; Fox Chase Cancer Center (FCCC) radiation oncology clinic in Philadelphia, Pennsylvania; and Veterans Affairs Hospitals (VA) in Seattle, Washington; San Antonio, Texas; and Augusta, Georgia. The main outcome of the trial was decisional conflict over 6 months, measured by the validated Decisional Conflict (DC) scale [9]. A total of 494 eligible cases participated in the original study. A detailed description of the study sample and procedures has been previously reported [8]. Background P3P was found to significantly reduce DC related to making LPC treatment decisions over 6 months, adjusting Table 1 Description of baseline participant characteristics and study measures across study sites Site p-value Augusta Philadelphia San Antonio Seattle UW-SCCA Seattle Puget Sound-VA (n=91) (n=88) (n=25) (n=225) (n=47) Median (range) Median (range) Median (range) Median (range) Median (range) Age 62 (45–78) 66 (43–79) 62 (52–77) 62 (40–86) 63 (52–78) 0.03 N (%) N (%) N (%) N (%) N (%) College education or higher 23 25.3 51 58.0 6 24.0 168 74.7 18 38.3 <.0001 Caucasian 39 42.9 84 95.5 12 48 214 95.1 40 85.1 <.0001 Income 35K or less 54 59.3 7 8.0 8 32.0 16 7.1 24 51.1 <.0001 Married/partnered 56 61.5 72 81.8 20 80.0 188 83.6 27 57.4 <.0001 Working (yes) 40 44.0 55 62.5 11 44.0 137 60.9 19 40.4 .005 Program access location (at Clinic) 82 90.1 9 10.2 9 36.0 37 16.4 13 27.7 <.0001 Having a treatment choice at baseline 51 56.0 41 46.6 15 60.0 96 42.7 33 70.2 0.003 Number of doctors consulted <.0001 0 58 63.7 9 10.2 9 36 52 23.1 18 38.3 1 27 29.7 24 27.3 11 44.0 83 36.9 13 27.7 > 1 6 6.6 55 62.5 5 20.0 90 40.0 16 34.0 Perceived knowledge 34 37.4 67 76.1 7 28.0 147 65.3 23 48.9 <.0001 Fair/A lot Some 26 28.6 14 15.9 9 36.0 57 25.3 8 17.0 None/little 31 34.1 7 8.0 9 36.0 21 9.3 16 34.0 Information sources (yes) Self 16 17.6 45 51.1 6 24.0 102 45.3 16 34.0 <.0001 Health care provider 79 86.8 58 65.9 22 88.0 166 73.8 43 91.5 0.0005 Media 49 53.8 77 87.5 16 64.0 192 85.3 23 48.9 <.0001 Other people 58 63.7 72 81.8 15 60.0 178 79.1 31 66.0 0.005 STAI M SD M SD M SD M SD M SD State anxiety 35.7 13.4 42.6 15.0 40.3 16.3 40.6 12.2 39.0 11.9 0.009 Trait anxiety 34.2 11.4 33.7 11.0 35.6 12.9 32.5 8.8 36.0 11.2 0.16 EPIC-SF questionnaire Urinary irritative 87.5 16.5 93.8 11.1 87.5 18.2 87.5 15.8 87.5 14.8 0.02 Urinary incontinence 100 16.2 100 10.4 91.8 16.8 100 12.8 100 13.9 0.02 Bowel symptoms 100 13.4 100 9.7 95.8 13.2 100 9.8 100 12.0 0.04 Sexual symptoms 61.8 32.6 72.3 30.6 30.5 29.4 78.5 26.9 66.7 28.5 <.0001 Hormonal symptoms 90 14.8 95 12.2 85 20.7 95 11.0 90 10.6 <.0001 Note: M = Mean, SD = standard deviation STAI = State Trait Anxiety Inventory (scores range from 20–80 with higher scores indicating more anxiety), EPIC-SF = Expanded Prostate Index Composite-Short Form (scores range from 0–100 with higher scores indicating better HRQOL); One-way ANOVA and Chi-square test were used for comparing means and proportions across sites. Informed • I know which options are available to me. Higher score = less informed Higher score = less informed • I know the benefits of each option. • I know the risks and side effects of each option. • I am clear about which benefits matter most to me. Participants The study sites were described in detail elsewhere [8] and summarized in Table 1. One participating site (SPI) en- rolled only 18 participants, compared to 25 or more at all other sites, and was therefore excluded due to small sample size. 476 cases from 5 sites were included in this analysis. Institutional Review Board approval was obtained at each site for the original trial, with the University of Washington/Fred Hutchinson Cancer Consortium as the lead IRB site, and all participants had completed written informed consent. ble 2 Four subscales and items of the decisional conflict scale used at baseline [8,9] Table 2 Four subscales and items of the decisional conflict scale used at baseline [8,9] Table 2 Four subscales and items of the decisional conflict scale used at baseline [8,9] Table 2 Four subscales and items of the decisional conflict scale used at baseline [8,9 Table 2 Four subscales and items of the decisional conflict scale used at baseline [8,9] Uncertainty • I am clear about the best choice for me. Higher score = greater uncertainty • I feel sure about what to choose. • This decision is easy for me to make. Informed • I know which options are available to me. Higher score = less informed • I know the benefits of each option. • I know the risks and side effects of each option. Values Clarity • I am clear about which benefits matter most to me. Higher score = lack of clarity about personal values • I am clear about which risks and side effects matter most. • I am clear about which is more important to me (the benefits or the risks and side effects). Support • I have enough support from others to make a choice. Higher score = lack of support • I am choosing without pressure from others. • I have enough advice to make a choice. Responses for each item range from 0 (strongly agree) to 4 (strongly disagree); adapted from [7]. • I am clear about the best choice for me. • I feel sure about what to choose. Higher score = greater uncertainty Higher score = greater uncertainty • This decision is easy for me to make. Values Clarity • I am clear about which risks and side effects matter most. Higher score = lack of clarity about personal values Higher score = lack of clarity about personal values • I am clear about which is more important to me (the benefits or the risks and side effects). Background Table 1 Description of baseline participant characteristics and study measures across study sites Site Page 3 of 9 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 for participant personal characteristics and baseline mea- sures [8]. of prostate cancer information sources that had been used prior to enrollment. This variable was re-coded into four dichotomous (yes/no) variables related to information source: self (books, pamphlets that I got myself), health care provider (books, pamphlets that my health care providers gave me), media (magazines, news- papers, Internet, television/videos), and other people (family members, others). In the primary multivariable analysis, even after control- ling for participant personal characteristics and other study measures, the study site at which participants received consultation remained a significant predictor of DC [8,10]. The purpose of this analysis was to explore further the factors that could potentially explain the influence of study site on DC, focusing on site characteristics and pre- intervention variables measured at baseline prior to entry into the study and receiving consultation at the study site. The DC questionnaire [9] was completed at baseline. Because the eligible participants all were scheduled for a consult about a care or treatment decision and had not made a final decision, only the first 4 DC subscales were presented at baseline and included in this analysis: uncer- tainty, informed, values clarity, and support (Table 2). Item score responses ranged from 0 (agree) to 4 (strongly disagree) with higher scores representing more conflict. Subscale scores were transformed 0 (no DC) to 100 (extremely high DC) [11]. The Expanded Prostate Cancer Index Composite (short form) (EPIC) was reported, meas- uring prostate specific, health related quality of life (HRQOL) [12]. Higher scores indicate better HRQOL in the domains of irritative urinary symptoms, urinary incon- tinence, hormone-related side effects, and bowel and sexual function. Item scores were transformed to a 0 to 100 scale and the average scores within each subscale were taken to create subscale scores [13]. The Spielberger State-Trait Anxiety Inventory (STAI) was used to assess baseline anxiety. Item scores for STAI are summated for each subscale (state and trait) and scores range from 20–80 with higher scores indicating more anxiety [14]. Responses for each item range from 0 (strongly agree) to 4 (strongly disagree); adapted from [7]. Measures Participants in the original RCT self-reported personal characteristics, concerns and preferences, all previously documented [2] as important to prostate cancer treat- ment decision making. Additional self-reported variables included the number of doctors consulted about prostate cancer treatment prior to study enrollment and level of perceived knowledge about prostate cancer and its treat- ment. Participants also were asked do you think you know which treatment you want (yes/no) and how many weeks has it been since your prostate biopsy. One item in the original trial asked the participant to select what type Additional data about the study site clinical processes were collected after the original trial and prior to the sec- ondary analysis during interviews with study site investigators. The investigators were asked to “Describe the typical practices and process a man with localized prostate cancer would follow from detection and biopsy to making a care decision’. Investigators were prompted to report Statistical methods Baseline patient characteristics and study measures were compared among sites. One-way ANOVA and Chi-square test were used for means and proportions, respectively. All analyses were conducted using SAS (version 9.2). For each of the four DC subscales, linear regression was used to model the relation between baseline DC scores and study site adjusting for personal characteristics (or factors) and the site level variables. We explored whether study site remained a significant variable associated with baseline DC scores after adjusting for additional baseline factors. First, univariate analysis was performed between each factor. Factors that were potentially associated with DC score, with significance levels (p-value) less than 0.2 were included in the multivariable model. Backward vari- able selection was used to identify significant predictors, where a variable was statistically significant if p-value ≤0.05 and kept in the model if p-value ≤0.2. Possible two-way in- teractions among remaining predictors were examined. Site level variables exhibited multi-collinearity with study site and were not included in the multivariable model. Figure 1 illustrates the mean scores with 95% confi- dence intervals (CI) for the four DC subscales measured at baseline; baseline decisional uncertainly (p < 0.001) and informed (p = 0.03) subscale scores were signifi- cantly different across sites. The effect of the baseline personal characteristics and reports of knowledge level, information sources, anxiety and symptoms on DC sub- scale scores were estimated in both univariate (Table 3) and multivariable (Table 4) analyses. Support • I have enough support from others to make a choice. Higher score = lack of support Higher score = lack of support • I am choosing without pressure from others. • I have enough advice to make a choice. Responses for each item range from 0 (strongly agree) to 4 (strongly disagree); adapted from [7]. Page 4 of 9 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 symptom, were significantly different across sites (Table 1). The men enrolled in Philadelphia at Fox Chase and at the Seattle UW/SCCA were predominately college-educated, Caucasian, working and had accessed the baseline P3P measures on a personal computer or tablet. In contrast, men enrolled in the Veterans Administration hospital sites (Augusta, San Antonio and Seattle) were predominately high school educated, not working, had accessed the base- line measures in the clinic on a study computer and about half were of minority race or ethnicity. Further, the major- ity of men in Philadelphia and Seattle UW/SCCA had already consulted with other physicians since the time of biopsy, had retrieved prostate cancer information them- selves and from media, and reported a higher level of perceived prostate cancer knowledge. specific information about the clinical process in general regarding number of consultations, length of time between biopsy and disclosure of results, and/or treatment decision making, type of specialists at the site, length of time be- tween consult visit, method of disclosing biopsy results, and if educational material was given to patients. Based on the responses, 5 categorical variables were created. Additional site level variables Univariate analyses revealed sites that typically disclosed the diagnosis in person in the clinic, provided more than one in-person visit, took greater than one month between biopsy to treatment decision, and did not conduct follow- up by telephone had enrolled participants with signifi- cantly less decisional conflict related to uncertainty. Sites that provided educational information (handouts, books), enrolled participants that reported significantly more Overview The participant characteristics, including demographic, re- ports of knowledge level, information source, anxiety and Figure 1 Mean scores together with 95% confidence interval (CI) describing the variation in the four DCS subscales measured at baseline across sites. Note: Higher mean scores indicate more Decisional Conflict; ANOVA testing mean difference across sites: Uncertainty subscale p<0.001; Informed subscale p = 0.03; values clarity subscale p = 0.12; support subscale p = 0.07. Figure 1 Mean scores together with 95% confidence interval (CI) describing the variation in the four DCS subscales measured at baseline across sites. Note: Higher mean scores indicate more Decisional Conflict; ANOVA testing mean difference across sites: Uncertainty subscale p<0.001; Informed subscale p = 0.03; values clarity subscale p = 0.12; support subscale p = 0.07. Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 Page 5 of 9 Page 5 of 9 Table 3 The influence of personal factors on decisional conflict subscale scores-results from the univariate analysis Variable Decisional uncertainty Informed subscale Values clarity subscale Support subscale Estimate p-value Estimate p-value Estimate p-value Estimate p-value Study center <.0001 0.03 0.13 0.07 Augusta −4.67 −2.72 −1.99 −7.00 Philadelphia 12.62 −1.10 2.60 −3.47 San Antonio −2.86 6.05 9.82 −7.02 Seattle UW-SCCA 13.66 5.37 2.61 −2.32 Age −0.24 0.17 0.87 0.62 0.81 Treatment preference at baseline (no vs. yes) 27.3 <.0001 16.6 <.0001 14.32 <.0001 8.40 <.0001 Education (college no vs. yes) −6.62 0.008 0.54 0.22 0.90 Income (>35K vs. <35k) 10.8 0.0002 3.64 0.16 0.63 0.60 Caucasian (no vs. yes) −14.5 <.0001 −4.39 0.12 0.52 −3.22 0.10 P3P access (Clinic vs. Not Clinic) −13.8 <.0001 −3.76 0.11 −5.72 0.007 −2.40 0.14 Married/partnered (no vs. yes) 0.93 0.28 0.58 4.51 0.01 Number of doctors consulted 0.0004 <.0001 0.003 0.002 0 vs. >1 13.55 7.94 5.64 1 vs. >1 5.54 2.10 −0.05 Working status (No vs. yes) −6.05 0.02 −3.08 0.16 0.27 0.86 Perceived knowledge 0.38 <.0001 <.0001 <.0001 Fair/A lot vs. some −19.52 −11.72 −3.87 None/little vs. Overview some 3.76 6.88 7.73 STAI State anxiety 0.75 <.0001 0.50 <.0001 0.41 <.0001 0.44 <.0001 Trait anxiety 0.31 0.01 0.35 0.001 0.35 0.0002 0.43 <.0001 *EPIC scale Urinary irritative symptoms −0.1 0.2 −0.1 0.18 0.17 0.61 Urinary incontinence symptoms 1.0 0.82 0.81 0.45 Bowel symptoms 0.1 0.39 0.62 0.65 0.92 Sexual symptoms 0.06 0.16 −0.08 0.02 −0.06 0.07 0.90 Hormonal symptoms −0.03 0.72 −0.2 0.02 −0.15 0.05 −0.16 0.005 Information Sources Self (no vs. yes) −8.16 0.001 7.32 0.001 4.89 0.01 0.84 Health care provider (no vs. yes) 4.08 0.17 7.27 0.005 7.10 0.002 4.22 0.02 Media (no vs. yes) −13.98 <.0001 0.56 0.57 0.81 Other people (no vs. yes) 0.22 0.25 0.70 0.59 Note: Higher subscale scores indicate more decisional conflict; Seattle/University of Washington VA is the reference group; STAI = State Trait Anxiety Inventory; EPIC = Expanded Prostate Index Composite-Short Form. Table 3 The influence of personal factors on decisional conflict subscale scores-results from the univariate analysis Variable Decisional uncertainty Informed subscale Values clarity subscale Support subscale Note: Higher subscale scores indicate more decisional conflict; Seattle/University of Washington VA is the reference group; STAI = State Trait Anxiety Inventory; EPIC = Expanded Prostate Index Composite-Short Form. Decisional uncertainty subscale decisional conflict related to uncertainty. Sites that dis- closed the diagnosis in person enrolled participants that reported higher scores on the informed subscale and those that had less than 1 month from biopsy to treatment decision and gave educational handouts had participants that reported lower DC on the informed subscale (data re- ported in Table 5). These site level variables were collinear with the overall study site variable and therefore not included in subsequent multivariable analysis. decisional conflict related to uncertainty. Sites that dis- closed the diagnosis in person enrolled participants that reported higher scores on the informed subscale and those that had less than 1 month from biopsy to treatment decision and gave educational handouts had participants that reported lower DC on the informed subscale (data re- ported in Table 5). These site level variables were collinear with the overall study site variable and therefore not included in subsequent multivariable analysis. The baseline mean scores on the decisional uncertainly subscale were significantly higher at the Philadelphia Fox Chase radiation oncology site and the Seattle UW/SCCA site (Figure 1). In univariate analysis (Table 3), not having a treatment preference at baseline, Caucasian race, college education, income greater than $35,000, accessing the P3P intervention at home, working, having higher STAI scores, and seeking information independently or from the media Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 Page 6 of 9 Page 6 of 9 Table 4 Factors that explain study site influence on decisional conflict-results from the multivariable analysis Variable Decisional uncertainty Informed subscale Values clarity subscale Support subscale Est. p-value Est. p-value Est. p-value Est. P-value Study center 0.47 0.003 0.52 0.002 Augusta −5.58 −6.69 0.01 Philadelphia 0.96 −0.19 0.95 San Antonio 0.73 −7.56 0.04 Seattle UW-SCCA 5.83 0.51 0.84 Treatment preference at baseline (no vs. yes) 24.5 <.0001 10.92 <.0001 10.44 <.0001 4.87 0.0005 Caucasian (No vs. Yes) −4.30 0.16 −4.23 0.13 Intervention Access (Clinic vs. Not Clinic) −6.33 0.02 −6.17 0.01 Married/Partnered (no vs. yes) 2.95 0.07 Number of doctors 0.006 0.002 0.14 0.05 0 vs. >1 −5.68 0.01 9.18 4.9 4.51 0.01 1 vs. >1 −7.52 0.001 5.29 2.0 1.17 0.47 Working status (no vs. yes) −2.85 0.15 −4.25 0.03 Perceived knowledge <.0001 <.0001 <.0001 Fair/A lot vs. some −16.55 −10.55 −7.00 <.0001 None/little vs. Support subscale were associated with significantly higher decisional un- certainty scores. Seeing more than one doctor also was associated with higher decisional uncertainty scores. In univariate analyses (Table 3), having seen more than one doctor pre-study, having a treatment decision at study entry, reporting more knowledge, and baseline hormonal symptoms were associated with lower DC related to sup- port needed to make a decision. Higher state and trait anxiety, being single, fewer pre-study consults, were sig- nificantly associated with conflict related to less support. When participant level variables were entered in the multivariable model, site was no longer a statistically significant (p = 0.47) predictor of DC (Table 4). Lower decisional uncertainty was significantly associated with a treatment choice at study entry and accessing the P3P program in the clinic. Higher uncertainty was significantly associated with having seen more than one doctor, higher state anxiety, and obtaining information from the media. Study site remained a significant factor (p = 0.002) after conducting the multivariable analysis (Table 4). Participants enrolled at the Augusta and San Antonio VA hospitals reported significantly less DC related to sup- port at baseline. Factors associated with lower support subscale scores, and therefore having enough support for the decision thus far, were: having a treatment preference at study entry, having seen more than one doctor pre- study, and reporting a higher level of prostate cancer knowledge. Higher state anxiety scores were significantly associated with higher DC support scores. Decisional uncertainty subscale When participant level variables were entered in the multivariable model, site was no longer a statistically significant (p = 0.47) predictor of DC (Table 4). Lower decisional uncertainty was significantly associated with a treatment choice at study entry and accessing the P3P program in the clinic. Higher uncertainty was significantly associated with having seen more than one doctor, higher state anxiety, and obtaining information from the media. Discussion In the multisite P3P trial, the study site where partici- pants received consultation served significantly differ- ent patient populations and study site was a significant factor associated with the main outcome of DC at 6 months. The current analysis explained what factors played a role in the significance of study site for two of the four DC subscales, uncertainty and values clarity. For the remaining two subscales included in this ana- lysis, informed and support, study site remained signifi- cant. Potentially unmeasured variables play a role in why study site is important for these subscales and fu- ture trials will measure study site specific variables as well as participant factors. Though all potential baseline variables trending towards significance in the univariate model were included in the multivariable analysis (Table 4), study site remained a significant factor (p = 0.003) for the informed subscale. The aspect of DC related to being informed was signifi- cantly lower at the Augusta site, meaning participants at this site reported a perception of being more informed. Participants who reported having a treatment preference at study entry, seeing more than one pre-study doctor, having sexual function issues, and higher prostate cancer knowledge reported being more informed. Not working and higher state anxiety scores were significantly associ- ated with a report of being less informed. The primary finding from our secondary analysis docu- ments that the characteristics of men who sought treatment at the sites were explanatory of baseline DC measures. Dis- proportionate numbers of men with certain characteristics were consulted at the various study sites. The results high- light how men from diverse backgrounds engage in the complexity of decision making for LPC treatment. Race was implicated in univarate analysis of uncertainty, but did not predict DC once entered into the multivariable model. For those men with higher socioeconomic status, such as those at the Philadelphia Fox Chase radiation oncology site and the Seattle UW/SCCA, with access to information and health care resources, who sought information independ- ently, and accessed Web-based information and multiple doctors to discuss treatment options (by default at the radi- ation site), DC related to uncertainty was higher. These men may have known the complexity of the LPC treatment decision at study entry, and therefore experienced more conflict. In contrast, men with lower socioeconomic status, such as those from the VA hospital sites, who did not Informed subscale V i bl h Variables that were associated in univariate analysis (Table 3) with lower scores on the informed subscale were not having a treatment decision at study entry, high state anxiety, accessing P3P at home, having less knowledge, and no pre-study sexual or hormonal symptoms. Higher scores on the informed subscale were significantly associated with seeking information independently or from a health care provider. Decisional uncertainty subscale some 3.03 5.41 2.45 0.26 STAI State 0.53 <.0001 0.39 <.0001 0.27 <.0001 0.36 <.0001 Trait −0.15 0.19 EPIC scale Sexual symptoms −0.07 0.04 Information source Self (no vs. yes) 2.53 0.21 Health care provider (no vs. yes) 3.09 0.17 3.94 0.07 Media (no vs. yes) −7.74 0.002 Note: Higher estimates indicate more decisional conflict; Seattle/Puget Sound Veteran’s Administration Hospital is the reference group; STAI = State Trait Anxiety Inventory; EPIC = Expanded Prostate Index Composite-Short Form. Table 4 Factors that explain study site influence on decisional conflict-results from the multivariable analysis study site influence on decisional conflict-results from the multivariable analysis Note: Higher estimates indicate more decisional conflict; Seattle/Puget Sound Veteran’s Administration Hospital is the reference group; STAI = State Trait Anxiety Inventory; *EPIC = Expanded Prostate Index Composite-Short Form. able 5 Univariate analysis of the relationship between decisional conflict and the additional site level i bl ll t d Table 5 Univariate analysis of the relationship between decisional conflict and the additional site level variables collected Site Variable Decisional uncertainty subscale Informed subscale Values clarity subscale Support subscale Est. p-value Est. p-value Est. P-value Est. P-value Number of in person visits (>1 vs. . 1) −14.5 0.008 2.97 0.55 7.55 0.09 −4.72 0.16 Diagnosis disclosure method .0001 0.09 0.96 0.85 In person vs. telephone −14.7 <.0001 −3.27 0.31 0.85 0.77 −0.12 0.96 N/A vs. telephone −1.0 0.75 −6.47 0.03 −0.11 1.0 −1.16 0.57 Time from Biopsy to treatment decision 0.0001 0.09 0.96 0.85 <1 mo vs. N/A 1.0 0.75 6.47 0.03 0.01 1.0 1.16 0.57 > 1 mo vs. N/A −13.6 0.001 3.21 0.40 0.86 0.10 1.04 0.69 Educational material given (no vs. yes) 7.16 0.008 5.03 0.04 −0.37 0.87 0.69 0.68 Telephone follow-up given (no vs. yes) −14.5 0.008 2.97 0.55 7.55 0.09 −4.72 0.16 Note: Higher estimates indicate more decisional conflict. 5 Univariate analysis of the relationship between decisional conflict and the additional site level les collected Page 7 of 9 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 were associated with significantly higher decisional un- certainty scores. Seeing more than one doctor also was associated with higher decisional uncertainty scores. Conclusions d d l h Individual characteristics of men seeking consultation for localized prostate cancer were associated with DC at baseline and men with these characteristics were en- rolled disproportionately at the various sites. While the original impact of the P3P intervention was positive des- pite these site differences, we now understand more of the influential baseline factors, notably information ac- cess and perceptions of knowledge about prostate cancer options. The modifiable factor of anxiety was identified as contributing to higher conflict at baseline. These find- ing will inform future development and implementation of the P3P and other decision support interventions. Clinical implications Cli i i h Having a preferred treatment choice at study entry and having higher perceived knowledge were important factors that contributed to lower DC across subscales. It is im- portant to recognize that having perceptions of a preferred treatment decision and having high perceived knowledge prior to having had LPC treatment related consultation does not equate with actual knowledge about LPC treat- ment options. A descriptive study [15] evaluating prostate cancer related knowledge in 109 men with and without a prostate cancer diagnosis from low income settings, found low to moderate levels of prostate cancer knowledge and comprehension in the sample. The findings that of the age appropriate men, fewer than half knew the various pros- tate cancer treatment options and less than a fourth knew the potential side effects of treatment, may help place our findings in context. Men with low socioeconomic status may perceive themselves as having adequate knowledge but still require more informational support related to prostate cancer and treatment. In a recent publication, Kaplan et al. [16] reported baseline factors that predicted DC scores in men with LPC from a VA clinic. They re- ported that lower prostate cancer knowledge was associ- ated with higher DC and uncertainty at baseline. In the P3P RCT, men at sites, such as the Seattle VA site, that had the lowest baseline DC scores, indicating men were the least conflicted, actually had the largest difference between control and intervention groups with regard to overall DC scores six months from enrollment [8]. Our secondary analysis helps us understand that participants who were not as prepared at baseline may have been less aware of the complexity of the decision and, the educa- tional and coaching component of the P3P intervention benefited these men over time as they engaged in the complex task of a shared decision. Clinicians who consult with men regarding management of LPC may use these findings to better support men and decrease DC. Anxiety could be assessed at baseline to help clinicians understand which patients are likely to feel most conflicted when making a treatment decision and then target support to specific patient needs. Clinical centers which serve a high proportion of men with no access to the Internet and who are typically not consulting with multiple clinicians may want to assure (not assume) that men have a full understanding of prostate cancer and options for treating or monitoring the condition. Values clarity subscale Not having a decision at baseline, accessing P3P at home, reporting less knowledge, higher STAI, and no hormonal symptoms were significantly associated with having higher values clarity scores, indicating more conflict in univariate analyses (Table 3). Seeking infor- mation independently or from the health care provider was associated with lower scores indicating more values clarity. Multivariable analyses (Table 4) indicated that study site was not significantly (p = 0.52) associated with values clarity. State anxiety was significantly asso- ciated with a lack of values clarity (higher subscale score). Lower values clarity subscale scores, indicating less conflict, were associated with having a treatment preference at study entry, accessing the study program at the study site and reporting a higher level of prostate cancer knowledge. Page 8 of 9 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 access information outside of the clinic and did not seek in- formation independently may have been less aware of the complexities and implications of the multiple treatment options available for LPC, and therefore experienced less conflict at study entry. significantly lower state anxiety in men with newly diag- nosed prostate cancer six weeks after randomization to an informational intervention. In 2012, the American College of Surgeons Commission on Cancer proposed a 2015 mandate for cancer care settings that the management of distress, that includes anxiety, should be a component of all patient care [18]. The significant influence of scores from the EPIC hor- monal symptom domain was an interesting finding in the univariate analysis for three of four DC scales, though not sustained in the multivariable model. Men at the San Antonio VA had significantly worse scores in this domain. Certainly, at diagnosis these men had not been exposed to any androgen suppressive therapies. Two of the items within this domain inquire about depression and energy; this may help explain the relationship between the EPIC hormonal domain scores and DC. Limitations to our findings are important to identify. Study site process variables were measured by investigator recall and the current analysis may not have included all necessary site information, potentially missing important contributions that unknown study site variables may have made to the results. Abbreviations P3P: Personal patient profile: Prostate; LPC: Localized prostate cancer; DC: Decisional conflict; STAI: State trait anxiety index. Competing interests h h d l h Competing interests The authors declare that they have no competing interests. References 1. American Cancer Society: Cancer Facts and Figures 2014. Atlanta: American Cancer Society; 2014. 1. American Cancer Society: Cancer Facts and Figures 2014. Atlanta: American Cancer Society; 2014. 2. Berry DL, Ellis WJ, Russell KJ, Blasko JC, Bush N, Blumenstein B, Lange PH: Factors that predict treatment choice and satisfaction with the decision in men with localized prostate cancer. Clin Genitourin Cancer 2006, 5:219. 2. Berry DL, Ellis WJ, Russell KJ, Blasko JC, Bush N, Blumenstein B, Lange PH: Factors that predict treatment choice and satisfaction with the decision in men with localized prostate cancer. Clin Genitourin Cancer 2006, 5:219. 3. Sommers BD, Beard CJ, D’Amico AV, Kaplan I, Richie JP, Zeckhauser RJ: Predictors of patient preferences and treatment choices for localized prostate cancer. Cancer 2008, 113:2058. 4. Davison BJ, Breckon E: Factors influencing treatment decision making and information preferences of prostate cancer patients on active surveillance. Patient Educ Couns 2012, 87:369. 4. Davison BJ, Breckon E: Factors influencing treatment decision making and information preferences of prostate cancer patients on active surveillance. Patient Educ Couns 2012, 87:369. 5. Holmboe ES, Concato J: Treatment decisions for localized prostate cancer. J Gen Intern Med 2000, 15:694. cancer. J Gen Intern Med 2000, 15:694. 6. Steginga SK, Occhipinti S, Gardiner RA, Yaxley J, Heathcote P: Making decisions about treatment for localized prostate cancer. BJU Int 2002, 89:255. 6. Steginga SK, Occhipinti S, Gardiner RA, Yaxley J, Heathcote P: Making decisions about treatment for localized prostate cancer. BJU Int 2002, 89:255. 7. Berry DL, Halpenny B, Wolpin S, Davison BJ, Ellis WJ, Lober WB, McReynolds J, Wulff J: Development and evaluation of the personal patient profile-prostate (P3P), a Web-based decision support system for men newly diagnosed with localized prostate cancer. J Med Internet Res 2010, 12:e67. 8. Berry D, Halpenny B, Hong F, Wolpin S, Lober W, Russell K, Ellis W, Govindarajulu U, Bosco J, Davison B: The Personal Patient Profile-Prostate decision support for men with localized prostate cancer: a multi-center randomized trial. Urol Oncol 2011:1012 9. O’Connor AM: Validation of a decisional conflict scale. Med Decis Mak 1995, 15:25. 10. Berry D, Hong F, Halpenny B, Wolpin S, Lober W, Russell K, Ellis W, Davison BJ, Barsevick A, Yang C: 1001 decisional conflict varies by institution for men making treatment decisions for localized prostate cancer. J Urol 2011, 185:e403. 11. O’Connor, A: User Manual-Decisional Conflict Scale 1993. Author details 1 1The Phyllis F. Cantor Center for Research in Nursing & Patient Care Services, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02115, USA. 2Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, 450 Brookline Ave, CLSB-11017, Boston, Massachusetts 02215, USA. doi:10.1186/s12955-014-0159-3 Cite this article as: Underhill et al.: When study site contributes to outcomes in a multi-center randomized trial: a secondary analysis of decisional conflict in men with localized prostate cancer. Health and Quality of Life Outcomes 2014 12:159. Received: 7 May 2014 Accepted: 9 October 2014 Received: 7 May 2014 Accepted: 9 October 2014 Acknowledgements Th h ld lik The authors would like to thank Barbara Halpenny MA and Traci Blonquist MS for their contributions to the preparation of this manuscript. We acknowledge the generous time and efforts of the research staff and clinicians of the P3P trial. 18. Cancer Program Standards 2010: Ensuring Patient-Centered Care [http://www.facs.org/cancer/coc/programstandards2012.pdf] Authors’ contributions MU ib d d If a man had high state, or situational, anxiety at study entry, he was more likely to have higher DC across all subscales. State anxiety is a modifiable factor that could be addressed by clinical teams providing consultation for LPC treatment. Davison and colleagues [17] reported Authors contributions MU contributed to data collection, data analysis, and manuscript preparation. MU has given final approval of the submitted manuscript. FH contributed to data analysis and manuscript preparation. FH has given final approval of the submitted manuscript. DB has contributed to study design, data collection, data analysis, and manuscript preparation. DB has given final approval of the submitted manuscript. All authors read and approved the final manuscript. Page 9 of 9 Page 9 of 9 Underhill et al. Health and Quality of Life Outcomes 2014, 12:159 http://www.hqlo.com/content/12/1/159 decision-making trial. Cancer 2014, 120:2721–2727. doi:10.1002/ cncr.28755. 17. Davison BJ, Degner LF: Empowerment of men newly diagnosed with prostate cancer. Cancer Nurs 1997, 20:187. 18. Cancer Program Standards 2010: Ensuring Patient-Centered Care [http://www.facs.org/cancer/coc/programstandards2012.pdf] doi:10.1186/s12955-014-0159-3 Cite this article as: Underhill et al.: When study site contributes to outcomes in a multi-center randomized trial: a secondary analysis of decisional conflict in men with localized prostate cancer. Health and Quality of Life Outcomes 2014 12:159. References See [https:// decisionaid.ohri.ca/eval.html#DecisionalConflictScale] (last checked 27 October 2009). 12. Szymanski KM, Wei JT, Dunn RL, Sanda MG: Development and validation of an abbreviated version of the expanded prostate cancer index composite instrument for measuring health-related quality of life among prostate cancer survivors. Urology 2010, 76:1245. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 13. Scoring Instructions for the Expanded Prostate cancer Index Composite Short Form (EPIC-26) [http://www.med.umich.edu/urology/research/EPIC/ EPIC-26-Scoring-1.2007.pdf] 14. Spielberger CD: State Trait Anxiety Inventory for Adults: Sampler Set: Manual, Test, Scoring Key; [form Y]; STAIS-AD. Melano Park, California: Mind Garden; 1983. 15. Wang DS, Jani AB, Tai CG, Sesay M, Lee DK, Goodman M, Echt KV, Kilbridge KE, Master VA: Severe lack of comprehension of common prostate health terms among low-income inner-city men. Cancer 2013 119:3204. 16. Kaplan AL, Crespi CM, Saucedo JD, Connor SE, Litwin MS, Saigal CS: Decisional conflict in economically disadvantaged men with newly diagnosed prostate cancer: baseline results from a shared
https://openalex.org/W2907571806	https://financetp.fa.ru/jour/article/download/783/529	Russian	null	Economic Mechanism and Cash Flows Modeling for Reverse Mortgage	Finansy: teoriâ i praktika	2,018	cc-by	10,100	АННОТАЦИЯ Исследуется экономический механизм обратной ипотеки — кредитного продукта, направленного на повышение уровня жизни граждан преклонного возраста, являющихся владельцами недвижимости. Изложена суть операции обратного ипо- течного кредитования, принципы действия механизмов использования принадлежащих пожилым людям объектов недви- жимости для обеспечения им дополнительного дохода. Приведены примеры использования системы обратного ипотеч- ного кредитования в США, Великобритании, Испании и Австралии. Авторы описывают существующие методики обратного ипотечного кредитования в России. На основе анализа опыта зарубежных стран дана оценка экономической целесо- образности вложения денежных средств в данный кредитный инструмент. С учетом фактора потребительского спроса имеется возможность рассчитать равновесную ставку — следовательно, найти координаты точки рыночного равновесия. Разработана математическая модель обратной ипотеки для случая с пожизненными аннуитетными платежами. Данная модель позволяет рассчитать ожидаемые выгоды заемщиков и кредиторов. Сделано (и реализовано в модели) два приме- чания, которые значительно отличают моделирование обратной ипотеки от других кредитных продуктов: 1) пожизненный обратный ипотечный кредит не имеет фиксированного срока окончания; 2) при взятии кредита такого типа заемщики рассматривают мотив не только потребления, но и накопления наследства. В результате построена модель, позволяющая рассчитать положение точек безубыточности и рыночного равновесия (относительно процентной ставки). Это поможет экономически заинтересованным агентам оценить потенциал рынка обратного ипотечного кредитования в России. Ключевые слова: обратное ипотечное кредитование; аннуитет; математическая модель; дополнительное пенсион- ное обеспечение; недвижимость; договор пожизненной ренты; таблицы смертности; системы страхования; Агентст- р р р р Ключевые слова: обратное ипотечное кредитование; аннуитет; математическая модель; дополнительное пенсион- ное обеспечение; недвижимость; договор пожизненной ренты; таблицы смертности; системы страхования; Агентст- во по реструктуризации ипотечных жилищных кредитов Для цитирования: Кузьмина Е. В., Янин А. А. Экономический механизм и моделирование денежных потоков при обратн дитовании. Финансы: теория и практика. 2018;22(6):106-120. DOI: 10.26794/2587-5671-2018-22-6-106-120 ОРИГИНАЛЬНАЯ СТАТЬЯ CC BY 4.0 BY 4.0 DOI: 10.26794/2587-5671-2018-22-6-106-120 УДК 334.01(045) JEL J14, G21 Экономический механизм и моделирование денежных потоков при обратном ипотечном кредитовании Е. В. Кузьминаa, А. А. Янинb следовательский университет Высшая школа экономики, Санкт-Петербург, Россия; b Санкт-Петербургский государственный университет, Санкт-Петербург, Россия a http://orcid.org/0000-0003-3843-8940; b https://orcid.org/0000-0002-3544-7879 Е. В. Кузьминаa, А. А. Янинb a Национальный исследовательский университет Высшая школа экономики, Санкт-Петербург, Россия; b Санкт-Петербургский государственный университет, Санкт-Петербург, Россия a http://orcid.org/0000-0003-3843-8940; b https://orcid.org/0000-0002-3544-7879 Economic Mechanism and Cash Flows Modeling for Reverse Mortgage E. V. Kuz’minaa, A. A. Yaninb a National Research University “Higher School of Economics”, St. Petersburg, Russia; b St. Petersburg State University, St. Petersburg, Russia a http://orcid.org/0000-0003-3843-8940; b https://orcid.org/0000-0002-3544-7879 E. V. Kuz’minaa, A. A. Yaninb a National Research University “Higher School of Economics”, St. Petersburg, Russia; b St. Petersburg State University, St. Petersburg, Russia a http://orcid.org/0000-0003-3843-8940; b https://orcid.org/0000-0002-3544-7879 ФИНАНСЫ И КРЕДИТ / FINANCE AND CREDIT ФИНАНСЫ И КРЕДИТ / FINANCE AND CREDIT ВВЕДЕНИЕ Большинство развитых стран, включая США, стра- ны ЕС и Японию, еще в середине 90-х гг. прошлого века столкнулись с таким явлением, как неуклон- ное старение населения. В нашей стране также происходят аналогичные процессы. Экономиче- ские проблемы, которые вызывает старение на- селения, являются весьма серьезными, поскольку ставят перед государством и обществом нелегкие вопросы. Особенность этого продукта заключается в том, что недвижимость служит залогом по кредиту. Его получает владелец недвижимости от кредито- ра в виде аннуитета, кредитной линии, единовре- менного платежа или их комбинации. При этом жилье переходит в собственность кредитора как залоговое имущество только после смерти креди- тополучателя. Эта операция, которая в США по- зиционируется как часть пенсионной системы [1], является, по сути, обратной для обычной ипотеки, при которой кредитополучатель делает первона- чальный взнос, получает кредит в виде недвижи- мости, а потом долгие годы выплачивает банку аннуитет. Снижение доходов пенсионеров означает об- щее падение уровня платежеспособного спроса, что, в свою очередь, приводит к сокращению сбыта товаров и услуг для хозяйствующих субъ- ектов и в конечном итоге к замедлению темпов экономического развития. Эти экономические факторы влекут за собой социальные и внутри- политические проблемы, требующие принятия неотложных мер. Наиболее кардинальными из них являются меры по повышению доли трудо- способной части населения: повышение пенси- онного возраста, стимулирование рождаемости, привлечение в страну трудоспособных иммиг- рантов. Однако эффективность этих подходов ограничена, и они сами по себе могут вызывать дополнительные трудности. В классическом варианте обратной ипотеки кредитополучатель всю оставшуюся жизнь получает аннуитет как добавку к пенсии, а его смерть или покидание жилища (например, переселение в дом для престарелых) означает наступление события, при котором обязанности банка считаются выпол- ненными, и наступает срок платежа по кредиту. Платеж осуществляется путем продажи залогового имущества. Из вырученной суммы погашается тело кредита и начисленные проценты, а оставшаяся сумма передается наследникам. Характерно, что кредитор не получает жилье в собственность ни в момент оформления кредита, ни в момент его погашения. Оно лишь служит обеспечением кредита, и кредитор в момент погашения получает обратно ровно ту сумму, которую он успел выдать креди- тополучателю до момента его смерти, и отдельно начисленные проценты на каждую выданную часть тела кредита. Итак, проблем много, подходов к их решению тоже немало, и при этом не существует единствен- ной универсальной «панацеи». Необходим целый комплекс мер, к числу которых относятся и те из них, что направлены на повышение уровня жиз- ни граждан пенсионного возраста. На наш взгляд, именно они, наряду с мерами по повышению ро- ждаемости, должны быть отнесены к числу наиболее приоритетных задач. ABSTRACT Economic mechanism and cash flows modeling for reverse mortgage. Finansy: teoriya i praktika = Finance: Theory and Practice. 2018;22(6):106-120. (In Russ.). DOI: 10.26794/2587-5671-2018-22-6-106-120 ABSTRACT The research is devoted to the economic mechanism of reverse mortgage — a credit product aimed at improving the standard of living of senior citizens, owners of real estate. The idea of the reverse mortgage has been given, as well as the mechanisms of use of real estate owned by senior citizens in order to provide them with additional income. The examples of reverse mortgage in the US, the UK, Spain and Australia have been given. The authors have also described the methods of reverse mortgage lending in Russia. Based on the analysis of international experience, the economic expediency of investing in this credit tool has been assessed. Considering consumer demand factor, it is possible to calculate the equilibrium rate and, therefore, to find the coordinates of the market equilibrium point. The authors have developed a mathematical model of reverse mortgage for the case of lifetime annuity payments. This model allows to calculate the expected benefits of borrowers and lenders. There have been done (and implemented) two notes that significantly distinguish reverse mortgage ФИНАНСЫ: ТЕОРИЯ И ПРАКТИКА / FINANCE: THEORY AND PRACTICE  Т. 22, № 6’2018 106 Е. В. Кузьмина, А. А. Янин modeling from other loan products: 1) a lifetime reverse mortgage does not have a fixed expiration date; 2) when taking a loan of this type, borrowers consider not only consumption, but also accumulation of inheritance. The model allows to calculate the position of break-even points and market equilibrium (relative to the interest rate). This will help economically interested agents to assess the potential of the reverse mortgage market in Russia. modeling from other loan products: 1) a lifetime reverse mortgage does not have a fixed expiration date; 2) when taking a loan of this type, borrowers consider not only consumption, but also accumulation of inheritance. The model allows to calculate the position of break-even points and market equilibrium (relative to the interest rate). This will help economically interested agents to assess the potential of the reverse mortgage market in Russia. Keywords: reverse mortgage lending; annuity; mathematical model; supplementary pensions; real estate; life annuity contract; mortality tables; insurance system; Agency for Restructuring of Housing Mortgage Loans For citation: Kuz’mina E.V., Yanin A. A. Economic mechanism and cash flows modeling for reverse mortgage. Finansy: teoriya i praktika = Finance: Theory and Practice. 2018;22(6):106-120. (In Russ.). DOI: 10.26794/2587-5671-2018-22-6-106-120 For citation: Kuz’mina E.V., Yanin A. A. ВВЕДЕНИЕ Заметим, что обратная ипотека — не единствен- ный вариант использования пожилыми людьми своего жилья для повышения уровня потребле- ния. Существуют также альтернативные варианты, к числу которых относится продажа жилья с по- следующей арендой, договор пожизненной ренты, поэтапная продажа жилья с последующей рентой и другие. Все эти варианты, на наш взгляд, являются для пожилых людей менее привлекательными, чем операция обратной ипотеки. В данной работе рассмотрены механизмы ис- пользования принадлежащих пожилым людям объектов недвижимости для обеспечения им до- полнительного дохода. Уделено особое внимание такому интересному, но малоизвестному в нашей стране финансовому продукту, как обратная ипо- тека. Предложена математическая модель, которая позволила бы оценить экономическую целесоо- бразность внедрения данного продукта. 107 financetp.fa.ru ФИНАНСЫ И КРЕДИТ / FINANCE AND CREDIT квартиру в надлежащем состоянии. Поэтому, когда она будет выставлена на продажу, ее стоимость может оказаться значительно ниже рыночной. Понятно, что кредитор не может повлиять ни на продолжительность жизни каждого конкретного кредитополучателя, ни на уровень цен на рынке не- движимости, ни на состояние заложенного объекта. Следовательно, единственным способом снижения риска является залоговая оценка объекта недви- жимости, которая влияет на сумму выдаваемого кредита по обратной ипотеке. На практике такая оценка не превышает 40% рыночной стоимости. РИСКИ, ХАРАКТЕРНЫЕ ДЛЯ ОПЕРАЦИИ ОБРАТНОЙ ИПОТЕКИ Как можно заметить, вариантов распорядиться своим жильем для повышения уровня благососто- яния на оставшиеся годы жизни у пожилых лю- дей имеется множество. Но большинство из них сопряжено с неприятным чувством утраты соб- ственности, которую можно было бы передать по наследству. Технология обратной ипотеки лише- на этого фундаментального недостатка, поскольку у наследников остается возможность расплатить- ся по кредиту и вступить в права наследства. Риски кредитора. Обратная ипотека имеет пре- имущество перед рассмотренными выше вариан- тами в том, что кредитор получает обратно в ито- ге только тело кредита и начисленные проценты и никакого «приза» в виде квартиры, доставшейся дешево в результате скорой смерти ее владельца после заключения договора. Недвижимость вы- ступает в роли залогового обеспечения, и не более. Таким образом, кредитор при обратной ипотеке нисколько не заинтересован в скором уходе из жизни кредитополучателя. Наоборот, чем дольше он живет, тем больший процентный доход получит кредитор. С другой стороны, для кредитора суще- ствует риск, что при высокой продолжительности жизни конкретного заемщика и неблагоприят- ной динамике цен на недвижимость тело кредита в какой-то момент может превысить продажную стоимость заложенной недвижимости. Понятно, что для кредитополучателя этот риск является по- ложительным, т. е. в терминах теории управления рисками он будет возможностью, а для кредитора это негативный риск [2]. И, следовательно, необхо- димо им управлять. Страхование как метод управления риском в за- падных странах активно применяется на практике. Более того, без страхования сделок по обратной ипотеке не бывает вовсе. В США, где обратная ипо- тека является частью пенсионной системы, работает государственная программа страхования убытков кредиторов [4]. Возможно также участие крупных страховых компаний в операциях по обратной ипотеке. Риски заемщика. К рискам заемщика относится, прежде всего, риск банкротства кредитора. В случае его наступления выплаты прекращаются на дли- тельное время или навсегда. Защитой заемщика от этого риска является механизм гарантированных выплат, который также реализован в США [4]. В слу- чае если финансовое учреждение, обслуживающее заем обратной ипотеки, не сможет осуществлять выплаты по договору, эту обязанность берет на себя гарант в лице государства. МЕЖДУНАРОДНЫЙ ОПЫТ ПРОВЕДЕНИЯ ОПЕРАЦИЙ ОБРАТНОЙ ИПОТЕКИ Американская модель обратной ипотеки После смерти владельца его жилье зачастую пе- реходит в собственность муниципальных властей, которые либо выставляют его на продажу, либо выделяют очередникам. Разумеется, так проис- ходит, если у владельца не объявятся наследники. Таким образом, развитый в США механизм обрат- ной ипотеки помогает владельцам недвижимости поднять свой уровень жизни, не лишаясь при этом жилья. Но, с другой стороны, в ряде случаев этот механизм способствует пополнению жилищного фонда муниципалитета, предназначенного для малообеспеченных категорий населения, выпол- няя, таким образом, важную социальную фун- кцию. Надо отметить, что в США механизм обрат- ной ипотеки является важной частью пенсионной системы, именно поэтому государственная под- держка играет столь важную роль. На этапе зало- говой оценки объекта недвижимости потенциаль- К методам управления рисками относятся сле- дующие: избежание риска, снижение риска, при- нятие риска на себя, сокращение потерь, передача риска другому субъекту и страхование [3]. Из всех перечисленных методов наиболее подходящими для данного случая являются методы сокращения потерь и страхование. Далее мы рассмотрим воз- можные варианты их применения. Метод сокращения потерь состоит в комплексе мероприятий, направленных на минимизацию потерь от наступления рискового события [3]. В дан- ном случае рисковым событием является превы- шение тела кредита над продажной стоимостью залогового имущества. Здесь еще приходится учи- тывать то, что владельцы недвижимости являются людьми престарелыми и, как правило, небогатыми. Следовательно, у них может не быть физических и материальных возможностей поддерживать свою ФИНАНСЫ: ТЕОРИЯ И ПРАКТИКА / FINANCE: THEORY AND PRACTICE  Т. 22, № 6’2018 108 Е. В. Кузьмина, А. А. Янин ного заемщика банк рассчитывает максимальную сумму кредита по договору обратной ипотеки. Кредит будет погашен через продажу квартиры после смерти заемщика, но если после продажи квартиры вырученной суммы не хватит для по- гашения кредита, то разницу банку-кредитору скомпенсирует государство, которое таким обра- зом, берет на себя риск, выполняя функцию стра- ховщика. Целью поддержки обратной ипотеки со стороны государства, осуществляемой в форме программы государственного страхования убыт- ков, является повышение ее привлекательности для кредиторов путем минимизации финансовых рисков. залоговой недвижимости, кредитор имеет право подать в HUD страховое требование о возмещении ему выплаченных сумм, после чего он освобождает- ся от обязательств по данному договору, а выплаты заемщику продолжит HUD 1. Защита преимуще- ственного права кредитора по сделке обратной ипотеки на объект недвижимости, являющийся предметом залога, всегда осуществляется на уровне законодательства штата, в котором располагается недвижимость. Британская модель обратной ипотеки Великобритания имеет более чем 40-летнюю исто- рию этого рынка. Его отличительной чертой явля- ется то, что до 2000 г. 1 Публикация об обратной ипотеке на портале Credits.ru. URL: http://credits.ru/publications/375564/obratnaya-ipoteka (дата обращения: 21.11.2018). МЕЖДУНАРОДНЫЙ ОПЫТ ПРОВЕДЕНИЯ ОПЕРАЦИЙ ОБРАТНОЙ ИПОТЕКИ доминирующим продуктом на нем была модель продажи, при которой про- давец передает право собственности на недви- жимость кредитору (покупателю) в обмен на еди- новременную денежную выплату и пожизненное право проживания. При этом покупатель заклю- чает с продавцом договор пожизненной аренды с безвозмездным проживанием до момента смерти продавца. Если до последнего финансового кризиса в США активными участниками рынка обратных ипотек были негосударственные финансовые организации, то после 2008 г. подавляющее большинство сделок совершается в рамках государственной программы HECM (Home Equity Conversion Mortgage, что можно перевести как «ипотека конверсии жилищного ка- питала»). Эта программа, представляющая собой партнерство государства с частным капиталом, была создана в 1989 г. как способ содействия гражданам, находящимся в возрасте старше 62 лет, в получении денежных средств за счет недвижимости, в кото- рой они проживают постоянно. В роли кредиторов выступают частные финансовые институты, но все операции строго регулируются специальными правилами, установленными Департаментом жи- лья и городского развития. Этот государственный орган, для обозначения которого часто использу- ется аббревиатура HUD (Department of Housing and Urban Development), курирует программу государ- ственного страхования обратных ипотек. Принци- пиальным моментом операций обратных ипотек, выдаваемых по программе HECM, является то, что все кредиты выдаются на пожизненный срок, при этом заемщик получает денежные средства в виде единовременной выплаты, аннуитета, ограничен- ного (фиксированного) числа выплат, кредитной линии, комбинации кредитной линии с фиксиро- ванным числом выплат и с аннуитетом. Заемщик имеет право поменять способ получения выплат оставшихся заемных средств. Государство в лице Федеральной жилищной администрации (Federal Housing Administration — сокращенно FHA) берет на себя функции страховщика. Этот орган входит в HUD и формирует специальный страховой фонд, из которого и осуществляются страховые выплаты. Что же касается кредитора, ему HUD гарантирует й В В случае если финансовое учреждение, обслуживающее заем обратной ипотеки, не сможет осуществлять выплаты по договору, эту обязанность берет на себя гарант в лице государства. Таким образом, кредитор становится собст- венником недвижимости, а бывший владелец — его арендатором. Конечно, такую схему нельзя в полной мере назвать обратной ипотекой, но с начала 2000-х гг. в Великобритании сущест- венно увеличилась доля классических обратных ипотек, которая превысила долю продуктов по схеме модели продаж. В 1991 г. в Великобритании была создана ассоциа- ция кредиторов Safe Home Income Plans (сокращенно — SHIP), которая включала в свой состав 22 постоянных члена. SHIP обслуживает 95% британского рынка, где действуют коммерческие банки, страховые компании Что же касается кредитора, ему HUD гарантирует возврат основной суммы кредита. МЕЖДУНАРОДНЫЙ ОПЫТ ПРОВЕДЕНИЯ ОПЕРАЦИЙ ОБРАТНОЙ ИПОТЕКИ В случае, когда она превысит 98% оценочной стоимости объекта 1 Публикация об обратной ипотеке на портале Credits.ru. URL: http://credits.ru/publications/375564/obratnaya-ipoteka (дата обращения: 21.11.2018). financetp.fa.ru 109 ФИНАНСЫ И КРЕДИТ / FINANCE AND CREDIT и мелкие кредитные организации, специализирую- щиеся на обратных ипотеках. право на пользование объектом залогового недви- жимого имущества. Заемщику не требуется вносить ежемесячные платежи в счет обслуживания и погашения займа. Он может в любой момент погасить свою задол- женность и расторгнуть договор обратной ипотеки. Заем может быть потрачен на различные цели, на- правленные на улучшение качества жизни граждан: оплата повседневных потребительских расходов, проведение дорогостоящих медицинских операций, улучшение жилищных условий, осуществление текущего и капитального ремонта, оплата услуг регулярного патронажа. Полученные средства зай- ма не могут быть направлены на цели, связанные с предпринимательской деятельностью. Надо отметить, что британское законодательство предусматривает специальные правила, согласно которым в процессе предварительной консультации потенциального заемщика с финансовым экспер- том и юристом участвуют наследники и другие материально заинтересованные лица [1]. Испанская модель обратной ипотеки Испания является вторым по величине рынком обратной ипотеки в Европейском союзе. Креди- ты обратной ипотеки выдаются коммерческими и сберегательными банками. Они предусматрива- ют в основном единовременную выплату и пред- лагаются на пожизненный или фиксированный срок. Испанский заемщик может при желании по- лучить и аннуитет, но приобретается такой про- дукт у страховых компаний. Схема оформления достаточно проста: пен- сионер оформляет кредит в банке под залог име- ющегося у него недвижимого имущества, после чего банк перечисляет ему кредит равными долями (или всю сумму единовременно) в течение срока кредитования. После смерти заемщика банк про- дает квартиру, погашает кредит, а оставшуюся от продажи объекта недвижимости часть денежных средств переводит наследникам. Договор обратной ипотеки базируется на статье 583 ГК РФ, согласно которой одна сторона (получатель ренты) передает другой стороне (плательщику ренты) в собствен- ность имущество, а плательщик ренты обязуется в обмен на полученное имущество периодически выплачивать получателю ренту в виде определен- ной денежной суммы либо предоставления средств на его содержание в иной форме. Австралийская модель обратной ипотеки Рынок обратных ипотек весьма развит и в Авс- тралии, где в 2005 г. была создана ассоциация SEQUAL, которая состоит из банков и финан- совых компаний, специализирующихся по сделкам обратной ипотеки. Члены ассоциации SEQUAL предоставляют потребителю единый список гарантий, основными из которых, так же как у HECM, являются отсутствие регресса, единообразие документации по сделке и реко- мендация консультации потенциального заем- щика с независимым финансовым экспертом и наследниками. МЕЖДУНАРОДНЫЙ ОПЫТ ПРОВЕДЕНИЯ ОПЕРАЦИЙ ОБРАТНОЙ ИПОТЕКИ Что касается оценки стоимости залоговой недви- жимости, то она проводится за счет АРИЖК, которое оплачивает услуги независимого оценщика. После того, как оценка объекта недвижимости произведе- на, рассчитывается размер займа, который зависит от залоговой оценки, а также от возраста заемщика и формы выплаты (ежемесячный аннуитет в пре- делах 10 лет или единовременная выплата в раз- мере 45–85% от стоимости жилья). После заключе- ния договора АРИЖК производит периодический контроль состояния заемщика и предмета ипотеки. Заметим, что полученные заемщиком денежные средства не являются его доходом, следовательно, не облагаются налогом. Таким образом, институт обратной ипотеки применяется как один из возможных финансовых инструментов дополнительного пенсионного обес- печения во многих странах с передовой экономикой. Обратная ипотека в Российской Федерации В России данный инструмент может занять опре- деленную нишу в линейке новых банковских про- дуктов. Разумеется, институт обратной ипотеки необходимо адаптировать под российские реалии, причем речь идет не только о процентных ставках, но и о системе страхования для всех сторон дого- вора обратной ипотеки. В Санкт-Петербурге программа обратной ипоте- ки действует с сентября 2015 г. в соответствии с за- коном Санкт-Петербурга от 19.11.2014 № 629–120. При заключении этого договора получатель ренты получает единовременную сумму в размере 10% от стоимости жилья, а затем всю жизнь получает ежемесячные рентные платежи в размере, установ- В настоящее время «обратная ипотека» в России действует в тестовом режиме. Ее инициатором стало Агентство по реструктуризации ипотечных жилищных кредитов (АРИЖК). Пенсионер получает заем под залог жилья. При этом он остается соб- ственником своей недвижимости на протяжении всей жизни, за ним также сохраняется пожизненное ФИНАНСЫ: ТЕОРИЯ И ПРАКТИКА / FINANCE: THEORY AND PRACTICE  Т. 22, № 6’2018 110 Е. В. Кузьмина, А. А. Янин ипотеки на основе расчета текущей приведенной стоимости ипотечного кредита. В работе выведе- на формула, по которой можно определить точку безубыточности обратного ипотечного кредита в реальных условиях с учетом нескольких видов риска кредитора. Затем, применяя социо-демо графические данные по Сингапуру, автор произво- дит оценку параметров модели, после чего следует вывод о том, что на сингапурском рынке возможно достижение прибыльности операции обратной ипотеки. Отличительной чертой данной работы является предположение о динамическом изме- нении ставки. Таким образом, автор использует в своей модели переменную процентную ставку по обратному ипотечному кредиту. Кроме того, в данной работе подробно описывается механизм формирования аннуитетных платежей по обратной ипотеке, который несколько отличается от стан- дартного механизма, используемого для обычной ипотеки. ленном договором ренты. ОБЗОР ЛИТЕРАТУРЫ И НАУЧНЫХ РАБОТ В основу представленной ниже математической модели легли несколько научных работ. ОБЗОР ЛИТЕРАТУРЫ И НАУЧНЫХ РАБОТ В основу представленной ниже математической модели легли несколько научных работ. S. Moulton et al. [7] анализируют риск банкрот- ства заемщика. Жилье остается в собственности заемщика до окончания срока выплат. Если заем- щик оказывается не в состоянии вовремя платить налоги, то взнос за него делает банк-кредитор, при этом размер взноса прибавляется к общему объему долга. Если остаточной стоимости зало- гового имущества не хватает для покрытия долга по налоговым выплатам, то заемщик объявляет- ся банкротом. В этом случае договор обратной ипотеки прекращается, а после продажи жилья с аукциона банк, используя механизм регресса, потребует покрытия своих расходов по оплате налоговых долгов заемщика. Авторы статьи ис- следуют влияние риска банкротства на прибыль банков и способы его минимизации. Кроме того, в статье исследуется влияние на поведение по- требителя различных способов выплат денеж- ных средств по договору обратного ипотечного кредитования. В статье H. Chen et al. [5] анализируется предло- жение продуктов обратной ипотеки на рынке США, а также соответствие рискам кредиторов установ- ленной банками наценки по платежам. В созданную авторами модель ценообразования включены все основные риски кредитора, которые затем учи- тываются при расчете текущей приведенной сто- имости обратного ипотечного кредита и размера страхового платежа. Авторы статьи делают вывод о том, что текущие процентные ставки по ипотеч- ным кредитам и размеры страховых взносов на рынке США в целом покрывают риски финансовых институтов. Структура модели, предложенная авто- рами данной статьи, взята за основу при разработке нашей модели. В статье B. Case, A. B. Schnare [3] содержится опи- сание американской программы HECM обратного ипотечного кредитования и рассматриваются не- сколько основных вопросов: • способы организации системы обратной ипотеки вообще и конкретно программы HECM; M. D. Hurd [8] исследует парадокс, не вписываю- щийся в стандартную микроэкономическую теорию, но обнаруживаемый эмпирическим путем. Суть этого парадокса состоит в накоплении людьми в развитых странах значительно большего объема материальных благ, чем они могут потребить. На основе этого автор делает вывод о существовании у людей экономических мотивов накопления на- следства. Хотя данная статья не связана непосред- ственно с обратной ипотекой, содержащиеся в ней идеи помогли при создании модели, поскольку институт обратной ипотеки предполагает исполь- зование недвижимости индивида, приобретение • характеристики заемщиков по обратной ипотеке и требования к ним со стороны кредито- ров; • мотивы, которыми руководствуются заем- щики при принятии решений об участии в про- грамме HECM. Выводы и положения данной статьи были ис- пользованы при построении уравнения спроса в нашей модели. Работа Y. K. МЕЖДУНАРОДНЫЙ ОПЫТ ПРОВЕДЕНИЯ ОПЕРАЦИЙ ОБРАТНОЙ ИПОТЕКИ Заключить договор ренты с городом, от лица которого выступает государст- венное бюджетное учреждение «Горжилобмен», могут граждане, достигшие возраста 75 лет. Как можно заметить, развитие системы обрат- ного ипотечного кредитования в России идет при- мерно теми же путями, что и в западных странах, что выражается в государственном регулировании и контроле, системе страхования рисков. Для более эффективного и надежного функционирования этой системы в нашей стране необходимо разработать стандарты раскрытия информации для анализа фи- нансовых продуктов, а также, по примеру западных стран, внедрить систему независимых консультан- тов для помощи потенциальным заемщикам на этапе подготовки к заключению договора. ОБЗОР ЛИТЕРАТУРЫ И НАУЧНЫХ РАБОТ В основу представленной ниже математической модели легли несколько научных работ. Tse [6] непосредственно посвящена моделированию финансовых продуктов обратной ипотеки. Автор исследует прибыльность обратной financetp.fa.ru 111 ФИНАНСЫ И КРЕДИТ / FINANCE AND CREDIT которой является популярным способом накопле- ния благ с целью передачи их по наследству. тетные платежи и кредитная линия. Моделирование кредитной линии требует наличия стохастического члена, отражающего желание i-го индивида взять определенную сумму денег в периоде n. Поскольку это порождает неопределенность, в данной работе при моделировании мы не будем рассматривать кредитную линию в качестве возможного способа выплаты маржи [10]. тетные платежи и кредитная линия. Моделирование кредитной линии требует наличия стохастического члена, отражающего желание i-го индивида взять определенную сумму денег в периоде n. Поскольку это порождает неопределенность, в данной работе при моделировании мы не будем рассматривать кредитную линию в качестве возможного способа выплаты маржи [10]. Изучение указанных выше научных публикаций помогло при формулировании постановки задач исследования, включая необходимость обращения особого внимания на риски экономических агентов, влияющих на ожидаемую прибыль и ожидаемую полезность операции, а также необходимость спе- цифицирования способа совершения платежей по договору обратной ипотеки в процессе моделирова- ния. Рассмотрим теперь основную математическую модель обратной ипотеки. Операция обратной ипотеки является примером annuity-due — способа платежей, т. е. такого формата, при котором платежи совершаются в начале пери- ода. Учитывая это обстоятельство, а также тот факт, что стандартная формула аннуитета необходима для вычисления размера платежа при заданном остатке займа (заметим, что в случае операции обратной ипотеки размер платежа известен, а оста- ток займа — нет), формула аннуитета была нами преобразована следующим образом (в предпо- ложении о помесячных аннуитетных выплатах): МОДЕЛИРОВАНИЕ ДЕНЕЖНЫХ ПОТОКОВ ПО ДОГОВОРУ ОБРАТНОЙ ИПОТЕКИ С ПОЖИЗНЕННЫМИ АННУИТЕТНЫМИ ПЛАТЕЖАМИ С ИСПОЛЬЗОВАНИЕМ ПРОГНОЗНОЙ МОДЕЛИ ПРОДОЛЖИТЕЛЬНОСТИ ЖИЗНИ ЗАЕМЩИКА 1 1 12 , 1 1 1 12 n b i L A i   + −     = ×     −     +   (2) Обратная ипотека представляет собой совершен- но новый продукт на финансовом рынке нашей страны. Внедрение нового вида услуг на свобод- ном рынке возможно только при условии наличия экономического интереса у всех сторон. Операция обратной ипотеки должна быть выгодна и заем- щикам, и кредиторам. Следовательно, существуют определенные факторы, стимулирующие участие в сделке потенциальных контрагентов, а также ограничения на параметры, действующие на ис- следуемом рынке. n — количество прошедших месяцев с начала выплаты аннуитетов. n — количество прошедших месяцев с начала выплаты аннуитетов. Учитывая формулу (2), мы вывели новую фор- мулу прибыли: ( ) 1 0 12 1 1 12 , 1 1 1 1 12 n n t n t i A n C r i − =       + −         π = − × −   − +   + ξ     ∑ ∑ (3) ( ) 1 0 12 1 1 12 , 1 1 1 1 12 n n t n t i A n C r i − =       + −         π = − × −   − +   + ξ     ∑ ∑ (3) ( ) ( ) ( ), N S I S S C π = + − − ∑ (1) (1) (3) где: S — тело кредита; S — тело кредита; I — процентная маржа; C — сопутствующие издержки при выдаче; C — сопутствующие издержки при выдаче; N (индекс суммирования) — количество выдан- ных обратных ипотечных кредитов. где: где: financetp.fa.ru (2) где: где: b L — общий остаток займа; b L — общий остаток займа; b L — общий остаток займа; b L — общий остаток займа; A — месячный аннуитетный платеж; i — годовая ставка; i — годовая ставка; Главным и непременным условием участия бан- ка-кредитора в операции обратного ипотечного кредитования является получение им по результа- там этой операции прибыли. В общем виде формула прибыли выглядит следующим образом: n — количество прошедших месяцев с начала выплаты аннуитетов. r — ставка дисконтирования; [ ] r — ставка дисконтирования; [ ] [ ] 1; n T ∈ , в первой сумме индексом суммиро- вания является n, а нижней и верхней границами суммирования, соответственно, значения 1 и Т, где Т — количество месяцев, на которые выдается кредит; Формула банковской прибыли выражает величи- ну суммарного превышения процентной надбавки над издержками по выдаче кредитов. Следующей задачей (в целях конкретизации формулы прибыли) является определение маржи I(S) [9]. Форма про- центной маржи полностью зависит от типа платежа. Математически все формы выплаты маржи являют- ся разновидностями двух видов платежей: аннуи- ξ — доля аннуитета с начисленными процен- тами в периоде t по отношению к общему объему займа. Математически все формы выплаты маржи являют- ся разновидностями двух видов платежей: аннуи- ФИНАНСЫ: ТЕОРИЯ И ПРАКТИКА / FINANCE: THEORY AND PRACTICE  Т. 22, № 6’2018 112 Е. В. Кузьмина, А. А. Янин Данные по смертности среди определенных возрастных групп населения обычно получают с по- мощью таблиц смертности. Однако у этого подхода имеется существенный недостаток: продолжитель- ность жизни вычисляется по историческим данным. Операция обратной ипотеки сильно растянута во времени на несколько периодов, поэтому кредитора интересует уровень смертности среди населения не в текущий момент, а в будущем. В периоде t дисконтируются только начисленные в нем аннуитетные платежи. Поэтому с учетом того, что результатом первой части формулы являет- ся чистый суммарный остаток долга, мы должны дисконтировать по действительной для периода t ставке только ту часть займа, которая были начи- слена в этом же периоде. Именно для этого в данной формуле и исполь- зуется переменная ξ . Расчет этой переменной яв- ляется нетривиальной математической задачей, поскольку при использовании формулы annuity due и постоянно возрастающей суммы долга функци- ональная связь между начисленным аннуитетом и суммой долга весьма трудно вычисляема. По этой причине допустимо использовать упрощенную формулу средней ставки дисконтирования, кото- рая выдает аналогичный результат при значениях ставки примерно до 40% годовых: Ожидаемую продолжительность жизни следует рассчитать с помощью модели Ли-Картера. Данная модель используется для предсказания уровней смертности для групп населения с помощью сто- хастического моделирования. ( ) , , ln , x t x x t x t m a b k e = + + (5) (5) где: д ln — натуральный логарифм, , x t m — показа- тель уровня смертности для возрастной группы х в периоде t; ( ) 1 0 12 1 1 . где: , U — матрица левых сингулярных векторов; x m — коэффициент смертности для х возраст- ной группы; V — матрица правых сингулярных векторов V* — сопряженно транспонированная матрица от матрицы V; x d — количество умерших в возрастной группе х; x Live — среднее количество живущих в воз- растной группе х. Σ — матрица сингулярных чисел (с сингулярными числами, которые находятся на главной диагонали). r — ставка дисконтирования; [ ] 1 n t t n r − = + ∑ ax — средние значения смертности по возраст- ным группам; Формула (3) справедлива для тех случаев, когда срок кредитования известен заранее и фиксиро- ван. Проблема расчета значения прибыли состоит, однако, в том, что наиболее распространенным и популярным видом аннуитетного платежа по операции обратной ипотеки является пожизнен- ный аннуитет. bx — изменение смертности для определенной возрастной группы; kt — вектор-тренд смертности для периода t; ux, t — нормально распределенная ошибка. , На начальный момент неизвестны все нахо- дящиеся справа векторы, поэтому их необходимо оценить по следующим формулам [7]: , На начальный момент неизвестны все нахо- дящиеся справа векторы, поэтому их необходимо оценить по следующим формулам [7]: Совершенно очевидно, что в момент взятия кредита продолжительность жизни заемщика — па- раметр неизвестный, и потому невозможно точно рассчитать денежный поток. Отсюда следует, что для приблизительной оценки ожидаемой прибыли по договору обратной ипотеки необходимо ис- пользовать такой параметр, как математическое ожидание продолжительности жизни заемщика [6]. Совершенно очевидно, что в момент взятия кредита продолжительность жизни заемщика — па- раметр неизвестный, и потому невозможно точно рассчитать денежный поток. Отсюда следует, что для приблизительной оценки ожидаемой прибыли по договору обратной ипотеки необходимо ис- пользовать такой параметр, как математическое ожидание продолжительности жизни заемщика [6]. Этот параметр вычисляется с помощью ко- эффициентов смертности (уровней смертности). В данном исследовании под этим термином мы будем понимать статистический показатель, вы считываемый по следующей формуле: ( ) , 1 1 ln , N x x t t a m N = = ×∑ (6) (6) где: где: N — общее количество периодов, для которых строится коэффициент смертности. N — общее количество периодов, для которых строится коэффициент смертности. Далее необходимо оценить член x t b k . Авторы модели Ли-Картера указывают на то, что оценку нельзя производить методом наименьших квадра- тов, предлагая использовать метод сингулярного разложения: Этот параметр вычисляется с помощью ко- эффициентов смертности (уровней смертности). В данном исследовании под этим термином мы будем понимать статистический показатель, вы считываемый по следующей формуле: , M U V = ∑ (7) (7) , x x x d m Live = (4) где: (4) M — разлагаемая матрица (в данном случае [ln( , x t m ) — x a ]); M — разлагаемая матрица (в данном случае [ln( , x t m ) — x a ]); где: где: xe — ожидаемая продолжительность жизни че- ловека, который находится в возрастной группе х; (9) γ — количество лет в возрастной категории (например, если смертность предсказывается для категорий по пять лет, то γ = 5); age — текущий возраст человека; j — индекс произведения; х — индекс возрастной группы; Х — максимальная возрастная группа, для ко- торой рассчитывается коэффициент смертности; где: д ( ) E π — математическое ожидание размера прибыли;  financetp.fa.ru 113 ФИНАНСЫ И КРЕДИТ / FINANCE AND CREDIT получает риск чрезмерного увеличения тела кре- дита: если объем кредита станет выше рыночной стоимости залогового имущества, то кредитор по- несет убыток. Увеличение длительности выплаты аннуитетов (т. е. увеличение продолжительности жизни), таким образом, несет двойной эффект: уве- личиваются начисляемые на тело кредита проценты, растет вероятность превышения тела кредита над рыночной стоимостью залога. В итоге модель Ли-Картера выдает матрицу (либо вектор) показателей уровня смертности населения для различных возрастных групп. После этого, возвращаясь к формуле (3), можно переписать ограничение на параметр n в следую- щем виде (для случая пожизненной ренты): ( ) ( ) ( ) age j age 1; , 1 , x x X x j x n E e E e age m = =   ∈ =   = + γ −ψ ∑∏ ãäå (8) ( ) ( ) ( ) age j age 1; , 1 , x x X x j x n E e E e age m = =   ∈ =   = + γ −ψ ∑∏ ãäå (8) (8) Предложим, исходя из этого, следующую фор- мулу прибыли: ( ) ( ) ( ) ( ) ( )  1 o 0 12 12 i 1 1 12 1 1 1 i 1 12 1 1 1 1 1 , n n n T t t E p A n p h C n r r − =       + −         = − − ×   −   +     × − γ − × − + π + ∑ ∑  (9) где: д ( ) E π — математическое ожидание размера прибыли;  ψ — корректирующий коэффициент. Математически коэффициент смертности не тождественен вероятности смерти (так как в числи- теле используется показатель «среднее количество живущих»). Однако он отличается от вероятности на константу, которую можно рассчитать по формуле γ — оценка общего размера займа «сверху» (по- лучаемая при превышении срока жизни заемщика над ожидаемым); n — длительность кредитования, соответству- ющая этой оценке; , x x Live O ψ = o C — операционные и другие виды издержек на выдачу кредита; где: Th — рыночная стоимость залогового имущества в периоде окончания кредитования; x O — количество людей, доживших до попадания в возрастную группу х. p — вероятность превышения общего баланса кредита над стоимостью обеспечения. Учитывая соотношение между параметрами, 1 ψ < , т. е. вероятность умереть меньше коэффи- циента смертности (для последней возрастной категории, однако, вероятность смерти равна еди- нице, а коэффициент смертности меньше единицы). Второе слагаемое является математическим ожиданием потерь банка. Второе слагаемое является математическим ожиданием потерь банка. Параметр p является функцией от уровня смер- тности, следовательно, 0 p m ∂ < ∂ [11]. Таким образом, в модели с появлением матема- тического ожидания продолжительности кредитова- ния начинает проявляться неопределенность. Кроме того, с увеличением длительности выплаты анну- итетов возрастает тело кредита. Обратная ипотека относится к кредитам без права регресса, т. е. заем- щик отвечает по своим обязательствам только теми активами, которые указываются в оформляемом при взятии кредита договоре. В случае с обратной ипотекой таким активом является жилье заемщика. Следовательно, кредитор имеет право покрывать свои издержки (и маржу) только в пределах сто- имости залогового имущества. Поэтому кредитор Длительность кредитования — не единственный аргумент этой функции. Вероятность превышения общего баланса кредита над стоимостью залогово- го обеспечения также зависит от цены залогового имущества. Будущая цена продажи жилья зависит от двух основных факторов: средняя рыночная цена за квадратный метр ( p h ) и износ жилья ( ch ). Как и в случае с длительностью кредитования, стоимость залогового имущества влияет как на вероятность потерь, так и на их величину. Таким образом, вероятность p представляет собой фун- кцию от нескольких аргументов: ФИНАНСЫ: ТЕОРИЯ И ПРАКТИКА / FINANCE: THEORY AND PRACTICE  Т. 22, № 6’2018 114 Е. В. Кузьмина, А. А. Янин c w ݓഥ 𝑦𝑦ଵ 𝑦𝑦ଶ 𝑐𝑐̅ Рис. 1 / Fig 1. Выбор оптимального потребления благ / Selection of optimal consumption of goods Источник / Source: составлено авторами / compiled by the authors. Рис. 1 / Fig 1. 2 Mortgage Conduct of Business Sourcebook. Гл. 3, 8, 9, 11, 12. URL: http://fsahandbook.info/FSA/handbook/LI/2003/2003_71. pdf (дата обращения: 20.11.2018). д ( ) E π — математическое ожидание размера прибыли;  Выбор оптимального потребления благ / Selection of optimal consumption of goods Источник / Source: составлено авторами / compiled by the authors. ( ) ( ) , , c w L f c w As p c p w = −λ − − (11) ( ) , , 0, 0, 0. p c p c p p p p f m h h m h h ∂ ∂ ∂ = < ∂ ∂ ∂ ïðè (10) ( ) ( ) , , c w L f c w As p c p w = −λ − − (11) где: L — функция Лагранжа; L — функция Лагранжа; Изучаемая формула не включает в себя взаимо- действие спроса и предложения, поэтому необходи- мо рассмотреть действующие на заемщика стимулы. λ — множитель Лагранжа; As — активы потребителя в денежном выраже- нии; As — активы потребителя в денежном выраже- нии; p — цены на материальные блага. д V — косвенная функция полезности. p — цены на материальные блага. 2 / Fig 2. Эффект обратного ипотечного кредита / Effect of reverse mortgage loan Источник / Source: составлено авторами / compiled by the authors. Тогда с помощью тождества Роя можно вывести маршаллианский спрос на потребительские блага: ленного количества w, чтобы получить больше c и оказаться на бюджетной границе в точке y2. Здесь проявляется проблема неделимости жи- лья: индивид не имеет возможности отказаться от части своего жилья: либо он продает w , либо ничего. По этой причине точка y2 (несмотря на то, что она находится на бюджетной границе) фактически является недостижимой в периоде t вследствие неделимости недвижимости. Теперь можно рассмотреть эффект обратного ипотеч- ного кредита. . c c V p D V As ∂ ∂ = −∂ ∂ (13) (13) Спрос на нормальное благо положительно за- висит от дохода, поэтому: 0. c D Ac ∂ > ∂ Отсюда, согла- сно функции маршаллианского спроса, можно сделать вывод о том, что с повышением дохода потребление благ также будет возрастать. Операция обратной ипотеки позволяет обойти ограничение неделимости объекта недвижимости, поскольку делает возможной операцию частич- ной конвертации неликвидного актива в деньги. Благодаря этому свойству, индивид-заемщик может достичь точки y3, которая по критерию полезности строго лучше начальной точки. Од- нако точка y3 все же хуже точки y2 вследствие того, что кредит уменьшает общий объем ак- тивов потребителя. Однако операция обратной ипотеки имеет еще одно важное преимущество: объект недвижимости — это не только наследст- во, но и благо потребления, поскольку индивид получает полезность от проживания в нем. По- скольку операция обратной ипотеки не лишает индивида-заемщика возможности проживать в своем объекте недвижимости, то этот объект не теряет своей полезности для него, как это было бы в случае продажи жилья). Заметим, что повышение объема ликвидных ак- тивов происходит за счет снижения объема нелик- видных активов. Причем вследствие существования банковской маржи выполняется нижеприведенное неравенство: T 1 . T t T t A L = < ∑ Заметим, что в любом периоде t общая сумма займа (фактически уменьшающая объем активов) превышает общую сумму полученных аннуитетов. Следовательно, операция обратной ипотеки, при прочих равных условиях, снижает общую сумму активов, которыми владеет индивид. Несмотря на это, операция обратной ипотеки может быть выгодна для заемщика вследствие двух качеств жилья: неделимость и низкая ликвидность. Предположим, что индивид владеет жильем, исполь- зуемым в двух качествах: наследство и место прожи- вания. Тогда может произойти следующая ситуация. Таким образом, обратная ипотека может быть выгодна при определенных предпочтениях по- требителей. p — цены на материальные блага. Согласно положениям микроэкономической теории основой потребительского выбора являются предпочтения индивида, которые можно выразить посредством функции полезности. Основным мо- тивом любого экономического субъекта является потребление благ, поэтому функция полезности представляет собой функцию от количества по- требляемых благ, т. е. услуг и товаров: U = f(c), где с — вектор потребляемых благ. Здесь следует заметить, что в формуле вместо дохода потребителя используются его активы: постановка задачи предполагает, что в каждом периоде потребитель делает выбор между потре- блением своих средств и накоплением наследства. В реальности имущество, используемое в каче- стве наследства, чаще всего обладает невысокой ликвидностью и покупается реже, чем один раз в период, поэтому индивиды осуществляют выбор другого рода: оставить часть своих активов в ка- честве имущества для наследования или продать их. Результатом решения проблемы максимизации является оптимальный набор параметров ( ) * , . c w Обратная ипотека оказывает на равновесие следу- ющее влияние: в каждом периоде к активам по- требителя добавляется сумма, равная аннуитетной выплате. Это должно оказать определенный эффект на потребление благ индивидом. На основе опти- мального набора благ можно вывести косвенную функцию полезности: Эмпирические данные демонстрируют тот факт, что люди часто накапливают больше материальных благ, чем могут потребить. У потребителей, таким образом, имеется еще один мотив — накопление наследства. Этот мотив состоит в том, что человек получает полезность от владения благами без их непосредственного потребления. Поскольку данный мотив особенно значим для пожилых людей (для которых и предназначена обратная ипотека), то его необходимо также рассмотреть: U = f(c, w), где w — вектор благ, накопленных в качестве наследст- ва. В каждом периоде потребитель решает задачу максимизации полезности 2: ( ) ( ) ( ) ( ) , , , , , , c w c w V p p Ac U c p As w p As = (12) где: ( ) ( ) ( ) ( ) , , , , , , c w c w V p p Ac U c p As w p As = (12) (12) где: д V — косвенная функция полезности. 115 financetp.fa.ru ФИНАНСЫ И КРЕДИТ / FINANCE AND CREDIT w c 𝑦𝑦ଵ 𝑦𝑦ଷ Рис. 2 / Fig 2. Эффект обратного ипотечного кредита / Effect of reverse mortgage loan Источник / Source: составлено авторами / compiled by the authors. c Рис. 2 / Fig 2. Эффект обратного ипотечного кредита / Effect of reverse mortgage loan Источник / Source: составлено авторами / compiled by the authors. Рис. p — цены на материальные блага. Мы предлагаем следующее условие осущест- вления индивидом обратного ипотечного займа в обобщенной форме: Допустим, заемщик имеет в периоде t наде- ление благами ( , , w c ), соответствующее точке y1. Индивид предпочел бы отказаться от опреде- ФИНАНСЫ: ТЕОРИЯ И ПРАКТИКА / FINANCE: THEORY AND PRACTICE  Т. 22, № 6’2018 116 Е. В. Кузьмина, А. А. Янин ( ) ( )( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( )( ) 0 0 1 1 ( 1 ( 1 , x T t t t T t t t t E e t t t t t age T E G m U c As A U c As m U w h U w h U c h m − = − = = + = −ψ +ρ × + − − − ψ +ρ × − − −γ − −ψ ∑ ∑ ∑  (14 В этом случае стандартное условие участия выглядит как ( ) E G alternative ≥ или, если другие альтернативы являются убыточными или недо- ступными, то ( ) 0 E G ≥ . (14) ( ) Вероятность того, что операция обратной ипоте- ки будет выгодна заемщику, повышается, благодаря следующим трем факторам: t age T = +∑ 1. ( ) ( ) MU c MU w  — если предельная по- лезность от потребления в начальной точке зна- чительно превышает предельную полезность от накопления наследства индивидом. В реальности такое может произойти, если у индивида, напри- мер, нет близких родственников. где: 3. ( ) ( ) 0 t U c h  — полезность от проживания в жилье для индивида весьма значительная. Теперь аналогично построим условие мотивации банковского агента: ( ) ( ) 1 1 12 [ 1 p 1 1 1 12 n i E D A n i       + −         = × − − ×   −   +     π ( ) ( ) ( )  ( ) ( ) ( ) ( ) ( ) 1 0 12 12 1 1 12 12 1 1 12 [ 1 p 1 1 1 12 1 1 1 1 1 1 1 ] , 1 1 n n b T t n t n t t o n i E D A n i p L h n r r Br r C N NB r B r − = − =       + −         = × − − ×   −   +     × − − × − + +   × +     +   − ≥ −   +   +     +   π ∑ ∑   (15) ( )  ( ) ( ) ( ) ( ) ( ) 1 0 12 12 1 1 12 12 1 1 1 1 1 1 1 ] , 1 1 n b T t n t n t t o n p L h n r r Br r C N NB r B r − = − = × − − × − + +   × +     +   − ≥ −   +   +     +   ∑ ∑   (15) • Третий член является ожиданием потерь полезности индивида от потери жилья. Если опе- рация обратной ипотеки имеет фиксированный срок выплат, то в том случае, когда индивид-заем- щик переживает этот срок, он теряет свой объект недвижимости и, следовательно, теряет и полез- ность от проживания в ней за каждый последую- щий год своей жизни вне дома. Разумеется, при системе пожизненной выплаты аннуитетов этот член равняется нулю. • Третий член является ожиданием потерь полезности индивида от потери жилья. 3. ( ) ( ) 0 t U c h  — полезность от проживания в жилье для индивида весьма значительная. Если опе- рация обратной ипотеки имеет фиксированный срок выплат, то в том случае, когда индивид-заем- щик переживает этот срок, он теряет свой объект недвижимости и, следовательно, теряет и полез- ность от проживания в ней за каждый последую- щий год своей жизни вне дома. Разумеется, при системе пожизненной выплаты аннуитетов этот член равняется нулю. (15) Ожидаемый выигрыш потребителя сравнивается с ожидаемыми выигрышами от других возможных источников финансирования: к примеру, в качестве альтернативного источника можно рассматривать продажу квартиры или взятие обычного потреби- тельского кредита. где: где: д B — номинал облигации;  r — процентная ставка доходности облигации в месячном выражении; N — количество приобретенных облигаций. 117 ρ — субъективная ставка дисконтирования; ρ — субъективная ставка дисконтирования; th — стоимость имущества; ( )  ( ) t U h — полезность от обладания жильем в пе- риоде t. ( ) t U h — полезность от обладания жильем в пе- риоде t. 2. Небольшое значение коэффициента смер- тности m — нетрудно увидеть, что величина ( ) 0. E G m ∂ < ∂ Теперь можно по порядку рассмотреть части формулы (14): • Первый член является дисконтированной суммой математического ожидания выигрышей от наличия обратной ипотеки. Индивид-заемщик получает аннуитетные выплаты, которые тратятся на потребление. Разница между полезностью с ан- нуитетами и без них представляет собой выигрыш заемщика от операции обратной ипотеки. Дан- ный выигрыш умножается на вероятность выжить заемщику в периоде t, дисконтируется и суммиру- ется по периоду кредитования. m ∂ Если потребитель конвертирует наследство в деньги, то он рассчитывает прожить несколько периодов, дабы воспользоваться полученными ликвидными средствами для потребления. Тради- ционно на рынках с несовершенной информацией возникает ситуация неблагоприятной селекции (adverse selection). На данном рынке возникает ред- кая ситуация благоприятной селекции. ( ) ( )  • Второй член представляет собой дискон- тированную сумму математического ожидания потерь заемщика. Полезность от мотива накопле- ния наследства зависит от стоимости этого акти- ва. При операции обратной ипотеки стоимость актива снижается на размер тела кредита, потому потери — это разница в стоимости недвижимо- сти в случае с обременением и без обременения. • Второй член представляет собой дискон- тированную сумму математического ожидания потерь заемщика. Полезность от мотива накопле- ния наследства зависит от стоимости этого акти- ва. При операции обратной ипотеки стоимость актива снижается на размер тела кредита, потому потери — это разница в стоимости недвижимо- сти в случае с обременением и без обременения. Эта разница тоже дисконтируется, умножается на вероятность смерти (так как польза от наличия наследства появляется только при наступлении смерти индивида-заемщика). • Второй член представляет собой дискон- тированную сумму математического ожидания потерь заемщика. Полезность от мотива накопле- ния наследства зависит от стоимости этого акти- ва. При операции обратной ипотеки стоимость актива снижается на размер тела кредита, потому потери — это разница в стоимости недвижимо- сти в случае с обременением и без обременения. Эта разница тоже дисконтируется, умножается на вероятность смерти (так как польза от наличия наследства появляется только при наступлении смерти индивида-заемщика). у р 3. ( ) ( ) 0 t U c h  — полезность от проживания в жилье для индивида весьма значительная. у р 3. ρ — субъективная ставка дисконтирования; ( ) ( ) 0 t U c h  — полезность от проживания в жилье для индивида весьма значительная. где: Q — количество домохозяйств, состоящих из людей возраста, подходящего для обратного ипо- течного кредитования; ϕ — доля тех из них, кто имеет подходящее имущество; Выводы G F — функция распределения чистых выигры- шей потребителей; В данной работе была построена математическая модель, позволяющая рассчитать получаемые бан- ком-кредитором денежные потоки по договору обратной ипотеки. Основанием для создания мо- дели являются функции мотивации экономических агентов. Мотивацией банковского агента является его ожидаемая прибыль, а мотивацией индивида- заемщика — ожидаемая полезность. Математи- ческая модель рассматривает выигрыши агентов, учитывая риски, которые они несут, типы платежей, которые совершаются по договору обратной ипо- теки, и различные эндогенно заданные параметры, которые являются индикаторами макроэкономи- ческой ситуации и состояния смежных рынков. Ре- зультатом проведенных с помощью модели расче- тов является ожидаемый чистый выигрыш банка. Это позволит рассмотреть экономическую целесо- образность вложения денежных средств в данный кредитный инструмент. Благодаря наличию в мо- дели фактора потребительского спроса, имеется возможность рассчитать равновесную ставку — следовательно, найти координаты точки рыночно- го равновесия. Возможно дальнейшее улучшение модели по следующим пунктам: подбор подходя- щих математических инструментов для получения оценок экзогенных параметров, рассмотрение бо- лее сложного случая совершения выплат по схеме кредитной линии, изучение характера зависимости от своих аргументов функции p. Z — некая константа, которая является индика- тором того, что не все потенциальные потребители услуги будут выбирать обратную ипотеку, даже если она для них выгодна. Эта константа может интерпретироваться по-разному (например, как уровень экономической рациональности объектов). Оптимальный выбор банковского агента мы выразим, если продифференцируем выражение в формуле (15) по процентной ставке: ( ) ( ) ( ) ( ) ( )  ( ) ( ) ( ) ( )   ( )  1 0 12 1 0 12 12 2 12 2 1 1 12 [ 1 1 1 1 12 1 1 1 1 1 1 ] [ 1 1 1 12 1 12 12 1 12 1 12 12 ]. financetp.fa.ru ФИНАНСЫ И КРЕДИТ / FINANCE AND CREDIT Заметим, что в данной модели процентная став- ка является основной переменной, которую может варьировать кредитор. Поскольку кредитор является лицом, коммерчески заинтересованным, он будет максимизировать свою прибыль по ставке i. Таким образом, коммерческий банк с помощью условия первого порядка может вывести оптимальную про- центную ставку. Согласно микроэкономической теории при известной функции спроса и получен- ных оценках экзогенных параметров необходимо приравнять выражение (17) к нулю, откуда может быть получена оценка i. Обычно доходность инвестиционного проекта сравнивают с доходностью альтернативного без- рискового вложения денежных средств. Причем в качестве такого безрискового вложения рассма- тривают вложение в государственные облигации. Для того чтобы сравнение было корректно, стоимос- ти проектов должны быть эквивалентны, потому BN = nAD. D — в данном случае есть не что иное, как количественное выражение спроса. Следовательно, спрос возможно выразить следующим образом: ( ) ( ) 1 , G D Q F alternative Z = ϕ× − − (16) (16) Формулы (16) и (17) образуют условие рыночного равновесия, поскольку коммерческий банк, обладая полной информацией о рынке, устанавливает ставку по операции обратной ипотеки, а индивиды-заем- щики предъявляют спрос на кредитный продукт (согласно своим функциям полезности). Выводы Следо- вательно, выводы, сделанные при помощи модели, могут быть использованы на практике банковскими и государственными агентами. Заметим, что для данного исследования, а именно для оценки па- раметров, присутствующих в модели, необходимо получить достаточное количество наблюдений; требуется следить за рынком потребительских и ипотечных кредитов на протяжении несколь- ких месяцев или лет (в зависимости от структуры данных). Поэтому модель имеет исключительную важность для будущих исследований. ЗАКЛЮЧЕНИЕ Резюмируя вышеизложенное, считаем необхо- димым подчеркнуть, что мировой опыт исполь- зования системы обратного ипотечного креди- тования свидетельствует об ее эффективности и привлекательности для пожилых людей с точки зрения улучшения их материального положения. Повышение благосостояния граждан преклонно- го возраста приведет к снижению социальной на- пряженности в обществе и к повышению уровня платежеспособного спроса на товары и услуги со стороны значительной части населения страны. Выводы n n T t t n o t t n n n n i E D p A n i i i p L h n r C D p A n r r i ni pA i r i ni i − = − =       + −     ∂ ∂     = × − − ×   ∂ ∂ −   +     × − − × + × − + × − × × + +   − + × + +     × − × +   − + × + +     × π ∑ ∑ (17) (17) Данная теоретическая модель имеет значитель- ный потенциал использования. В ней присутствуют необходимые условия для расчета объема рынка, ожидаемых выигрышей агентов и прочих важных ФИНАНСЫ: ТЕОРИЯ И ПРАКТИКА / FINANCE: THEORY AND PRACTICE  Т. 22, № 6’2018 118 Е. В. Кузьмина, А. А. Янин Очевидно, что этот фактор позитивно отразится на темпах экономического роста. Эти чрезвы- чайно важные в условиях кризиса социально- экономические последствия внедрения в эко- номическую жизнь страны операции обратной ипотеки означают необходимость ее всемерного стимулирования и регулирования со стороны го- сударства. Именно поэтому инициативы Агент- ства по ипотечному жилищному кредитованию заслуживают внимания и поддержки. Разумеется, речь не может идти о слепом и бездумном копи- ровании института обратной ипотеки западных стран. Его правовая и экономическая адаптация к российским реалиям потребует внимательного и детального осмысления результатов проведения «пилотного проекта». Законодателю потребуется разработать дополнительные правовые акты, до- полнить перечень банковских операций и прин- ципы банковского обслуживания в Российской Федерации, нормы и принципы наследственного права. Наконец, необходимо формировать си- стему повышения финансовой грамотности на- селения страны, одним из ключевых элементов которой могла бы стать система частных и госу- дарственных финансовых консультантов для фи- зических лиц. экономических показателей, на основе которых можно принимать управленческие решения. Следо- вательно, выводы, сделанные при помощи модели, могут быть использованы на практике банковскими и государственными агентами. Заметим, что для данного исследования, а именно для оценки па- раметров, присутствующих в модели, необходимо получить достаточное количество наблюдений; требуется следить за рынком потребительских и ипотечных кредитов на протяжении несколь- ких месяцев или лет (в зависимости от структуры данных). Поэтому модель имеет исключительную важность для будущих исследований. экономических показателей, на основе которых можно принимать управленческие решения. Список источников 1. Zedeck S. et al. APA dictionary of statistics and research methods. Washington, DC: American Psychological Association; 2014. 434 p. 1. Zedeck S. et al. APA dictionary of statistics and research methods. Washington, DC: American Psychological Association; 2014. 434 p. 2. Полиди Т. Д., Копейкин А., Семенюк А. Г., Языков А. Обратная ипотека: перспективы пр в России. М.: Фонд «Институт экономики города»; 2014. 14 c. ди Т. Д., Копейкин А., Семенюк А. Г., Языков А. Обратная ипотека: перспективы применени сии. М.: Фонд «Институт экономики города»; 2014. 14 c. 3. Case B., Schnare A. B. Preliminary evaluation of the HECM reverse mortgage program. Real Estate Economics. 1994;22(2):301–346. DOI: 10.1111/1540–6229.00636 3. Case B., Schnare A. B. Preliminary evaluation of the HECM reverse mortgage program. Real Estate Economics. 1994;22(2):301–346. DOI: 10.1111/1540–6229.00636 4. Raifner U., Clerc-Renaud S., Pérez-Carrillo E.F., Tiffe A., Knobloch M. Study on equity release schemes in the EU. 2009. URL: https://www.researchgate.net/publication/259295497_Study_on_equity_release_schemes_in_ EU_Part_I_General_Report (дата обращения: 21.11.2018). 4. Raifner U., Clerc-Renaud S., Pérez-Carrillo E.F., Tiffe A., Knobloch M. Study on equity release schemes in the EU. 2009. URL: https://www.researchgate.net/publication/259295497_Study_on_equity_release_schemes_in_ EU_Part_I_General_Report (дата обращения: 21.11.2018). 5. Chen H., Cox S. H., Wang S. S. Is the home equity conversion mortgage in the United States sustainable? Evidence from pricing mortgage insurance premiums and non-recourse provisions using the conditional Esscher transform. Insurance: Mathematics and Economics. 2010;46(2):371–384. DOI: 10.1016/j. insmatheco.2009.12.003i 6. Tse Y. K. Modelling reverse mortgages. Asia Pacific Journal of Management. 1995;12(2):79–95. DOI: 10.1007/ BF01734387 7. Moulton S., Haurin D. R., Shi W. An analysis of default risk in the Home Equity Conversion Mortg program. Journal of Urban Economics. 2015;90:17–34. DOI: 10.1016/j.jue.2015.08.002 program. Journal of Urban Economics. 2015;90:17–34. DOI: 10.1016/j.jue.2015.08.002 8. Hurd M. D. Mortality risk and bequests. Econometrica: Journal of the Econometric Society. 1989;57(4):779–813. DOI: 10.2307/1913772 8. Hurd M. D. Mortality risk and bequests. Econometrica: Journal of the Econometric Society. 1989;57( DOI: 10.2307/1913772 9. Lee R. D., Carter L. R. Modeling and forecasting US mortality. Journal of the American Statistical Association. 1992;87(419):659–671. DOI: 10.2307/2290201 10. Nakajima M., Telyukova I. A. Reverse mortgage loans: A quantitative analysis. The Journal of Finance. 2017;72(2):911–950. DOI: 10.1111/jofi.12489 11. Lee Y. T., Wang C. W., Huang H. C. On the valuation of reverse mortgages with regular tenure payments. Insurance: Mathematics and Economics. 2012;51(2):430–441. DOI: 10.1016/j.insmatheco.2012.06.008 119 financetp.fa.ru Е. В. Кузьмина, А. А. Янин References 1. Zedeck S. et al. APA dictionary of statistics and research methods. Washington, DC: American Psychological Association; 2014. 434 p. 2. Polidi T. D., Kopeikin A., Semenyuk A. G., Yazykov A. Reverse mortgage: Prospects for use in Russia. Moscow: Foundation “Institute for Urban Economics”; 2014. 14 p. (In Russ.). 3. Case B., Schnare A. B. Preliminary evaluation of the HECM reverse mortgage program. Real Estate Economics. 1994;22(2):301–346. DOI: 10.1111/1540–6229.00636 4. Raifner U., Clerc-Renaud S., Pérez-Carrillo E.F., Tiffe A., Knobloch M. Study on equity release schemes in the EU. 2009. URL: https://www.researchgate.net/publication/259295497_Study_on_equity_release_schemes_in_ EU_Part_I_General_Report (accessed 21.11.2018). 5. Chen H., Cox S. H., Wang S. S. Is the home equity conversion mortgage in the United States sustainable? Evidence from pricing mortgage insurance premiums and non-recourse provisions using the conditional Esscher transform. Insurance: Mathematics and Economics. 2010;46(2):371–384. DOI: 10.1016/j. insmatheco.2009.12.003 6. Tse Y. K. Modelling reverse mortgages. Asia Pacific Journal of Management. 1995;12(2):79–95. DOI: 10.1007/ BF01734387 7. Moulton S., Haurin D. R., Shi W. An analysis of default risk in the Home Equity Conversion Mortgage (HECM) program. Journal of Urban Economics. 2015;90:17–34. DOI: 10.1016/j.jue.2015.08.002 8. Hurd M. D. Mortality risk and bequests. Econometrica: Journal of the Econometric Society. 1989;57(4):779–813. DOI: 10.2307/1913772 9. Lee R. D., Carter L. R. Modeling and forecasting US mortality. Journal of the American Statistical Association. 1992;87(419):659–671. DOI: 10.2307/2290201 10. Nakajima M., Telyukova I. A. Reverse mortgage loans: A quantitative analysis. The Journal of Finance. 2017;72(2):911–950. DOI: 10.1111/jofi.12489 11. Lee Y. T., Wang C. W., Huang H. C. On the valuation of reverse mortgages with regular tenure payments. Insurance: Mathematics and Economics. 2012;51(2):430–441. DOI: 10.1016/j.insmatheco.2012.06.008 ИНФОРМАЦИЯ ОБ АВТОРАХ Елена Валерьевна Кузьмина — кандидат экономических наук, доцент финансового департамента НИУ «ВШЭ», Санкт-Петербург, Россия elena.kuzmina@autorambler.ru Андрей Алексеевич Янин — магистрант, Высшая школа менеджмента, Санкт-Петербургский государст- венный университет, Санкт-Петербург, Россия andreyyanin1995@mail.ru Елена Валерьевна Кузьмина — кандидат экономических наук, доцент финансового департамента НИУ «ВШЭ», Санкт-Петербург, Россия elena.kuzmina@autorambler.ru Андрей Алексеевич Янин — магистрант, Высшая школа менеджмента, Санкт-Петербургский государст- венный университет, Санкт-Петербург, Россия andreyyanin1995@mail ru ФИНАНСЫ: ТЕОРИЯ И ПРАКТИКА / FINANCE: THEORY AND PRACTICE  Т. 22, № 6’2018 120
https://openalex.org/W1976825733	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0113810&type=printable	English	null	Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation	PloS one	2,014	cc-by	15,792	Abstract This is an open-access article distributed under the terms of the Creative Commons Att unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are wit Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant Funding: Oklahoma Center for the Advancement of Science and Technology HR12-008 AFW, University of Oklahoma College of Medicine Alumni Association AFW, and National Eye Institute EY021497 BPC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ter for the Advancement of Science and Technology HR12-008 AFW, University of Oklahoma College of Medicine Alumni Association stitute EY021497 BPC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * Email: allan-wiechmann@ouhsc.edu Allan F. Wiechmann1,2, Brian P. Ceresa3, Eric W. Howard1 1 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 2 Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 3 Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky, United States of America Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation Allan F. Wiechmann1,2, Brian P. Ceresa3, Eric W. Howard1 Abstract Background and Objectives: The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium. Methodology/Principal Findings: Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1- MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohisto- chemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime. Conclusions/Significance: MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals. Received September 24, 2014; Accepted October 31, 2014; Published November 20, 2014 Received September 24, 2014; Accepted October 31, 2014; Published November 20, 2014 Copyright: 2014 Wiechmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Wiechmann et al. Citation: Wiechmann AF, Ceresa BP, Howard EW (2014) Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation. PLoS ONE 9(11): e113810. doi:10.1371/journal.pone.0113810 November 2014 \| Volume 9 \| Issue 11 \| e113810 Tissue preparation Whole eye globes were removed from the frogs and immersion- fixed for a total of one hour at 4uC in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. Midway through the fixation period, the anterior segments (cornea, iris, ciliary body and lens) were dissected free of the posterior segments. This protocol was employed to minimize disturbance of the CE prior to fixation, yet achieve sufficient preservation of the cornea. The mechanism of activation of MMP-2 is perhaps the best understood of the entire family of zinc-dependent MMPs [26–29]. The most potent means of activation of MMP-2 occurs through formation of a ternary complex with membrane type 1 (MT1)- MMP (also called MMP-14) and tissue inhibitor of MMP-2 (TIMP-2) [26–28]. TIMP-2 bound to the membrane-anchored MT1-MMP acts as a receptor for pro-MMP-2. Binding of pro- MMP-2 to TIMP-2 (bound to MT1-MMP) enables adjacent active molecules of MT1-MMP to cleave and activate the MMP-2. After MT1-MMP is activated, it is rapidly internalized from the cell surface [27,28]. MMP-2 activity is highly dependent on the levels of TIMP-2; low (equimolar) levels of TIMP-2 are required for MMP-2 activation, whereas a higher (two-fold) level of TIMP-2 inhibits MMP-2 activation [28,30]. For preparation of cryostat sections, corneas were dissected free of the rest of the anterior segments and rinsed three times with 0.1 M phosphate-buffered saline (PBS), pH 7.4. Isolated corneas were transferred to 30% sucrose in phosphate buffer for 16–20 hr at 4uC, and then mounted in Tissue-Tek O.C.T. mounting matrix (Sakura Finetek, Torrance, CA). Sagittal 10 mm sections were cut on a cryostat microtome and collected onto glass slides. Sections were rinsed three times with PBS, and then placed into 0.01 M sodium citrate buffer with 0.05% Tween-20 (Sigma) for epitope retrieval treatment. Slides were incubated at 100uC for 10 min, allowed to cool to room temperature (RT) for 20 min, then rinsed three times with PBS and incubated in blocking buffer (2% bovine serum albumin [BSA; Sigma] 0.2% Triton X-100 [Sigma], and 0.04% sodium azide in PBS) for at least one hour. We chose the Xenopus laevis model because our previous studies on circadian events in the Xenopus eye established the foundation for this present investigation, and circadian rhythms have been particularly well-studied in this model [31–33]. Also, since Xenopus are aquatic, there are fewer confounding issues of nocturnal eyelid closure and daytime dryness as occurs in terrestrial mammals. Introduction breakdown of corneal barrier integrity is a health concern for millions of people. Disruption of the integrity of the CE barrier contributes to incapacitating disorders such as herpetic infection and corneal erosions, and the very prevalent disorder of keratoconjunctivitis (dry eye syndrome) [1–4]. The uniquely transparent cornea comprises much of the anterior surface of the eye and is incessantly challenged by environmental assaults during the daytime. A non-keratinizing stratified squamous epithelium and thin layer of tear film cover the corneal surface. The single thin layer of cells at the tear/surface interface of the corneal epithelium (CE) provides the most crucial barrier to ocular infection. Deterioration of sight due to The basal layer of cells is the proliferative layer of the CE, and as the basal CE cells divide (on a cyclic rhythm) [5–8], they give rise to daughter cells that are displaced apically. These epithelial cells continue to be displaced apically as additional cells are November 2014 \| Volume 9 \| Issue 11 \| e113810 1 PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e113810 Circadian Regulation of Tight Junctions in Corneal Epithelium Circadian Regulation of Tight Junctions in Corneal Epithelium Circadian Regulation of Tight Junctions in Corneal Epithelium Circadian Regulation of Tight Junctions in Corneal Epithelium generated from the basal epithelium. The addition of new cells from the basal layer is balanced by the shedding of living epithelial cells at the CE surface. In the healthy cornea, desquamation of surface CE cells is not dependent upon apoptosis as was once thought [9], indicating that the living surface cells must actively dissociate themselves (i.e.; disconnect their junctional complexes) from neighboring cells. Tissue preparation Additionally, the functions of MMPs have been particularly well-studied in this species in which they were originally discovered [20,21,34]. The purpose of this project was to determine if; 1) there are day/night changes in the pattern of expression of tight junction proteins in Xenopus laevis CE, 2) if any diurnal changes in the pattern of tight junction protein expression correlate negatively with local expression of MMP proteins, and 3) and if areas of surface cell desquamation are associated with the presence of MMP at or near the surface epithelium. Our data suggest that discrete clusters of surface CE are subjected to intercellular detachment and subsequent desquamation, and that this process is mediated via MMP activity associated with tight junction protein dissociation. Furthermore, this mosaic pattern of MMP expression, tight junction degradation and cell surface desquamation occurs preferentially during the nighttime, suggest- ing a circadian influence on CE surface cell homeostatic turnover. For preparation of corneas for use in whole mount immuno- cytochemistry, fixed anterior segments were placed individually into shallow-bottomed 2.0 ml microfuge tubes. They were rinsed three times with PBS and then transferred into 0.01 M sodium citrate buffer/0.05% Tween-20 for epitope retrieval treatment. Specimens were incubated in a water bath at a starting temperature of 70uC which increased progressively to 80uC during a course of 10 min. The temperature range was selected to afford some epitope retrieval, yet minimize distortion of the tissues. Epitope retrieval was necessary in this study due to the relatively lower reactivity of Xenopus proteins with antibodies generated against mammalian proteins. The anterior segments were kept intact during this procedure as they helped to lessen distortion of the corneas during the heating process. Tissues were allowed to cool to RT for 30 min, then rinsed three times with PBS and incubated in BSA blocking buffer for at least two hours. The corneas were dissected free of the remaining anterior segment components during the PBS washing steps. Animals Post-metamorphic Xenopus laevis (African clawed frogs) were obtained from Xenopus Express (Brooksville, FL) and maintained in aquaria at 20uC on a daily lighting schedule of 12 hr dark: 12 hr light for a minimum of two weeks. Frogs were deeply anesthetized by immersion in 0.5% triciane methanesulfonate (MS-222; Sigma, St. Louis, MO) in buffered water and killed by decapitation at specific times during the light/dark cycle. The transmembrane tight junction proteins occludin and claudin extend extracellularly from the lateral plasma membranes to form covalent bonds with complementary proteins on neighboring cells. This cross-linking creates a paracellular barrier that regulates the permeability of the epithelium, which is especially tight in the CE. Occludin and claudin have been shown to be somewhat unanticipated targets for cleavage by matrix metalloproteinases (MMPs) in a wide range of pathological conditions and tissues [10–19]. MMPs were named as such based on initial observations in Xenopus laevis in which they degraded extracellular matrix proteins such as collagen [20,21], but it has become increasingly apparent that the array of protein targets of MMP cleavage extend far beyond extracellular matrix proteins [22–24]. The vast majority of studies on the role MMPs in adult tissues have focused on their responses to pathological situations [11–13,19,25]. In this study, we propose a novel role for MMP-2 (and its binding partners) in the normal homeostasis of epithelial renewal and turnover. Ethics statement Animals were cared for in accordance with the guidelines of the Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center (Protocol number 12-076-T). In situ zymography procedures MMP activity in Xenopus CE was analyzed by in situ zymography, as described by Hadler-Olsen et al. [35] DQ gelatin (Molecular Probes) is a fluorescein-gelatin conjugate that is so heavily labeled with fluorescein that the fluorescence is quenched. This substrate is digested by gelatinases and collagenases in tissue sections to yield fluorescent peptides that can be visualized by fluorescence microscopy. Corneas were obtained from frogs late in the dark period 2 hrs before lights on, and early in the light period 2 hrs after lights on. Isolated corneas were mounted in O.C.T. mounting matrix, and sagittal 10 mm sections were cut on a cryostat microtome and collected onto glass slides. Unfixed corneal cryostat sections were air-dried and stored at -80uC until time of use. Whole corneas were mounted (CE surface up) onto glass slides by making 4–5 slits from peripheral to mid-cornea with scissors and then gently compressing the tissue under weighted coverslips after the mounting matrix was applied to achieve a flat-mounted cornea. Coverslips were mounted onto the slides with tissue sections or whole corneas with Prolong Gold Antifade reagent containing DAPI (Life Technologies, Eugene, OR). For double-label immunocytochemistry, the same procedure was followed as described for the primary antibody incubations, except that after the first labeling procedure, the tissues or sections were incubated with 5 mg/ml of a second primary antibody that was raised in a species different from that used in the first immunolabeling. The primary antibody was labeled with 2 mg/ml goat anti-rabbit or goat anti-mouse antibody conjugated to AlexaFluor 488 (green) or AlexaFluor 568 (red), using the same conditions as described for the first immunolabeling, and then mounted onto glass slides and coverslips. In all of the corneal whole mount experiments, images from only the central region of the cornea were captured so that similar regions among samples were analyzed. Thawed sections were rinsed once with reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.2 mM sodium azide, pH 7.7), and then incubated in a humidified chamber at RT for 30 min in reaction buffer containing 1 mg/ml DQ-gelatin with or without 20 mM EDTA or 1 mM 1,10-phenanthroline (Sigma) as negative controls. EDTA and phenanthroline inhibit MMP activity, and are often employed to determine the specificity of the gelatinase activity [35,36]. MMP-2 and TIMP are co-localized in Xenopus surface corneal epithelium sections Cryostat sections of Xenopus cornea immunolabeled for either MMP-2 or TIMP-2 revealed that both proteins were expressed mainly in the squamous surface layer of cells in the CE (Figure 1). The experiments with corneal sections preceded the use of corneal whole mounts in this study, and during the course of the study, we altered the timing of harvesting the corneas. The corneas used for the cryostat section experiments were obtained either late in the dark period (2 hr before lights on) or early in the light period (2 hrs after lights on), so the time points were only four hours apart. These were the same time points used in the in situ zymography experiments, but later when using corneal whole mounts, we 5.0 software, San Diego, CA). Significance was assumed for p values less than 0.05. 5.0 software, San Diego, CA). Significance was assumed for p values less than 0.05. the Xenopus proteins. The primary antibodies used in this study are listed in Table 1. For negative controls, tissue sections or whole corneas were incubated in blocking buffer lacking the primary antibody. Following incubation with the primary antibody, sections or whole corneas were rinsed three times in PBS, and incubated in 2 mg/ml of goat anti-rabbit or goat anti-mouse antibody conjugated to AlexaFluor 488 (green; Molecular Probes, Eugene, OR) or AlexaFluor 568 (red) in PBS for 2 hr at RT. Sections or whole corneas were rinsed three times in PBS. In situ zymography procedures The sections were then rinsed three times in PBS, and placed into 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for 10 min at RT. The sections were rinsed three times in PBS, and coverslips were mounted onto the slides with Prolong Gold Antifade reagent containing DAPI. The presence of fluorescence in the corneal sections was analyzed by confocal microscopy. The immunolabeled sections and corneal whole mounts were viewed by confocal microscopy, using an Olympus FluoView 1000 laser-scanning confocal microscope (Center Valley, PA). The pinhole (confocal aperture diameter) conditions were fixed at 105 mm in all of the images generated in this study. The objective lenses used in this study were an Olympus PlanApo N 606/1.42 Oil lens (‘/0.17/FN26.5), and UPlanSApo 1006/1.40 Oil lens (‘/0.17/FN26.5). The control specimens were examined under identical conditions to the appropriate non-control specimens. In all cases, image scale was calibrated and brightness and contrast were adjusted as necessary to highlight specific labeling. Confocal immunocytochemistry procedures Cryostat sections or whole corneas were incubated with 5 mg/ ml of primary antibody in BSA blocking buffer for three days at 10uC. This prolonged incubation period was necessary because of the relatively lower reactivity of the mammalian antibodies with November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 2 Circadian Regulation of Tight Junctions in Corneal Epithelium Circadian Regulation of Tight Junctions in Corneal Epithelium 5.0 software, San Diego, CA). Significance was assumed for p values less than 0.05. 5.0 software, San Diego, CA). Significance was assumed for p values less than 0.05. Circadian Regulation of Tight Junctions in Corneal Epithelium Circadian Regulation of Tight Junctions in Corneal Epithelium Figure 1. MMP-2 and TIMP are co-localized in Xenopus leavis surface corneal epithelium sections. Cryostat sections of corneas obtained during the late dark period (NIGHT: two hrs before lights on) or early light period (DAY; two hrs after lights on) were immunolabeled with MMP-2 and or/TIMP-2 antibodies and analyzed with confocal microscopy. (A) At nighttime, MMP-2 immunoreactivity (red) was localized in cell clusters mainly to the surface layer of CE cells (arrow), with some labeling also present in the underlying sub-superficial layer of cells. Immunolabeling in the deeper layers of the CE was very sparse. (B) As negative controls, corneal sections obtained at night were processed for immunohistochemistry in the absence of primary antibody, and no specific immunoreactivity was observed (arrow). (C) At early daytime, intensity of MMP-2 immunoreactivity (red) was generally diminished relative to the night time levels (arrow). (D) At late nighttime, TIMP-2 immunoreactivity (green) was localized in cell clusters mainly to the surface layer of CE cells (arrow), with some labeling also present in the underlying sub-superficial layer of cells. Immunolabeling in the deeper layers of the CE was very sparse. (E) As negative controls, corneal sections obtained during the daytime were processed for immunohistochemistry in the absence of primary antibody, and no specific immunoreactivity was observed (arrow). (F) At early daytime, intensity of TIMP-2 immunoreactivity (green) was generally higher relative to the nighttime levels (arrow), and some labeling also present in the underlying sub-superficial layer of cells, inverse of the temporal pattern observed with MMP-2 in A and C. (G) Double-label immunocytochemistry of MMP-2 (red) and TIMP-2 (green) of nighttime corneas revealed co-localization of the two proteins. Yellow indicates regions of co-localization of the red and green signal (arrows). (H) During the early daytime, MMP-2 (red) and TIMP-2 (green) displayed a similar yellow co-localization (arrow) as seen at nighttime. Note that the two proteins were either expressed together (arrow) or not at all (arrowhead). Sections were stained with DAPI, which stained the nuclei blue. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g001 immunolabeling of these proteins was not continuous throughout the cornea; they were present sporadically in clusters of surface cells. Some intermittent immunolabeling for both proteins was also observed in the layer of sub-superficial cells directly below the surface layer. Circadian Regulation of Tight Junctions in Corneal Epithelium When present in the scattered cell clusters, the intensity of immunolabeling for MMP-2 was generally higher late at night (Figure 1A) than early in the day (Figure 1C), whereas TIMP-2 labeling intensity was generally lower late at night (Figure 1D) than early in the day (Figure 1F). Sections incubated without primary antibody displayed no specific immunoreactivity (Figure 1B and 1E). Double-label immunocytochemistry of MMP- 2 and TIMP-2 revealed that the two proteins co-localized to the same cells in the surface CE (Figure 1G and 1H). Moreover, they were either expressed together or neither protein was expressed. MMP gelatinase activity is present in Xenopus surface corneal epithelium (F) At early daytime, intensity of TIMP-2 immunoreactivity (green) was generally higher relative to the nighttime levels (arrow), and some labeling also present in the underlying sub-superficial layer of cells, inverse of the temporal pattern observed with MMP-2 in A and C. (G) Double-label immunocytochemistry of MMP-2 (red) and TIMP-2 (green) of nighttime corneas revealed co-localization of the two proteins. Yellow indicates regions of co-localization of the red and green signal (arrows). (H) During the early daytime, MMP-2 (red) and TIMP-2 (green) displayed a similar yellow co-localization (arrow) as seen at nighttime. Note that the two proteins were either expressed together (arrow) or not at all (arrowhead). Sections were stained with DAPI, which stained the nuclei blue. Scale bar = 20 mm. doi:10 1371/journal pone 0113810 g001 Figure 1. MMP-2 and TIMP are co-localized in Xenopus leavis surface corneal epithelium sections. Cryostat sections of corneas obtained during the late dark period (NIGHT: two hrs before lights on) or early light period (DAY; two hrs after lights on) were immunolabeled with MMP-2 and or/TIMP-2 antibodies and analyzed with confocal microscopy. (A) At nighttime, MMP-2 immunoreactivity (red) was localized in cell clusters mainly to the surface layer of CE cells (arrow), with some labeling also present in the underlying sub-superficial layer of cells. Immunolabeling in the deeper layers of the CE was very sparse. (B) As negative controls, corneal sections obtained at night were processed for immunohistochemistry in the absence of primary antibody, and no specific immunoreactivity was observed (arrow). (C) At early daytime, intensity of MMP-2 immunoreactivity (red) was generally diminished relative to the night time levels (arrow). (D) At late nighttime, TIMP-2 immunoreactivity (green) was localized in cell clusters mainly to the surface layer of CE cells (arrow), with some labeling also present in the underlying sub-superficial layer of cells. Immunolabeling in the deeper layers of the CE was very sparse. (E) As negative controls, corneal sections obtained during the daytime were processed for immunohistochemistry in the absence of primary antibody, and no specific immunoreactivity was observed (arrow). (F) At early daytime, intensity of TIMP-2 immunoreactivity (green) was generally higher relative to the nighttime levels (arrow), and some labeling also present in the underlying sub-superficial layer of cells, inverse of the temporal pattern observed with MMP-2 in A and C. (G) Double-label immunocytochemistry of MMP-2 (red) and TIMP-2 (green) of nighttime corneas revealed co-localization of the two proteins. MMP gelatinase activity is present in Xenopus surface corneal epithelium In situ zymography was performed on unfixed Xenopus corneal sections, in which a quenched fluorescently-labeled gelatin complex is cleaved by endogenous active gelatinases (i.e.; MMP- 2 and MMP-9) to un-quench the fluorescent signal. Therefore, the presence of green fluorescence indicates the presence of gelatinase enzyme activity. High levels of presumptive MMP activity were present in the surface layers of the CE, especially late at night (Figure 2A). The endogenous MMP activity appeared to be lower early in the light period (Figure 2B), thus supporting the hypothesis that MMP activity declines during the light period. Inclusion of a specific inhibitor of MMP-2 and MMP-9 (phenanthroline) in the incubation mixture blocked most of the gelatinase activity (Figure 2C), confirming that most of the fluorescent signal is due to the presence of MMP activity. In addition, the presence of the zinc chelator EDTA blocked the gelatinase activity (data not shown), also suggesting that the observed gelatinase activity is due to MMP. Figure 1. MMP-2 and TIMP are co-localized in Xenopus leavis surface corneal epithelium sections. Cryostat sections of corneas obtained during the late dark period (NIGHT: two hrs before lights on) or early light period (DAY; two hrs after lights on) were immunolabeled with MMP-2 and or/TIMP-2 antibodies and analyzed with confocal microscopy. (A) At nighttime, MMP-2 immunoreactivity (red) was localized in cell clusters mainly to the surface layer of CE cells (arrow), with some labeling also present in the underlying sub-superficial layer of cells. Immunolabeling in the deeper layers of the CE was very sparse. (B) As negative controls, corneal sections obtained at night were processed for immunohistochemistry in the absence of primary antibody, and no specific immunoreactivity was observed (arrow). (C) At early daytime, intensity of MMP-2 immunoreactivity (red) was generally diminished relative to the night time levels (arrow). (D) At late nighttime, TIMP-2 immunoreactivity (green) was localized in cell clusters mainly to the surface layer of CE cells (arrow), with some labeling also present in the underlying sub-superficial layer of cells. Immunolabeling in the deeper layers of the CE was very sparse. (E) As negative controls, corneal sections obtained during the daytime were processed for immunohistochemistry in the absence of primary antibody, and no specific immunoreactivity was observed (arrow). Data analysis Red or green channels of confocal images were converted to grayscale in Photoshop for quantitative analysis. Relative image intensities of four representative areas of each group were measured using Image J software. Statistical analysis was performed using the unpaired Student’s t-test (GraphPad Prism Table 1. List of Primary Antibodies. Antigen Manufacturer (catalog #) Host Claudin-4 Invitrogen (364800) rabbit Claudin-4 Invitrogen (329400) mouse MMP-2 Invitrogen (436000) mouse MT1-MMP, hinge region Millipore (AB6004) rabbit Occludin Invitrogen (711500) rabbit Occludin Invitrogen (331500) mouse TIMP-2 Millipore (AB2965) rabbit TIMP-2 Millipore (MAB13446) mouse doi:10.1371/journal.pone.0113810.t001 PLOS ONE \| www.plosone.org 3 November 2014 \| Volume 9 \| Issue 11 \| e113810 Table 1. List of Primary Antibodies. Table 1. List of Primary Antibodies. Table 1. List of Primary Antibodies. Antigen Manufacturer (catalog #) Host Claudin-4 Invitrogen (364800) rabbit Claudin-4 Invitrogen (329400) mouse MMP-2 Invitrogen (436000) mouse MT1-MMP, hinge region Millipore (AB6004) rabbit Occludin Invitrogen (711500) rabbit Occludin Invitrogen (331500) mouse TIMP-2 Millipore (AB2965) rabbit TIMP-2 Millipore (MAB13446) mouse doi:10.1371/journal.pone.0113810.t001 PLOS ONE \| www.plosone.org 3 November 2014 \| Volume 9 \| Issue 11 \| e113810 November 2014 \| Volume 9 \| Issue 11 \| e113810 3 MMP gelatinase activity is present in Xenopus surface corneal epithelium Yellow indicates regions of co-localization of the red and green signal (arrows). (H) During the early daytime, MMP-2 (red) and TIMP-2 (green) displayed a similar yellow co-localization (arrow) as seen at nighttime. Note that the two proteins were either expressed together (arrow) or not at all (arrowhead). Sections were stained with DAPI, which stained the nuclei blue. Scale bar = 20 mm. doi:10 1371/journal pone 0113810 g001 Figure 2. In situ zymography demonstrates MMP gelatinase activity in Xenopus surface corneal epithelium. The presence of green fluorescence with confocal microscopy indicates the presence of gelatinase enzyme activity in unfixed cornea sections obtained during the late dark period (NIGHT: two hrs before lights on) or early light period (DAY; two hrs after lights on). (A) Late at night, MMP activity (green) was present in the surface layers of the CE (arrow) with some labeling also present in the deeper layers of the CE. In the early daytime, MMP activity (green) in the surface CE (arrow) appeared to be lower early in the light period with some labeling persisting in the deeper layers of the cornea. (C) Inclusion of a specific inhibitor of MMP-2 and MMP-9 (1.0 mm phenanthroline) in the incubation mixture blocked most of the gelatinase activity. Sections were stained with DAPI, which stained the nuclei blue. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g002 Figure 2. In situ zymography demonstrates MMP gelatinase activity in Xenopus surface corneal epithelium. The presence of green fluorescence with confocal microscopy indicates the presence of gelatinase enzyme activity in unfixed cornea sections obtained during the late dark period (NIGHT: two hrs before lights on) or early light period (DAY; two hrs after lights on). (A) Late at night, MMP activity (green) was present in the surface layers of the CE (arrow) with some labeling also present in the deeper layers of the CE. In the early daytime, MMP activity (green) in the surface CE (arrow) appeared to be lower early in the light period with some labeling persisting in the deeper layers of the cornea. (C) Inclusion of a specific inhibitor of MMP-2 and MMP-9 (1.0 mm phenanthroline) in the incubation mixture blocked most of the gelatinase activity. Sections were stained with DAPI, which stained the nuclei blue. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g002 doi:10.1371/journal.pone.0113810.g001 obtained them at time points that were 12 hours apart in the diurnal cycle, as will be described in that section. Occludin and claudin-4 are co-localized on surface corneal epithelium lateral membranes and are disrupted at night At the late afternoon time point (3 hrs before lights off) occludin and claudin-4 were co-localized to the lateral membranes of the surface CE as predicted (Figure 3A–C). The immunolabeling pattern showed continuous bands encircling the apical region of the lateral membranes of the surface layer of CE cells. Although the degree of co-localization was very high, subtle differences in relative intensities of occludin and claudin-4 labeling were often apparent (Figure 3C). This suggests that the relative expression of the two proteins is not precisely the same in all cells, although they are generally quite consistent. Figure 3. Tight junction proteins occludin and claudin-4 are co- expressed in Xenopus corneal epithelium lateral membranes and are disrupted at night. Double-label confocal immunocyto- chemistry was performed on whole flat-mounted preparations of Xenopus corneas that were obtained from animals in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, occludin (green) was uniformly primarily localized to the lateral membranes of the surface CE. (B) The same specimen as in A was labeled for the presence of claudin-4 (red), and was also uniformly primarily localized to the lateral membranes of the surface CE. (C) Merged green/red images from A and B demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal. (D) In the late night, occludin (green) was localized to the CE surface cell lateral membranes as during the day, but the pattern of labeling was often interrupted in some clusters of cells (arrows). (E) The same specimen as in D was labeled for the presence of claudin-4 (red), and was also localized to the CE surface cell lateral membranes, but the pattern of labeling was also interrupted in the same clusters of cells (arrows). (F) Merged green/red images from D and F demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal, with a similar level of disruption a discrete loci. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. Figure 3. Tight junction proteins occludin and claudin-4 are co- expressed in Xenopus corneal epithelium lateral membranes and are disrupted at night. Differential expression of tight junction proteins and MMPs in whole-mounted corneas Immunocytochemistry was performed on flatmount prepara- tions of Xenopus corneas that were obtained from animals in the late afternoon (9 hours after lights on during a 12L:12D cycle; CT09), and in the late night (3 hours before lights on; CT21). These times points (12 hrs apart) were selected based on our prediction that maximal tight junction disruption would occur late in the dark period (having a more prolonged period exposed to nighttime signals), and that tight junctions would be least disrupted late in the day, since they were further temporally separated from the nighttime signaling. Other intermediate time points were also analyzed and determined to exhibit a range of intermediate characteristics of the late evening and late afternoon time points (data not shown). The most obvious day/night differences were between the late night and late afternoon time points, which are reported here. Occludin and claudin-4 are co-localized on surface corneal epithelium lateral membranes and are disrupted at night Double-label confocal immunocyto- chemistry was performed on whole flat-mounted preparations of Xenopus corneas that were obtained from animals in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, occludin (green) was uniformly primarily localized to the lateral membranes of the surface CE. (B) The same specimen as in A was labeled for the presence of claudin-4 (red), and was also uniformly primarily localized to the lateral membranes of the surface CE. (C) Merged green/red images from A and B demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal. (D) In the late night, occludin (green) was localized to the CE surface cell lateral membranes as during the day, but the pattern of labeling was often interrupted in some clusters of cells (arrows). (E) The same specimen as in D was labeled for the presence of claudin-4 (red), and was also localized to the CE surface cell lateral membranes, but the pattern of labeling was also interrupted in the same clusters of cells (arrows). (F) Merged green/red images from D and F demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal, with a similar level of disruption a discrete loci. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. In contrast, at the late evening time point (3 hrs before lights on) occludin and claudin-4 immunolabeling often displayed a disrup- tion of the daytime pattern in small discrete clusters of cells (Figure 3D–F). The loss of occludin and claudin-4 on some lateral membranes suggested that the tight junction barrier was disrupted at these specific locations. There appeared to be more variation in the relative degree of co-localization and differential expression of occludin and claudin-4 on the intact lateral membranes compared to what was observed during the day (Figure 3C, F). MMP gelatinase activity is present in Xenopus surface corneal epithelium obtained them at time points that were 12 hours apart in the diurnal cycle, as will be described in that section. The MMP-2 and TIMP-2 immunolabeling appeared to be associated both with the plasma membrane and cytoplasm of surface CE cells (Figure 1). It is important to note that the November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 4 Circadian Regulation of Tight Junctions in Corneal Epithelium Figure 3. Tight junction proteins occludin and claudin-4 are co- expressed in Xenopus corneal epithelium lateral membranes and are disrupted at night. Double-label confocal immunocyto- chemistry was performed on whole flat-mounted preparations of Xenopus corneas that were obtained from animals in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, occludin (green) was uniformly primarily localized to the lateral membranes of the surface CE. (B) The same specimen as in A was labeled for the presence of claudin-4 (red), and was also uniformly primarily localized to the lateral membranes of the surface CE. (C) Merged green/red images from A and B demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal. (D) In the late night, occludin (green) was localized to the CE surface cell lateral membranes as during the day, but the pattern of labeling was often interrupted in some clusters of cells (arrows). (E) The same specimen as in D was labeled for the presence of claudin-4 (red), and was also localized to the CE surface cell lateral membranes, but the pattern of labeling was also interrupted in the same clusters of cells (arrows). (F) Merged green/red images from D and F demonstrate a high degree of co-localization in the surface cell CE lateral membranes, as indicated by the yellow signal, with a similar level of disruption a discrete loci. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. November 2014 \| Volume 9 \| Issue 11 \| e113810 Occludin and claudin-4 expression appear inversely associated with the presence of MMP-2 on the corneal epithelium surface and exhibit day/night changes Flat-mounted whole corneas were double-labeled for the presence of occludin and MMP-2 immunoreactivity at late daytime (3 hrs before lights off) and nighttime points (3 hrs before lights on). At the late afternoon time point, occludin immunolabel- ing of the lateral membranes was generally intact (Figure 4A) as described earlier (Figure 3A). In some daytime specimens, we occasionally observed some sparse scattered MMP-2 immunore- activity located in both the cytoplasm and/or lateral membranes of the surface CE, which was sometimes co-localized with occludin (Figure 4A). These discrete sites of MMP-2 immunoreactivity often appeared to coincide with some loss of occludin immuno- labeling of the lateral membranes. m doi:10.1371/journal.pone.0113810.g003 m doi:10.1371/journal.pone.0113810.g003 membranes. In clusters bordering these occludin-negative cell clusters, the pattern of occludin labeling was often disrupted, insofar as the cells had occludin on their lateral membranes, but the cells were clearly not attached to their neighboring cells (Figure 4B). Concomitant with this disruption of intercellular occludin attachments between the lateral membranes of neigh- boring cells, MMP-2 immunoreactivity was quite elevated in both the cytoplasm and lateral membranes of these cells (Figure 4B, C). Many of these MMP-2 immunoreactive cells displaced upward- folding flaps of membrane, indicative of cells that were in the process of desquamation (Figure 4B, C). At the late night time point, most of the surface CE cells exhibited intact occludin labeling, although there were many clusters of surface CE cells that lacked occludin immunoreactivity in their lateral membranes (Figure 4B), as described earlier (Figure 3D). They were considered to be the former sub-superficial layer of CE cells that had been only recently exposed to the corneal surface and had not yet expressed occludin in their lateral In the cell clusters that were almost devoid of occludin labeling on their lateral membranes, intense occludin immunoreactivity was often observed in perinuclear intracytoplasmic compartments (Figure 4C). We speculate that this intracytoplasmic occludin may represent newly-synthesized occludin that has not yet been November 2014 \| Volume 9 \| Issue 11 \| e113810 November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org 5 Circadian Regulation of Tight Junctions in Corneal Epithelium Figure 4. Occludin expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Occludin and claudin-4 expression appear inversely associated with the presence of MMP-2 on the corneal epithelium surface and exhibit day/night changes (B) During the late night, most of the surface CE cells exhibited intact occludin (green) labeling on their lateral membranes (double asterisks), but there were also many clusters of surface CE cells that lacked occludin immunoreactivity in their lateral membranes (single asterisk). In neighboring cells of some occludin-negative clusters, there was a gap between the occludin-labeled lateral membranes, indicating the disruption of tight junctions between the cells (arrows). MMP-2 (red) was very often expressed in the cells that exhibited gaps between the lateral membranes. Also, in these areas of high MMP-2 expression, upward-folding flaps of surface cells were lifting from the CE surface, representing cells in the act of desquamation (arrowheads). (C) In some cell clusters that were almost devoid of occludin labeling on their lateral membranes at nighttime (presumptive former sub-superficial cells; asterisks), intense occludin (green) immunoreactivity was observed in perinuclear intracytoplasmic compartments. The neighboring surface CE cells express high levels of MMP-2 (red) that are in the process of desquamation (arrows). There was also considerable co-localization (yellow) of occludin and MMP-2 on the lateral membranes between neighboring cells. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g004 transported to the plasma membrane, and that these cells may represent a population of sub-superficial cells recently exposed to the surface because of the recent desquamation of the overlying surface CE cells, and are undergoing a rapid terminal maturation process. Often, the bordering clusters of surface CE cells exhibited very high levels of MMP-2 immunoreactivity in their cytoplasm and lateral membranes, and were obviously in the process of desquamation (Figure 4C). Therefore, at nighttime in discrete clusters of surface CE cells, there was disruption of occludin labeling of the lateral membranes, a concomitant high incidence of MMP-2 immunoreactivity, and subsequent surface cell desqua- mation. Figure 5. Claudin-4 expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, claudin-4 (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. Occludin and claudin-4 expression appear inversely associated with the presence of MMP-2 on the corneal epithelium surface and exhibit day/night changes Flat- mounted whole corneas were double-labeled for localization of occludin and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, occludin (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. (B) During the late night, most of the surface CE cells exhibited intact occludin (green) labeling on their lateral membranes (double asterisks), but there were also many clusters of surface CE cells that lacked occludin immunoreactivity in their lateral membranes (single asterisk). In neighboring cells of some occludin-negative clusters, there was a gap between the occludin-labeled lateral membranes, indicating the disruption of tight junctions between the cells (arrows). MMP-2 (red) was very often expressed in the cells that exhibited gaps between the lateral membranes. Also, in these areas of high MMP-2 expression, upward-folding flaps of surface cells were lifting from the CE surface, representing cells in the act of desquamation (arrowheads). (C) In some cell clusters that were almost devoid of occludin labeling on their lateral membranes at nighttime (presumptive former sub-superficial cells; asterisks), intense occludin (green) immunoreactivity was observed in perinuclear intracytoplasmic compartments. The neighboring surface CE cells express high levels of MMP-2 (red) that are in the process of desquamation (arrows). There was also considerable co-localization (yellow) of occludin and MMP-2 on the lateral membranes between neighboring cells. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g004 Figure 4. Occludin expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Flat- mounted whole corneas were double-labeled for localization of occludin and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, occludin (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. Occludin and claudin-4 expression appear inversely associated with the presence of MMP-2 on the corneal epithelium surface and exhibit day/night changes (B) During the late night, most of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted (arrows) and was accompanied by high levels of MMP-2 immunoreactivity. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g005 Similar to the inverse pattern of occludin and MMP-2 association at the surface CE at nighttime, claudin-4 and MMP- 2 expression appeared to be inversely associated and exhibited day/night changes (Figure 5). During the daytime, claudin-4 immunoreactivity (Figure 5A) was very comparable to the intact occludin labeling of the surface CE lateral membranes observed during the day (Figure 4A) which was relatively unperturbed. Some occasional scattered MMP-2 immunoreactivity was exhib- ited in both the cytoplasm and lateral membranes during the day, and the membrane localization was either co-localized with claudin-4 or occurred in the absence of claudin-4 immunoreac- tivity (Figure 5A). This pattern is identical to the inverse association seen with occludin and MMP-2 (Figure 4A). The sites of membrane-localized MMP-2 were often associated with some disruption of claudin-4 membrane localization (Figure 5A). At nighttime, claudin-4 immunolabeling present in clusters of the CE surface cell lateral membranes was disrupted in the same pattern as occludin (Figure 4B, C); membrane labeling was sometimes discontinuous or absent (Figure 5B). Claudin-4 membrane label- ing was also not contiguous, insofar as there was clear separation of neighboring cells even when claudin-4 was present on the membrane of both cells (Figure 5B). The disruption of intercellular attachment and claudin-4 on the lateral membranes was also associated with high expression levels of MMP-2 at nighttime. Figure 5. Claudin-4 expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, claudin-4 (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. Discussion The corneal epithelium (CE) is a stratified epithelium that provides a major protective barrier to pathogens and environ- mental assaults on the eye. Disruption of the CE barrier function by disease or trauma and subsequent infections may be due in part to degradation of interconnecting tight junctions at the corneal surface by an induced secretion of matrix metalloproteinases Figure 6. Preservation of occludin expression is positively associated with TIMP-2 on surface CE and exhibits day/night changes. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and TIMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) During the daytime, occludin (green) immunolabeling of the CE surface cell lateral membranes was generally intact. Small clusters of surface and sub-superficial CE cells exhibited TIMP-2 immunoreactivity in intracytoplasmic compartments (arrows). (B) At nighttime, TIMP-2 (red) immunoreactivity in the surface CE was very low, and occludin (green) immunoreactivity was disrupted (arrows) in some cell clusters (asterisks). (C) A cornea retrieved in the late afternoon (DAY) had an unidentified pathology in which almost all CE was eroded, leaving the stromal layer at the corneal surface. A small patch of about 20 CE cells persisted as a single monolayer above the stromal layer. The surviving CE cells displayed occludin (green) immunolabeling on their lateral membranes, and very intense immunolabeling of TIMP-2 (red) in their cytoplasm (arrows). There was some high non-specific green labeling of the connective tissue surface. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g006 Figure 6. Preservation of occludin expression is positively associated with TIMP-2 on surface CE and exhibits day/night changes. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and TIMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) During the daytime, occludin (green) immunolabeling of the CE surface cell lateral membranes was generally intact. Small clusters of surface and sub-superficial CE cells exhibited TIMP-2 immunoreactivity in intracytoplasmic compartments (arrows). (B) At nighttime, TIMP-2 (red) immunoreactivity in the surface CE was very low, and occludin (green) immunoreactivity was disrupted (arrows) in some cell clusters (asterisks). Preservation of occludin expression appears positively associated with TIMP-2 on surface corneal epithelium and exhibits day/night changes Membrane type 1-MMP is expressed in cytoplasmic compartments of surface corneal epithelium cells and associates with lateral membranes at nighttime y g g In contrast to the inverse association of occludin immunolabel- ing on CE surface cell lateral membranes with MMP-2 expression, TIMP-2 appeared to correlate positively with the preservation of occludin junction integrity. Although the ternary complex binding of TIMP-2 with MMP-2 and MT1-MMP is needed for maximal activation of MMP-2, a higher level of TIMP-2 inhibits MMP-2 activity [27,28,30]. High expression levels of TIMP-2 may therefore represent areas in which potential MMP-2-induced tight junction protein cleavage is attenuated. During the daytime, small clusters of surface and sub-superficial CE cells exhibited TIMP-2 immunoreactivity in intracytoplasmic compartments both in the surface layer of CE cells and in the sub-superficial layer of cells directly underneath the surface layer (Figure 6A). These clusters of cells were typically associated with intact occludin lateral membrane immunolabeling, as was seen in most of the CE during the daytime. At nighttime however, TIMP-2 immunoreactivity in the surface CE was very low, most notably in areas in which occludin immunolabeling of the surface CE lateral membranes was disrupted (Figure 6B). Membrane type-1 MMP (MT1-MMP; MMP-14) is the membrane-bound binding partner that forms a ternary complex with TIMP-2 and MMP-2 to cleave the pro-MMP-2 into the enzymatically-active MMP-2 [27,28]. MT1-MMP was present in most, but not all surface CE cells, and was localized to cytoplasmic compartments both during the day and at night (Figure 7). At nighttime, however, MT1-MMP was often also localized to the surface CE cell lateral membranes (Figure 7B, C). The presence of MT1-MMP lateral membrane labeling was often associated with areas of tight junction disruption and surface cell desquamation as assessed by the diminished claudin-4 immunoreactivity. MMP-2 and TIMP-2 expression is significantly higher and lower at nighttime, respectively, in the surface corneal epithelium p Quantitative analysis of day/night differences in MMP-2, TIMP-2, MT1-MMP, occludin, and claudin-4 expression revealed statistically significant differences for MMP-2 and TIMP-2, but not for MT1-MMP (Figure 8). MMP-2 expression during the night was about 10-fold higher than during the day (P,0.01) whereas TIMP-2 expression during the night was about 85% lower compared to daytime levels (P,0.01). MT1-MMP expres- sion levels were approximately equivalent during the day and night, although as noted above in figure 7, much of the labeling at night was present on the lateral membranes. The expression levels of occludin and claudin-4 proteins were about 50–70% lower at nighttime than during the day, but the difference in occludin expression was not statistically significant although day/night change in claudin-4 expression was significant (P,0.05). During the course of this study, we occasionally encountered some frog corneas in which the CE was eroded due to unknown pathologies or injuries. These corneas were typically discarded, since the CE was generally absent. However, in one specimen that was immunolabeled for occludin and TIMP-2, we observed a small patch of CE cells resting above the stromal layer (Figure 6C). The CE cells in this patch of about 20 cells existed as a monolayer; the epithelium was no longer stratified. These surviving CE cells displayed robust occludin labeling on their lateral membranes, and extraordinarily intense immunolabeling of TIMP-2 in their perinuclear cytoplasm (Figure 6C). This fortuitous observation suggests that elevated TIMP-2 expression may aid in the preservation of tight junction integrity in pathologies or trauma to the CE. Occludin and claudin-4 expression appear inversely associated with the presence of MMP-2 on the corneal epithelium surface and exhibit day/night changes (B) During the late night, most of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted (arrows) and was accompanied by high levels of MMP-2 immunoreactivity. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g005 Figure 5. Claudin-4 expression on Xenopus surface CE is disrupted at night, and is inversely associated with MMP-2 expression. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and MMP-2 immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, claudin-4 (green) immunolabeling of the lateral membranes of the surface CE was generally intact, and some sporadic labeling of MMP-2 (red) was also present on the lateral membranes and/or cytoplasm (arrows) which was sometimes co-localized (yellow) with occludin. (B) During the late night, most of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted (arrows) and was accompanied by high levels of MMP-2 immunoreactivity. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g005 November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Circadian Regulation of Tight Junctions in Corneal Epithelium Preservation of occludin expression appears positively associated with TIMP-2 on surface corneal epithelium and exhibits day/night changes Discussion MT1-MMP (green) labeling, when present, was observed almost exclusively in cytoplasmic compartments of the surface CE. (B, C) Two examples are presented to illustrate that during the late night, a majority of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted. Also at nighttime, MT1-MMP labeling was present both in the cytoplasm and intermittently associated with the surface CE cell lateral membranes (arrows). The membrane co-localization of the red claudin-4 and green MT1-MMP resulted in a merged yellow labeling of the lateral membranes in some areas (arrows). Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. d i 0 3 /j l 0 38 0 00 m doi:10.1371/journal.pone.0113810.g007 health of the cornea, and it is clear that the surface epithelium does not peel off together as contiguous sheets of cells [42]. There is experimental evidence in mammalian models that demonstrates that living, non-apoptotic surface CE cells are shed into the tear fluid either singly or as small clusters [6,9]. Based on our observations of the Xenopus cornea, we propose that the cells of the surface CE are arranged into small mosaic clusters that differentiate, function, and desquamate in unison, but in a temporal sequence that is often out of phase with neighboring cell groups. (MMPs) as part of an inflammatory response [17,37–41]. In contrast, we propose that regulated disruption of intercellular attachments of the living surface CE cells is required for normal homeostatic cell turnover. We hypothesize that daily desquama- tion of surface CE cells requires cleavage of transmembrane junctional proteins by MMPs secreted by CE cells in a circadian manner, whereas over-expression of MMPs in pathological conditions can be detrimental to the protective barrier function of the CE. The concept that surface cell turnover occurs temporally as discrete mosaic clusters profoundly affects the methodological approaches we can use to effectively document the progression of periodic surface cell desquamation. The surface layer of cells of the CE represents a very small sub-population (probably less than 5%) of the entire population of CE cells. This is based on counting the number of cell nuclei in the entire thickness of CE, compared to the number cell nuclei at the surface (data not shown). Discussion (C) A cornea retrieved in the late afternoon (DAY) had an unidentified pathology in which almost all CE was eroded, leaving the stromal layer at the corneal surface. A small patch of about 20 CE cells persisted as a single monolayer above the stromal layer. The surviving CE cells displayed occludin (green) immunolabeling on their lateral membranes, and very intense immunolabeling of TIMP-2 (red) in their cytoplasm (arrows). There was some high non-specific green labeling of the connective tissue surface. Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g006 November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org 7 Circadian Regulation of Tight Junctions in Corneal Epithelium Figure 7. Membrane type 1-MMP is expressed in surface CE cells and associates with lateral membranes at nighttime. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and membrane type 1-MMP (MT1-MMP) immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, claudin-4 (red) immunolabeling of the surface CE cell lateral membranes was generally intact and uniform. MT1-MMP (green) labeling, when present, was observed almost exclusively in cytoplasmic compartments of the surface CE. (B, C) Two examples are presented to illustrate that during the late night, a majority of the surface CE cells exhibited intact claudin-4 (green) labeling on their lateral membranes, but there were also many clusters of surface CE cells in which the claudin-4 immunoreactivity was disrupted. Also at nighttime, MT1-MMP labeling was present both in the cytoplasm and intermittently associated with the surface CE cell lateral membranes (arrows). The membrane co-localization of the red claudin-4 and green MT1-MMP resulted in a merged yellow labeling of the lateral membranes in some areas (arrows). Specimens were stained with the blue nuclear DAPI stain. Scale bar = 20 mm. doi:10.1371/journal.pone.0113810.g007 Figure 7. Membrane type 1-MMP is expressed in surface CE cells and associates with lateral membranes at nighttime. Flat-mounted whole corneas were double-labeled for localization of claudin-4 and membrane type 1-MMP (MT1-MMP) immunoreactivity in the late afternoon (DAY; 9 hours after lights on in a 12L:12D cycle) and in the late night (NIGHT; 3 hours before lights on). (A) In the late afternoon, claudin-4 (red) immunolabeling of the surface CE cell lateral membranes was generally intact and uniform. Discussion Furthermore, we estimate that less than 10% of the surface layer of cells (i.e.; less than 0.5% of the total CE cells) are undergoing the final sequence of events that leads to imminent desquamation during the peak time period. Therefore, our in vivo analysis of surface CE had to be conducted primarily by observing the range of behaviors of the various clusters or populations of cells at the CE surface. Moreover, the inherently subjective nature of these analyses precluded the utility of more objective independent quantitative measurements, such as Western blotting or gel zymography. The necessary descriptive nature of the data presented in this report is a reflection of these methodological constraints. However, quantitative analyses of confocal images were performed to ascertain some statistically significant day/night differences in MMP-2 and TIMP-2 expression. Methodological approach of mosaic corneal epithelium cell turnover During the course of this study, it became apparent that the surface CE cells are not all behaving synchronously. It is generally appreciated that cells of the surface layer of the CE do not detach all at the same time; this would logically be detrimental to the Figure 8. MMP-2 and TIMP-2 expression levels are inversely correlated during the light/dark cycle in surface corneal epithelium. Representative confocal images (N = 4) of each sample group were analyzed for day/night changes in immunolabeling intensity. MMP-2 immunolabeling intensity was significantly higher at nighttime than during the day (P,0.01), whereas TIMP-2 intensity was significantly lower at nighttime compared to the day (P,0.01; Two- tailed Student’s t-test). MT1-MMP levels appeared unchanged between the light and dark cycle. The lower nighttime levels of occludin was not statistically significant, although the day/night change in claudin-4 expression was significant (P,0.05). doi:10.1371/journal.pone.0113810.g008 Figure 8. MMP-2 and TIMP-2 expression levels are inversely correlated during the light/dark cycle in surface corneal epithelium. Representative confocal images (N = 4) of each sample group were analyzed for day/night changes in immunolabeling intensity. MMP-2 immunolabeling intensity was significantly higher at nighttime than during the day (P,0.01), whereas TIMP-2 intensity was significantly lower at nighttime compared to the day (P,0.01; Two- tailed Student’s t-test). MT1-MMP levels appeared unchanged between the light and dark cycle. The lower nighttime levels of occludin was not statistically significant, although the day/night change in claudin-4 expression was significant (P,0.05). doi:10.1371/journal.pone.0113810.g008 Circadian Regulation of Tight Junctions in Corneal Epithelium Circadian Regulation of Tight Junctions in Corneal Epithelium borders throughout all layers of the CE [52], so was not a subject of this study. There are several types of claudin, and claudin-4 is one of the most consistently and highly-expressed members of the claudin family in CE [53,54], so it was chosen for inclusion in this study. It is generally considered that occludin is ubiquitously expressed in all tight junctions, but that expression of various claudin types tends to be more tissue-specific [55–58]. Phosphor- ylation of occludin and claudin can cause changes in tight junction barrier permeability [10,59–62], and may potentially contribute to dissociation of tight junction attachments. The possibility that phosphorylation of tight junction proteins contributes to the transient dissociation of surface face cells during CE desquamation may warrant further investigation. they desquamate from the surface and that they are able to separate from their neighboring cells because of regulated cleavage of tight junction proteins by MMPs secreted by CE cells. Conversely, in some pathological conditions, apoptosis of the surface CE layer does indeed occur [42,44,45]. The derangement of the CE barrier function in various corneal disorders is due to dysfunction, death, or loss of the surface layer of CE cells. For example, herpetic infection can result in stromal keratitis, leading to corneal melting, neovascularization, ulceration and perforation [1–3]. Over-expression of MMP occurs in CE cells after corneal damage [37,39,41], and MMPs are over- expressed in the CE in the areas of ulcerative keratitis that occurs in response to human immunodeficiency virus type 1 (HIV-1) infection. Over-expression of corneal MMP-2 is a characteristic of patients with ulcerative keratitis [40,41]. The precedent for extracellular cleavage of transmembrane junctional proteins and subsequent degradation of barrier function by secreted MMPs has been established in many other fields of study [10–19,63]. For example, there is partial co-localization of the metalloprotease meprinb and E-cadherin at the lateral membranes in cultured MDCK cells, and Meprinb cleaves E- cadherin extracellularly and weakens intercellular adhesion [63]. MMP-7 cleaves occludin extracellularly and increases paracellular permeability in cultured human vagina-ectocervical epithelial cells and decreases intercellular adhesion in MCDK cells [13,64]. MMP-2 or -9 degrades claudin-5 and/or occludin in tight junctions of cultured mouse brain microvascular endothelial cells, degrading barrier integrity and increasing paracellular permeabil- ity [65–67]. Expression of MMPs in the corneal epithelium p p MMPs have been localized to the mammalian cornea, including the CE and tear fluid. In mice, MMP-1, 9, 10, 12, 13 and TIMP-1 RNA is up-regulated in the migrating CE after wounding, and MMP-3 protein is increased in the superficial layers of the CE after wounding [68]. In rabbits, MMP-1, 2, 7, 8 and 9 protein expression is increased in the CE after UV irradiation [69]. Most MMPs are localized diffusely throughout CE, but the MMP-7 labeling is more pronounced in the superficial layers [69]. MMP-2 and MT1-MMP (MMP-14) protein is expressed in multiple layers of the normal and keratoconus human CE, with MT1-MMP appearing mostly in the basal layer and MMP-2 more diffuse throughout the CE [70]. MMP-1, 2, 8, 9 have been shown to be present in the normal human CE [71,72], with elevated levels of MMP-1, 2, 3, 7, 8 and 9 in melted corneas (keratolysis) [71]. RNA encoding MMP-1, 3, 9, 10, 11, and 13 are up- regulated by inflammatory cytokines in human CE [47,72–75], and MMP-9 is elevated in the tear fluid of patients with dysfunctional tear syndrome [76]. MMP-9 and TIMP-1 are produced by the human CE and are present in tear fluid [72,76]. In human tear fluid, MMP-9 is increased 200-fold and TIMP-1 is increased 4-fold upon wakening, resulting in a diurnal variation of MMP-9 activity in human tears [77]. These observations support the concept that recurrent conditions such as corneal erosions and corneal epithelial ulcerations which are exacerbated upon waking may be a result in part to increased degradative actions of MMPs that occur during the preceding night. This is also in agreement with our hypothesis that MMPs are up-regulated in a circadian fashion to cleave junctional proteins late at night to enable the surface cells to desquamate in the early morning in conjunction with the mild abrasion due to eyelid blinking. Circadian Regulation of Tight Junctions in Corneal Epithelium In a hypertensive rat model, induced ischemia causes increased MMP-2 secretion and degradation claudin-5 and occludin in endothelial tight junctions, leading to breakdown of the blood-brain barrier [19]. The disease known as Dry Eye Syndrome (DES), or kerato- conjunctivitis sicca, is a common ocular disorder in which ocular surface inflammation is considered to be both a cause and a consequence of corneal damage [1] including loss of CE barrier function. The disruption of the CE barrier function in DES is due in part to dysfunctional CE cells that lose their interconnecting tight junctions [46]. There is evidence that secretion of MMP-9 is increased in DES, and cleaves the tight junction occludin protein, resulting in breakdown of the CE barrier [47,48]. Altered CE barrier function is the cause of ocular irritation and visual morbidity in dry eye disease, and MMP-9 plays an important role in the disruption of CE barrier function in DES. MMP-9 knockout protects the CE from experimentally-induced dry eye desquamation and occludin breakdown in mice [48]. Compared to MMP-9 knockout mice, wild-type mice show greater desquamation of the surface CE and an increase in cleavage of occludin in the CE. Therefore, increased MMP-9 activity on the ocular surface in response to dryness disrupts CE barrier function which appears to be the result of accelerated loss of the surface CE cells mediated by proteolytic cleavage of occludin and other transmembrane junctional proteins. Therapeutic targeting of MMP-9 activity in patients with corneal erosions may be a promising route to enhance the recovery from these disorders [48]. The scenarios cited above depict a pattern of MMP participation in a range of corneal disorders in which, in response to an initial insult, MMPs convert from low constitutive expression to unchecked induced expression that causes pervasive damage to corneal tissues. Matrix metalloproteinase expression in corneal disease p p In some epithelia, surface cells undergo programmed cell death (apoptosis) prior to their desquamation from the surface [43]. However, under normal conditions, the desquamation of corneal surface cells does not require apoptosis as was once considered [9]. The living surface cells must actively dissociate themselves from neighboring cells, and we suggest that degradation of their junctional complexes is required for this to occur. We hypothesize that in the healthy cornea, surface cells are intact and alive when November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org 8 November 2014 \| Volume 9 \| Issue 11 \| e113810 Diurnal changes in tight junction proteins and MMP distribution in Xenopus corneal epithelium In this and other studies, MMP-2 immunoreactivity is most often observed in intracytoplasmic compartments because most of the secreted enzyme is not attached to the cell at the time of fixation. Therefore, much of the MMP-2 immunoreactivity that we have observed in CE cells is more a reflection of intracellular MMP-2 synthesis and/or accumulation, than secretion and activation of the enzyme itself. However, we routinely did observe some MMP-2 localization to the CE surface cell lateral membranes, especially during the nighttime, and often co- localized with occludin or claudin-4 (Figures 4 and 5). Further- more, this localization of MMP-2 on the lateral membranes was very often correlated to; 1) diminished tight junction protein expression, 2) separation of neighboring cell membranes, and 3) surface cell desquamation (Figures 4 and 5). Thus, MMP-2 immunoreactivity on CE lateral membranes may reflect the transient formation of the ternary complex with TIMP-2 and MT1-MMP on the plasma membrane, which would result in activation of secreted MMP-2. Our observation that MMP-2 is localized to lateral membranes far more frequently during the nighttime than during the day suggests that there is a diurnal rhythm of MMP-2 secretion and activation with highest levels occurring at nighttime in the Xenopus CE. Quantitative measurements of immunolabeling intensity of confocal images support the direct observations of higher MMP- 2 expression at night, and lower expression levels of TIMP-2 during the day (Figure 8). Absolute expression levels of MT1- MMP were relatively unchanged during the light/dark cycle (Figure 8). As predicted, the lower levels of occludin and claudin-4 immunolabeling intensities observed in the confocal images were not strongly supported by quantitative analysis (Figure 8). As described earlier, the loss of tight junction proteins at nighttime appeared in a mosaic patchwork pattern, with most of the corneal epithelium relatively unperturbed (Figure 3). Therefore, within study groups there was wide variation in the amount of intact occludin or claudin-4, reflective of the non-uniform pattern of MMP-2 expression on the surface CE at nighttime. Although most of the surface CE cells exhibited intact occludin labeling at nighttime, there were many clusters of surface CE cells that lacked occludin immunoreactivity in their lateral membranes (Figures 4 and 5). We speculate that these occludin-negative surface cells are former sub-superficial layer of CE cells that had been only recently exposed to the corneal surface and had not yet begun to express occludin in their lateral membranes. Circadian Regulation of Tight Junctions in Corneal Epithelium the Xenopus laevis CE, and display diurnal differences in their abundance and location. Importantly, we have observed an association of MMP-2 expression with degradation of occludin and claudin-4 on the surface cell lateral membranes. The use of confocal scanning microscopy in this study provides a higher level of resolution than was afforded by the standard light or fluorescence microscopy used in previous studies. The trio of MMP-2, TIMP-2, and MT1-MMP was chosen as the focus of this study since their ternary interactions and mechanisms of action are particularly well-understood compared to most other members of the MMP family [26–29]. In addition to MMP-2, TIMP-2, and MT1-MMP, we also observed MMP-9 immunolabeling of the Xenopus CE (data not shown). There may certainly be other members of the MMP family involved in CE cell turnover, and future unbiased approaches will be likely needed for a more complete understanding of the role of MMPs in CE surface cell desquamation. activation of MMP-2 [26–29]. We speculate that after MMP-2 is secreted, and the surface cells in the area have desquamated, any residual MMP-2 needs to be inactivated to protect the newly- forming tight junctions from cleavage. TIMPs may be secreted by the former sub-superficial cells to inhibit residual MMP activity and thus provide a permissive environment for transmembrane junctional proteins in cytoplasmic compartments to move to the cell surface. The necessary orderly sequence of MMP-2 secretion and surface cell desquamation, followed by TIMP-2 secretion logically requires some temporal signals. g y q p g In contrast to the MMP-2 (and TIMP-2; see figure 6) located in intracytoplasmic compartments representing inactive pro-enzyme, intracytoplasmic MT1-MMP may represent recently activated and internalized enzyme. After maturation in the Golgi apparatus, newly-synthesized MT1-MMP is rapidly transported to the plasma membrane [78]. However, cell surface localization of MT1-MMP is often not observed immunocytochemically because the enzyme on the plasma membrane is rapidly activated and internalized into endosomal compartments [26,78–80]. This is consistent with our observations that most MT1-MMP immunoreactivity is present in the cytoplasm in both the day and night (Figure 7). Conversely, inactive MT1-MMP is known to accumulate on plasma membranes presumably in anticipation of an increase in pro- MMP-2 secretion [27]. In addition to the intracytoplasmic localization of MT1-MMP, we did also observe significant MT1- MMP immunoreactivity on the lateral membranes at nighttime, but not during the day in surface CE cell clusters (Figure 7). Diurnal changes in tight junction proteins and MMP distribution in Xenopus corneal epithelium We presume that the surface CE cells that were overlying these cells were very recently desquamated. In surface CE clusters bordering occludin- negative cell clusters, the occludin labeling on the lateral membranes was often disrupted. Although the cells had occludin on their lateral membranes, the cells were clearly not attached to their neighboring cells (Figure 4). This pattern suggested that the extracellular attachments between occludin molecules of neigh- boring cells were severed but that the occludin had not yet been internalized. MMP-2 immunoreactivity was quite elevated in both the cytoplasm and lateral membranes of these occludin-positive cells that were detached from their neighboring cells, suggestive of a causative association. Circadian Regulation of Tight Junctions in Corneal Epithelium This may represent increased accumulation of inactive MT1-MMP on the plasma membrane at nighttime to be available to activate increased pro-MMP-2 secretion at night. November 2014 \| Volume 9 \| Issue 11 \| e113810 Expression of occludin and claudin in surface corneal epithelium tight junctions The CE surface squamous cells are joined together by tight and adhering junctions (zonula occludens and zonula adherens, respectively) on their lateral membranes. The adhering junctions provide most of the mechanical attachment between neighboring cells, and tight junction formation is preceded by and is dependent upon formation of adhering junctions [49]. The major transmem- brane attachment protein of the adhering junction is cadherin, and is expressed in all layers of the CE (i.e.; is not specific for the surface layer) [50], which is one reason why we did not focus our attention on the potential role of MMPs in degrading adhering junctions. Disruption of adhering junctions is obviously necessary for cell dissociation, and should be a subject of future study. Tight junctions comprise the major component of the para- cellular permeability barrier as well as protection from infection and pathogen invasion in the CE. In the CE, tight junctions are present only on the surface layer of cells and are composed mostly of occludin and claudin [51]. Junctional adhesion molecule (JAM) is also a component of tight junctions, but is also expressed at cell In this report, we have shown that MMP-2, TIMP-2, and MT1- MMP proteins are expressed predominantly by the surface layer of November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org 9 Circadian Regulation of Tight Junctions in Corneal Epithelium References Gingras D, Beliveau R (2010) Emerging concepts in the regulation of membrane-type 1 matrix metalloproteinase activity. Biochim Biophys Acta 1803: 142–150. 8. Refsum SB, Haskjold E, Bjerknes R, Iversen OH (1991) Circadian variation in cell proliferation and maturation. A hypothesis for the growth regulation of the rat corneal epithelium. Virchows Arch B Cell Pathol Incl Mol Pathol 60: 225– 230. 27. Osenkowski P, Toth M, Fridman R (2004) Processing, shedding, and endocytosis of membrane type 1-matrix metalloproteinase (MT1-MMP). J Cell Physiol 200: 2–10. 28. Sato H, Takino T (2010) Coordinate action of membrane-type matrix metalloproteinase-1 (MT1-MMP) and MMP-2 enhances pericellular proteolysis and invasion. Cancer Sci 101: 843–847. 9. Lomako J, Lomako WM, Decker SJ, Carraway CA, Carraway KL (2005) Non- apoptotic desquamation of cells from corneal epithelium: putative role for Muc4/sialomucin complex in cell release and survival. J Cell Physiol 202: 115– 124. 29. Sternlicht MD, Werb Z (2001) How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 17: 463–516. 10. Alexander JS, Elrod JW (2002) Extracellular matrix, junctional integrity and matrix metalloproteinase interactions in endothelial permeability regulation. J Anat 200: 561–574. 30. Brew K, Dinakarpandian D, Nagase H (2000) Tissue inhibitors of metallopro- teinases: evolution, structure and function. Biochim Biophys Acta 1477: 267– 283. J 11. Covington MD, Burghardt RC, Parrish AR (2006) Ischemia-induced cleavage of cadherins in NRK cells requires MT1-MMP (MMP-14). Am J Physiol Renal Physiol 290: F43–51. 31. Cahill GM, Besharse JC (1990) Circadian regulation of melatonin in the retina of Xenopus laevis: limitation by serotonin availability. J Neurochem 54: 716– 719. 12. Cowden Dahl KD, Symowicz J, Ning Y, Gutierrez E, Fishman DA, et al. (2008) Matrix metalloproteinase 9 is a mediator of epidermal growth factor-dependent e-cadherin loss in ovarian carcinoma cells. Cancer Res 68: 4606–4613. 32. Cahill GM, Besharse JC (1991) Resetting the circadian clock in cultured Xenopus eyecups: regulation of retinal melatonin rhythms by light and D2 dopamine receptors. J Neurosci 11: 2959–2971. 13. Gorodeski GI (2007) Estrogen decrease in tight junctional resistance involves matrix-metalloproteinase-7-mediated remodeling of occludin. Endocrinology 148: 218–231. 33. Cahill GM, Grace MS, Besharse JC (1991) Rhythmic regulation of retinal melatonin: metabolic pathways, neurochemical mechanisms, and the ocular circadian clock. Cell Mol Neurobiol 11: 529–560. 14. Huang W, Eum SY, Andras IE, Hennig B, Toborek M (2009) PPARalpha and PPARgamma attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities. Implications for circadian regulation of corneal epithelial cell homeostasis We suggest that tight and adhering junctions of the surface layers of the CE are assembled and dissembled on a daily basis in discrete clusters to enable surface cells to be periodically shed yet maintain the critical barrier function of the CE. The mosaic CE cell clusters display varying degrees of tight junction protein and MMP expression, but there is a strong tendency for a majority surface CE cells to exist in particular states of function at particular times of day, such as the tendency to desquamate preferentially in the early morning, compared to other the times during the diurnal cycle. Our study was intended to provide support for the hypothesis that degradation of junctional complexes, which leads to CE surface cell desquamation, is mediated via MMPs produced by CE cells which are potentially influenced by nocturnal circadian signals as part of a renewal cycle. Higher levels of TIMP-2 expression in the surface CE during the day (Figure 6) is consistent with the concept of diminished MMP-2 during the day, since high levels of TIMP-2 inhibits We have previously reported that receptors for the circadian signaling molecule melatonin are located on the lateral mem- branes of the Xenopus surface CE [81]. These observations led us November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org November 2014 \| Volume 9 \| Issue 11 \| e113810 10 Circadian Regulation of Tight Junctions in Corneal Epithelium Circadian Regulation of Tight Junctions in Corneal Epithelium to the current study and hypothesis. Furthermore, MMP-2 and the Mel1a melatonin receptor are co-localized to the surface cells of Xenopus CE (unpublished results). Changes in MMP-2 expression and/or activity may perhaps be a downstream consequence of nighttime melatonin receptor activation in the surface CE. Interestingly, melatonin regulates the expression and secretion of MMP-9 in several experimental systems [82–85], suggesting a potential mechanism for a diurnal rhythm of MMP expression and tight junction breakdown. Inhibition of MMP activity by melatonin often appears to be due to up-regulation of tissue inhibitors of MMPs (i.e.; TIMP-1) [85,86], which are known to protect CE barrier function in response to infection and trauma [38,87–89]. superficial cells anticipate the need for forming new tight junctions by pre-accumulating tight junction proteins prior to reaching the air/surface interface [90,91], and may be mediated by a nighttime circadian signal. References vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat. J Cereb Blood Flow Metab 27: 697–709. vessels is reversed by synthetic matrix metalloproteinase inhibitor in focal ischemia in rat. J Cereb Blood Flow Metab 27: 697–709. 1. Barabino S, Dana MR (2007) Dry eye syndromes. Chem Immunol Allergy 92: 176–184. J 20. Brinckerhoff CE, Matrisian LM (2002) Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol 3: 207–214. 2. Liesegang TJ, Melton LJ, 3rd, Daly PJ, Ilstrup DM (1989) Epidemiology of ocular herpes simplex. Incidence in Rochester, Minn, 1950 through 1982. Arch Ophthalmol 107: 1155–1159. that became a prince. Nat Rev Mol Cell Biol 3: 207–214. 21. Gross J, Lapiere CM (1962) Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc Natl Acad Sci U S A 48: 1014–1022. 21. Gross J, Lapiere CM (1962) Collagenolytic activity in amphib p 3. Streilein JW, Dana MR, Ksander BR (1997) Immunity causing blindness: five different paths to herpes stromal keratitis. Immunol Today 18: 443–449. culture assay. Proc Natl Acad Sci U S A 48: 1014–1022. 22. McCawley LJ, Matrisian LM (2001) Matrix metalloproteinases: they’re not just for matrix anymore! Curr Opin Cell Biol 13: 534–540. 4. Thomas J, Rouse BT (1997) Immunopathogenesis of herpetic ocular disease. Immunol Res 16: 375–386. for matrix anymore! Curr Opin Cell Biol 13: 534–540. 23. Morrison CJ, Butler GS, Rodriguez D, Overall CM (2009) Matrix metallopro- teinase proteomics: substrates, targets, and therapy. Curr Opin Cell Biol 21: 645–653. 5. Fogle JA, Yoza BK, Neufeld AH (1980) Diurnal rhythm of mitosis in rabbit corneal epithelium. Albrecht Von Graefes Arch Klin Exp Ophthalmol 213: 143–148. 24. Rodriguez D, Morrison CJ, Overall CM (2010) Matrix metalloproteinases: what do they not do? New substrates and biological roles identified by murine models and proteomics. Biochim Biophys Acta 1803: 39–54. 6. Fullard RJ, Wilson GS (1986) Investigation of sloughed corneal epithelial cells collected by non-invasive irrigation of the corneal surface. Curr Eye Res 5: 847– 856. and proteomics. Biochim Biophys Acta 1803: 39–54. 25. Sounni NE, Noel A (2005) Membrane type-matrix metalloproteinases and tumor progression. Biochimie 87: 329–342. 7. Haskjold E, Refsum SB, Bjerknes R (1989) Circadian variation in the mitotic rate of the rat corneal epithelium. Cell divisions and migration are analyzed by a mathematical model. Virchows Arch B Cell Pathol Incl Mol Pathol 58: 123– 127. 26. Acknowledgments We thank Jennifer Graef and Tiffany Nguyen for excellent technical assistance, and Dr. Jody Summers (University of Oklahoma) for generously providing use of the confocal microscope. Implications for circadian regulation of corneal epithelial cell homeostasis Circadian regulation of MMP activity and subsequent tight junction protein cleavage may represent a novel mechanism of corneal epithelial cell turnover and renewal. Author Contributions In order to maintain CE tissue homeostasis, the sequence of barrier breakdown, subsequent loss of surface cells, and re- establishment of a new surface epithelial barrier, needs to be temporally orchestrated. We propose that this sequence of events occurs preferentially during the night, when the CE has some protection by the closed eyelid during sleep. The underlying sub- Conceived and designed the experiments: AFW BPC EWH. Performed the experiments: AFW. Analyzed the data: AFW BPC EWH. Contributed reagents/materials/analysis tools: AFW. Wrote the paper: AFW. Critically reviewed manuscript: BPC EWH. Conceived and designed the experiments: AFW BPC EWH. Performed the experiments: AFW. Analyzed the data: AFW BPC EWH. Contributed reagents/materials/analysis tools: AFW. Wrote the paper: AFW. Critically reviewed manuscript: BPC EWH. Circadian Regulation of Tight Junctions in Corneal Epithelium Estil S, Primo EJ, Wilson G (2000) Apoptosis in shed human corneal cells. Invest Ophthalmol Vis Sci 41: 3360–3364. 46. Yeh S, Song XJ, Farley W, Li DQ, Stern ME, et al. (2003) Apoptosis of ocular surface cells in experimentally induced dry eye. Invest Ophthalmol Vis Sci 44: 124–129. p p 73. Kim HS, Shang T, Chen Z, Pflugfelder SC, Li DQ (2004) TGF-beta1 stimulates production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) by human corneal epithelial cells. Exp Eye Res 79: 263–274. 47. Luo L, Li DQ, Doshi A, Farley W, Corrales RM, et al. (2004) Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface. Invest Ophthalmol Vis Sci 45: 4293–4301. ( ) y p p y 74. Li DQ, Lokeshwar BL, Solomon A, Monroy D, Ji Z, et al. (2001) Regulation of MMP-9 production by human corneal epithelial cells. Exp Eye Res 73: 449–459. 75. Li DQ, Shang TY, Kim HS, Solomon A, Lokeshwar BL, et al. (2003) Regulated expression of collagenases MMP-1, -8, and -13 and stromelysins MMP-3, -10, and -11 by human corneal epithelial cells. Invest Ophthalmol Vis Sci 44: 2928– 2936. 48. Pflugfelder SC, Farley W, Luo L, Chen LZ, de Paiva CS, et al. (2005) Matrix metalloproteinase-9 knockout confers resistance to corneal epithelial barrier disruption in experimental dry eye. Am J Pathol 166: 61–71. 76. Chotikavanich S, de Paiva CS, Li de Q, Chen JJ, Bian F, et al. (2009) Production and activity of matrix metalloproteinase-9 on the ocular surface increase in dysfunctional tear syndrome. Invest Ophthalmol Vis Sci 50: 3203–3209. 49. Forster C (2008) Tight junctions and the modulation of barrier function in disease. Histochem Cell Biol 130: 55–70. 50. Scott RA, Lauweryns B, Snead DM, Haynes RJ, Mahida Y, et al. (1997) E- cadherin distribution and epithelial basement membrane characteristics of the normal human conjunctiva and cornea. Eye (Lond) 11 (Pt 5): 607–612. 77. Markoulli M, Papas E, Cole N, Holden BA (2012) The diurnal variation of matrix metalloproteinase-9 and its associated factors in human tears. Invest Ophthalmol Vis Sci 53: 1479–1484. 51. Ban Y, Dota A, Cooper LJ, Fullwood NJ, Nakamura T, et al. (2003) Tight junction-related protein expression and distribution in human corneal epithelium. Exp Eye Res 76: 663–669. p 78. Zucker S, Drews M, Conner C, Foda HD, DeClerck YA, et al. Circadian Regulation of Tight Junctions in Corneal Epithelium Clarke H, Soler AP, Mullin JM (2000) Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC- PK1 epithelial cell sheets. J Cell Sci 113 (Pt 18): 3187–3196. 85. Swarnakar S, Mishra A, Ganguly K, Sharma AV (2007) Matrix metallopro- teinase-9 activity and expression is reduced by melatonin during prevention of ethanol-induced gastric ulcer in mice. J Pineal Res 43: 56–64. 60. Cordenonsi M, Turco F, D’Atri F, Hammar E, Martinucci G, et al. (1999) Xenopus laevis occludin. Identification of in vitro phosphorylation sites by protein kinase CK2 and association with cingulin. Eur J Biochem 264: 374–384. 86. Paul S, Sharma AV, Mahapatra PD, Bhattacharya P, Reiter RJ, et al. (2008) Role of melatonin in regulating matrix metalloproteinase-9 via tissue inhibitors of metalloproteinase-1 during protection against endometriosis. J Pineal Res 44: 439–449. 61. Dorfel MJ, Huber O (2012) Modulation of tight junction structure and function by kinases and phosphatases targeting occludin. J Biomed Biotechnol 2012: 807356. 62. Hirase T, Kawashima S, Wong EY, Ueyama T, Rikitake Y, et al. (2001) Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms. J Biol Chem 276: 10423–10431. 87. Kenney MC, Chwa M, Alba A, Saghizadeh M, Huang ZS, et al. (1998) Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas. Curr Eye Res 17: 238–246. 88. Paterson CA, Wells JG, Koklitis PA, Higgs GA, Docherty AJ (1994) Recombinant tissue inhibitor of metalloproteinases type 1 suppresses alkali- burn-induced corneal ulceration in rabbits. Invest Ophthalmol Vis Sci 35: 677– 684. 63. Huguenin M, Muller EJ, Trachsel-Rosmann S, Oneda B, Ambort D, et al. (2008) The metalloprotease meprinbeta processes E-cadherin and weakens intercellular adhesion. PLoS One 3: e2153. 64. Lynch CC, Vargo-Gogola T, Matrisian LM, Fingleton B (2010) Cleavage of E- Cadherin by Matrix Metalloproteinase-7 Promotes Cellular Proliferation in Nontransformed Cell Lines via Activation of RhoA. J Oncol 2010: 530745. 89. Yuan X, Mitchell BM, Wilhelmus KR (2009) Expression of matrix metallopro- teinases during experimental Candida albicans keratitis. Invest Ophthalmol Vis Sci 50: 737–742. 65. Chen F, Ohashi N, Li W, Eckman C, Nguyen JH (2009) Disruptions of occludin and claudin-5 in brain endothelial cells in vitro and in brains of mice with acute liver failure. Hepatology 50: 1914–1923. 90. Sokol JL, Masur SK, Asbell PA, Wolosin JM (1990) Layer-by-layer desquamation of corneal epithelium and maturation of tear-facing membranes. Circadian Regulation of Tight Junctions in Corneal Epithelium (1998) Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the catalytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1- MMP). J Biol Chem 273: 1216–1222. 52. Chen L, Ebihara N, Fujiki K, Murakami A (2007) Expression and distribution of junctional adhesion molecule-1 in the human cornea. Jpn J Ophthalmol 51: 405–411. 79. Jiang A, Lehti K, Wang X, Weiss SJ, Keski-Oja J, et al. (2001) Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis. Proc Natl Acad Sci U S A 98: 13693–13698. 53. Ortiz-Melo MT, Sanchez-Guzman E, Gonzalez-Robles A, Valdes J, Gomez- Flores E, et al. (2013) Expression of claudins -2 and -4 and cingulin is coordinated with the start of stratification and differentiation in corneal epithelial cells: retinoic acid reversibly disrupts epithelial barrier. Biol Open 2: 132–143. 80. Remacle A, Murphy G, Roghi C (2003) Membrane type I-matrix metallopro- teinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface. J Cell Sci 116: 3905–3916. 54. Yoshida Y, Ban Y, Kinoshita S (2009) Tight junction transmembrane protein claudin subtype expression and distribution in human corneal and conjunctival epithelium. Invest Ophthalmol Vis Sci 50: 2103–2108. 81. Wiechmann AF, Hollaway LR, Rada JA (2009) Melatonin receptor expression in Xenopus laevis surface corneal epithelium: diurnal rhythm of lateral membrane localization. Mol Vis 15: 2384–2403. 55. Koval M (2013) Differential pathways of claudin oligomerization and integration into tight junctions. Tissue Barriers 1: e24518. 82. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Gonzalez MJ, Reiter RJ (2007) Relation of nocturnal melatonin levels to serum matrix metalloproteinase- 9 concentrations in patients with myocardial infarction. Thromb Res 120: 361– 366. 56. Krug SM, Schulzke JD, Fromm M (2014) Tight junction, selective permeability, and related diseases. Semin Cell Dev Biol. 57. Van Itallie CM, Anderson JM (2013) Claudin interactions in and out of the tight junction. Tissue Barriers 1: e25247. 83. Esposito E, Mazzon E, Riccardi L, Caminiti R, Meli R, et al. (2008) Matrix metalloproteinase-9 and metalloproteinase-2 activity and expression is reduced by melatonin during experimental colitis. J Pineal Res 45: 166–173. j 58. Van Itallie CM, Anderson JM (2014) Architecture of tight junctions and principles of molecular composition. Semin Cell Dev Biol. 84. Ganguly K, Maity P, Reiter RJ, Swarnakar S (2005) Effect of melatonin on secreted and induced matrix metalloproteinase-9 and -2 activity during prevention of indomethacin-induced gastric ulcer. J Pineal Res 39: 307–315. 59. Circadian Regulation of Tight Junctions in Corneal Epithelium Circadian Regulation of Tight Junctions in Corneal Epithelium 39. Matsubara M, Girard MT, Kublin CL, Cintron C, Fini ME (1991) Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea. Dev Biol 147: 425–439. 67. Liu J, Jin X, Liu KJ, Liu W (2012) Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contrib- ute to blood-brain barrier damage in early ischemic stroke stage. J Neurosci 32: 3044–3057. g 40. Smith VA, Hoh HB, Easty DL (1999) Role of ocular matrix metalloproteinases in peripheral ulcerative keratitis. Br J Ophthalmol 83: 1376–1383. 68. Gordon GM, Austin JS, Sklar AL, Feuer WJ, LaGier AJ, et al. (2011) Comprehensive gene expression profiling and functional analysis of matrix metalloproteinases and TIMPs, and identification of ADAM-10 gene expression, in a corneal model of epithelial resurfacing. J Cell Physiol 226: 1461–1470. in peripheral ulcerative keratitis. Br J Ophthalmol 83: 1376–13 41. Yang YN, Bauer D, Wasmuth S, Steuhl KP, Heiligenhaus A (2003) Matrix metalloproteinases (MMP-2 and 9) and tissue inhibitors of matrix metallopro- teinases (TIMP-1 and 2) during the course of experimental necrotizing herpetic keratitis. Exp Eye Res 77: 227–237. p g in a corneal model of epithelial resurfacing. J Cell Physiol 226: 1461– 69. Ardan T, Cejkova J (2012) Immunohistochemical expression of matrix metalloproteinases in the rabbit corneal epithelium upon UVA and UVB irradiation. Acta Histochem 114: 540–546. 42. Ren H, Wilson G (1996) Apoptosis in the corneal epithelium. Invest Ophthalmol Vis Sci 37: 1017–1025. 70. Collier SA, Madigan MC, Penfold PL (2000) Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas. Curr Eye Res 21: 662–668. 43. Rosenblatt J, Raff MC, Cramer LP (2001) An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr Biol 11: 1847–1857. y 71. Brejchova K, Liskova P, Hrdlickova E, Filipec M, Jirsova K (2009) Matrix metalloproteinases in recurrent corneal melting associated with primary Sjorgen’s syndrome. Mol Vis 15: 2364–2372. 44. Chen W, Zhang X, Zhang J, Chen J, Wang S, et al. (2008) A murine model of dry eye induced by an intelligently controlled environmental system. Invest Ophthalmol Vis Sci 49: 1386–1391. j g y 72. Sobrin L, Liu Z, Monroy DC, Solomon A, Selzer MG, et al. (2000) Regulation of MMP-9 activity in human tear fluid and corneal epithelial culture supernatant. Invest Ophthalmol Vis Sci 41: 1703–1709. p 45. References FASEB J 23: 1596–1606. 34. Fu L, Das B, Mathew S, Shi YB (2009) Genome-wide identification of Xenopus matrix metalloproteinases: conservation and unique duplications in amphibians. BMC Genomics 10: 81. 15. Monea S, Jordan BA, Srivastava S, DeSouza S, Ziff EB (2006) Membrane localization of membrane type 5 matrix metalloproteinase by AMPA receptor binding protein and cleavage of cadherins. J Neurosci 26: 2300–2312. 35. Hadler-Olsen E, Kanapathippillai P, Berg E, Svineng G, Winberg JO, et al. (2010) Gelatin in situ zymography on fixed, paraffin-embedded tissue: zinc and ethanol fixation preserve enzyme activity. J Histochem Cytochem 58: 29–39. 16. Partridge CA, Jeffrey JJ, Malik AB (1993) A 96-kDa gelatinase induced by TNF- alpha contributes to increased microvascular endothelial permeability. Am J Physiol 265: L438–447. 36. Mook OR, Van Overbeek C, Ackema EG, Van Maldegem F, Frederiks WM (2003) In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin. J Histochem Cytochem 51: 821–829. y 17. Sivak JM, Fini ME (2002) MMPs in the eye: emerging roles for matrix metalloproteinases in ocular physiology. Prog Retin Eye Res 21: 1–14. 37. Fini ME, Yue BY, Sugar J (1992) Collagenolytic/gelatinolytic metalloproteinases in normal and keratoconus corneas. Curr Eye Res 11: 849–862. 18. Williams H, Johnson JL, Jackson CL, White SJ, George SJ (2010) MMP-7 mediates cleavage of N-cadherin and promotes smooth muscle cell apoptosis. Cardiovasc Res 87: 137–146. 38. Kernacki KA, Chunta JL, Barrett RP, Hazlett LD (2004) TIMP-1 role in protection against Pseudomonas aeruginosa-induced corneal destruction. Exp Eye Res 78: 1155–1162. 19. Yang Y, Estrada EY, Thompson JF, Liu W, Rosenberg GA (2007) Matrix metalloproteinase-mediated disruption of tight junction proteins in cerebral November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org 11 November 2014 \| Volume 9 \| Issue 11 \| e113810 Circadian Regulation of Tight Junctions in Corneal Epithelium Invest Ophthalmol Vis Sci 31: 294–304. 66. Feng S, Cen J, Huang Y, Shen H, Yao L, et al. (2011) Matrix metalloproteinase- 2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins. PLoS One 6: e20599. 91. Wolosin JM, Chen M (1993) Ontogeny of corneal epithelial tight junctions: stratal locale of biosynthetic activities. Invest Ophthalmol Vis Sci 34: 2655– 2664. November 2014 \| Volume 9 \| Issue 11 \| e113810 PLOS ONE \| www.plosone.org 12
https://openalex.org/W2134571742	https://europepmc.org/articles/pmc3280836?pdf=render	English	null	Manufacture of a Polyaniline Nanofiber Ammonia Sensor Integrated with a Readout Circuit Using the CMOS-MEMS Technique	Sensors	2,009	cc-by	4,855	Sensors 2009, 9, 869-880; doi:10.3390/s90200869 Sensors 2009, 9, 869-880; doi:10.3390/s90200869 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS Manufacture of a Polyaniline Nanofiber Ammonia Sensor Integrated with a Readout Circuit Using the CMOS-MEMS Technique Mao-Chen Liu 1, Ching-Liang Dai 1,, Chih-Hua Chan 1 and Chyan-Chyi Wu 2 Keywords: Ammonia sensor, CMOS, Polyaniline, readout circuit. 1. Introduction Ammonia (NH3) is emitted by human activity. For instance, production of refrigeration systems and fertilizers emits ammonia. The worldwide ammonia emission resulting from domestic animals and industrial combustion is about 20-35 Tg/year and 2.1-8.1 Tg/year, respectively [1]. Ammonia is a toxic gas with exposure limit values of 25 ppm for a period of 8 h and of 35 ppm for a period of 10 min [2]. Ammonia sensors are important devices in many industrial, agricultural and biomedical applications. For instance, Boeker et al. [3] proposed an ammonia sensor based on a quartz crystal microbalance transducer for monitoring the ammonia concentration in agricultural emissions. Moos et al. [4] developed an ammonia gas sensor with a selective thick film of zeolites for automotive exhaust gas applications. Pushkarsky et al. [5] presented a laser-based photoacoustic ammonia sensor for industrial ammonia gas measurement. Huang et al. [6] developed polyaniline nanofibers using interfacial polymerization at an aqueous and organic interface, and the resulting polyaniline, which had high surface area and porosity, could enhance properties in applications such as chemical sensors. With the same synthetic method, Virji et al. [7] fabricated polyaniline nanofiber gas sensors with performances that were better than conventional polyaniline. Thereby, the ammonia sensor in this work adopts polyaniline as a sensitive material. Microelectromechanical systems (MEMS) technology was applied to fabricate various microsensors. The advantages of microsensors include small size, high performance, low cost and easy mass-production. Many studies have recently utilized MEMS technology to manufacture ammonia sensors. For instance, Lee et al. [8] fabricated a micro ammonia gas sensor using a bulk micromachining technique. The sensor that was resistive type composed of a polyaniline film, a SU-8 adhesion layer and an interdigital Pt electrode, where the polyaniline film was the ammonia sensing film. The ammonia sensor had a sensitivity of about 40% at 50 ppm ammonia. Mitzner et al. [9] utilized a post-CMOS process to manufacture a micro-hotplate-based gas sensor array. The post- CMOS process adopted XeF2 to etch a silicon substrate to obtain the micro-hotplate array, and a SnO2/Pt sensing film was sputtered on this micro-hotplate forming a resistive-type ammonia sensor. Llobet et al. [10] utilized silicon process technology to make an ammonia sensor, in which the sensor consisted of a heater, an interdigital electrode, a temperature meter and a sensing film. Tungsten oxide, coated on the interdigital electrode, was adopted as an ammonia sensing film. Li et al. Mao-Chen Liu 1, Ching-Liang Dai 1,, Chih-Hua Chan 1 and Chyan-Chyi Wu 2 1 Department of Mechanical Engineering, National Chung Hsing University, Taichung, 402 Taiwan, ROC; E-Mails: d9361301@mail.nchu.edu.tw; libra_louis@hotmail.com; 1 Department of Mechanical Engineering, National Chung Hsing University, Taichung, 402 Taiwan, ROC; E-Mails: d9361301@mail.nchu.edu.tw; libra_louis@hotmail.com; 2 Department of Mechanical and Electro-Mechanical Engineering, Tamkang University, Tamsui, 251 Taiwan, ROC; E-Mail: ccwu@mail.tku.edu.tw 2 Department of Mechanical and Electro-Mechanical Engineering, Tamkang University, Tamsui, 251 Taiwan, ROC; E-Mail: ccwu@mail.tku.edu.tw * Author to whom correspondence should be addressed; E-Mail: cldai@dragon.nchu.edu.tw; Tel.: +886-4-22840433; Fax: +886-4-22877170 Received: 13 December 2008; in revised form: 15 January 2009 / Accepted: 9 February 2009 / Published: 10 February 2009 Abstract: This study presents the fabrication of a polyaniline nanofiber ammonia sensor integrated with a readout circuit on a chip using the commercial 0.35 m complementary metal oxide semiconductor (CMOS) process and a post-process. The micro ammonia sensor consists of a sensing resistor and an ammonia sensing film. Polyaniline prepared by a chemical polymerization method was adopted as the ammonia sensing film. The fabrication of the ammonia sensor needs a post-process to etch the sacrificial layers and to expose the sensing resistor, and then the ammonia sensing film is coated on the sensing resistor. The ammonia sensor, which is of resistive type, changes its resistance when the sensing film adsorbs or desorbs ammonia gas. A readout circuit is employed to convert the resistance of the ammonia sensor into the voltage output. Experimental results show that the sensitivity of the ammonia sensor is about 0.88 mV/ppm at room temperature. Keywords: Ammonia sensor, CMOS, Polyaniline, readout circuit. Sensors 2009, 9 870 Sensors 2009, 9 Sensors 2009, 9 technique is their ability to integrate with circuits as a system on a chip (SOC) because of their compatibility with the CMOS process. The ammonia sensors, proposed by Lee et al. [8], Mitzner et al. [9], Llobet et al. [10], Li et al. [11] and Connolly et al. [12], had no integration with circuitry on a chip. In this work, we employ the CMOS-MEMS technique to fabricate a polyaniline nanofiber ammonia sensor integrated with a readout circuit on a chip, which can reduce the volume and cost and enhance the performance. The area of the integrated ammonia sensor chip is about 0.6 mm2. 2. Structure of the Integrated Ammonia Sensor Figure 1 shows the structure of the integrated chip, which contains an ammonia sensor and readout circuit. The ammonia sensor is composed of a sensing resistor and an ammonia sensing film. The sensing resistor is a polysilicon winding line. The ammonia sensing film, which is polyaniline prepared by a chemical polymerization method, is coated on the sensing resistor. A silicon dioxide layer is located between the sensing resistor and the polyaniline film. The sensing resistor is 2 m wide, 0.4 m thick and 9,000 m long. Figure 1. Schematic structure of the ammonia sensor integrated with readout circuit. Figure 2 illustrates the energy band diagram of the ammonia sensor. When the polyaniline surface contacts air, holes are produced on this surface. As shown in Figure 2(a), an accumulation of holes is formed on the surface of the polyaniline film, so that the valence band edge of polyaniline bends upward and is close to the Fermi level of polyaniline. An accumulation of electrons at the oxide- polysilicon interface is formed [16], which causes the conduction band edge of polysilicon to bend downward and be close to the Fermi level of polysilicon. When polyaniline interacts with ammonia, the following reaction occurs [17]: + + 3 4 PANIH +NH PANI+NH  1. Introduction [11] presented a micro gas with piezoresistive SiO2 cantilever beam fabricated by a surface and bulk micromachining process. An ammonia sensing layer of 11-mercaaptoundecanoic acid was coated on the piezoresistive cantilever beams. The sensor was packaged with a linear amplifier, and its output voltage was about 7 V in 1 ppm ammonia gas. Connolly et al. [12] reported a porous SiC ammonia sensor produced by silicon process technology, in which the sensor was a capacitive type. The sensing film of SiC was deposited by plasma enhanced chemical vapor deposition (PECVD) and was then made porous by electrochemical etching in 73% HF. The use of a commercial CMOS process to fabricate MEMS devices is known as the CMOS- MEMS technique [13]. Several microdevices manufactured by the CMOS-MEMS technique need a post-process to coat the functional films or release the suspended structures. For instance, the suspended structures of the CMOS-MEMS switches [14] and pressure sensors [15] were released by a wet etching post-process. The main advantage of microdevices fabricated by the CMOS-MEMS 871 Sensors 2009, 9 Sensors 2009, 9 a distance with the Fermi level of polyaniline, so that the resistance of polyaniline increases. At the same time, the accumulation of electrons at the oxide-polysilicon interface is reduced, which leads to the conduction band edge of polysilicon to reduce bending and have a distance with the Fermi level of polysilicon, so that the resistance of polysilicon increases. Figure 2. Energy band diagram of the sensor in (a) air and (b) ammonia. EF is the Fermi level, Ec-PANi is the conduction band of polyaniline, Ev- PANi is the valence band of polyaniline, EFi-PANi is the intrinsic Fermi level of polyaniline, Ec-PolySi is the conduction band of polysilicon, Ev-PolySi is the valence band of polysilicon, and EFi-PolySi is the intrinsic Fermi of polysilicon. Figure 3. Readout circuit for the ammonia sensor. Figure 3 presents the readout circuit for the ammonia sensor. The readout circuit consists of a Wheatstone circuit, an operational amplifier and resistances. As shown in Figure 3, the Wheatstone circuit is constituted by the sensing resistor (Rs) and three resistances (R1, R2 and R3). The sensing resistor changes its resistance as the sensing film adsorbs or desorbs ammonia gas. The readout circuit is utilized to convert the resistance of the ammonia sensor into the voltage output. In this design, R1=10 k, R2=10 k, R3=10 k, R4=100 , R5=10 k, R6=10 k and R7=10 k are adopted. Figure 4 illustrates the operational amplifier circuitry for the readout circuit, where Vdd represents a voltage power supply and Vss is the ground. Figure 3. Readout circuit for the ammonia sensor. Figure 3. Readout circuit for the ammonia sensor. Figure 3 presents the readout circuit for the ammonia sensor. The readout circuit consists of a Wheatstone circuit, an operational amplifier and resistances. As shown in Figure 3, the Wheatstone circuit is constituted by the sensing resistor (Rs) and three resistances (R1, R2 and R3). The sensing resistor changes its resistance as the sensing film adsorbs or desorbs ammonia gas. The readout circuit is utilized to convert the resistance of the ammonia sensor into the voltage output. In this design, R1=10 k, R2=10 k, R3=10 k, R4=100 , R5=10 k, R6=10 k and R7=10 k are adopted. Figure 4 illustrates the operational amplifier circuitry for the readout circuit, where Vdd represents a voltage power supply and Vss is the ground. Figure 3 presents the readout circuit for the ammonia sensor. + + 3 4 PANIH +NH PANI+NH  (1) According to Equation (1), the H+ holes of polyaniline reduce as polyaniline interacts with NH3. As shown in 2(b), the accumulation of holes at the surface of polyaniline is reduced when polyaniline is exposed to ammonia, resulting in the valence band edge of reduced polyaniline bending and having 872 3. Fabrication of the Integrated Ammonia Sensor The commercial 0.35 m CMOS process of Taiwan Semiconductor Manufacturing Company (TSMC) was used to fabricate the integrated chip with ammonia sensor and readout circuit. In order to expose the sensing resistor and coat the ammonia sensing film, the integrated chip requires a post- process after completion of the CMOS process. The post-process contains two main steps: (1) etching the sacrificial layers to expose the sensing resistor, and (2) coating the ammonia sensing film on the sensing resistor. Figure 7 shows the process flow of the integrated chip. Figure 7(a) illustrates the cross-section of the integrated chip after completion of the CMOS process. In the ammonia sensor, the polysilicon layer is adopted as the sensing resistor, and the metal and via layers are used as the sacrificial layers. The materials of the metal and via layers are aluminum (Al) and tungsten (W), respectively. The sacrificial layers are removed from the ammonia sensor, exposing the sensing resistor. As shown in Figure 7(b), the chip is immersed in two etchants: one is an Al etchant with phosphoric acid, nitric acid, acetic acid and deionized water in a ratio of 14:1:2:3. The other is a W etchant with sulfuric acid and hydrogen peroxide in a ratio of 2:1. Figure 8 displays the photograph of the integrated ammonia sensor after the wet etching process. Then, the chip is put in an oven at 300C for 8 h, so that a thin silicon dioxide layer is formed on the polysilicon surface. Finally, the polyaniline film is coated on the sensing resistor as shown in Figure 7(c). Sensors 2009, 9 Sensors 2009, 9 Figure 6. Simulated results of the readout circuit. Figure 6. Simulated results of the readout circuit. Figure 6. Simulated results of the readout circuit. 3. Fabrication of the Integrated Ammonia Sensor Sensors 2009, 9 The readout circuit consists of a Wheatstone circuit, an operational amplifier and resistances. As shown in Figure 3, the Wheatstone circuit is constituted by the sensing resistor (Rs) and three resistances (R1, R2 and R3). The sensing resistor changes its resistance as the sensing film adsorbs or desorbs ammonia gas. The readout circuit is utilized to convert the resistance of the ammonia sensor into the voltage output. In this design, R1=10 k, R2=10 k, R3=10 k, R4=100 , R5=10 k, R6=10 k and R7=10 k are adopted. Figure 4 illustrates the operational amplifier circuitry for the readout circuit, where Vdd represents a voltage power supply and Vss is the ground. 873 Sensors 2009, 9 Figure 4. Design of the operational amplifier circuit. Figure 4. Design of the operational amplifier circuit. HSPICE, which is a professional circuit simulation software, was used to simulate the operational amplifier and the readout circuit. Figure 5 shows the simulated results of the frequency response for the operational amplifier. As shown, the operational amplifier has a dc open loop gain of approximately 97 dB. Figure 4. Design of the operational amplifier circuit. HSPICE, which is a professional circuit simulation software, was used to simulate the operational amplifier and the readout circuit. Figure 5 shows the simulated results of the frequency response for the operational amplifier. As shown, the operational amplifier has a dc open loop gain of approximately 97 dB. Figure 5. Frequency response of the operational amplifier. Figure 5. Frequency response of the operational amplifier. Figure 6 depicts the simulated results of the readout circuit. In this simulation, the input voltage Vin is 3 V, the resistance of the ammonia sensor Rs varies from 23 k to 26 k. The output voltage of the readout circuit varies from 2.294 V to 2.372 V as the resistance of the ammonia sensor changes from 23 k to 26 k. Figure 5. Frequency response of the operational amplifier. Figure 6 depicts the simulated results of the readout circuit. In this simulation, the input voltage Vin is 3 V, the resistance of the ammonia sensor Rs varies from 23 k to 26 k. The output voltage of the readout circuit varies from 2.294 V to 2.372 V as the resistance of the ammonia sensor changes from 23 k to 26 k. Sensors 2009, 9 Figure 6. Simulated results of the readout circuit. 3. Sensors 2009, 9 Fabrication of the Integrated Ammonia Sensor 874 3. Fabrication of the Integrated Ammonia Sensor The polyaniline nanofibers [6,18] are prepared by a chemical polymerization method according to the following steps: (1) 8 mL HCl is mixed with 100 mL deionized water and stirred until a homogenous solution is obtained; (2) the solution of 1 mL C6H5NH2 is added into the HCl solution with stirring until the mixing solution becomes homogenous; (3) 2.4 g (NH4)2S2O8 is dissolved in 50 mL deionized water by stirring vigorously; (4) the (NH4)2S2O8 solution is added into the C6H5NH2/HCl solution and stirred until a homogeneous mixing solution is obtaned; (5) the mixing solution of (NH4)2S2O8/C6H5NH2/HCl is aged at room temperature for 120 h, producing a blackish green sediment; (6) the resulting product is filtered, and a precision micro dropper is used to drop the product on the ammonia sensor chip, followed by calcination in air at 110C for 1 h. Sensors 2009, 9 875 Sensors 2009, 9 Figure 7. Process flow of the ammonia sensor; (a) after the CMOS process, (b) etching sacrificial layers, and (c) coating the sensing film. y , ( ) g g The surface morphology of the polyaniline film is measured by a scanning electron microscope (JEOL JSM-6700F). Figure 9 shows a scanning electron microscope image of the polyaniline film. The sensing film, which is composed of many polyaniline nanofibers as shown in Figure 9, is porous and has a large surface area. The elements of the polyaniline nanofiber film are measured by an energy dispersive spectrometer (OXFORD INCA ENERGY 400), and the measured results are shown in Figure 10. The polyaniline nanofiber film contains 63 wt% C, 28 wt% O, 8.5 wt% S and 0.5 wt% Cl. The surface morphology of the polyaniline film is measured by a scanning electron microscope (JEOL JSM-6700F). Figure 9 shows a scanning electron microscope image of the polyaniline film. The sensing film, which is composed of many polyaniline nanofibers as shown in Figure 9, is porous and has a large surface area. The elements of the polyaniline nanofiber film are measured by an energy dispersive spectrometer (OXFORD INCA ENERGY 400), and the measured results are shown in Figure 10. The polyaniline nanofiber film contains 63 wt% C, 28 wt% O, 8.5 wt% S and 0.5 wt% Cl. The surface morphology of the polyaniline film is measured by a scanning electron microscope (JEOL JSM-6700F). Figure 9 shows a scanning electron microscope image of the polyaniline film. 3. Fabrication of the Integrated Ammonia Sensor The sensing film, which is composed of many polyaniline nanofibers as shown in Figure 9, is porous and has a large surface area. The elements of the polyaniline nanofiber film are measured by an energy dispersive spectrometer (OXFORD INCA ENERGY 400), and the measured results are shown in Figure 10. The polyaniline nanofiber film contains 63 wt% C, 28 wt% O, 8.5 wt% S and 0.5 wt% Cl. Figure 8. photograph of the integrated ammonia sensor chip after the wet etching process. igure 8. photograph of the integrated ammonia sensor chip after the wet etching process 876 Sensors 2009, 9 Sensors 2009, 9 Figure 9. Scanning electron microscope image of polyaniline nanofiber film. Figure 9. Scanning electron microscope image of polyaniline nanofiber film. Figure 10. Elements of polyaniline nanofiber film measured by energy dispersive spectrometer. Figure 10. Elements of polyaniline nanofiber film measured by energy dispersive spectrometer. 4. Results and Discussion A power supply, an oscilloscope, an LCR meter and a test chamber were utilized to measure the performance of the ammonia sensor chip. In order to characterize the variation of resistance in the sensing part, the ammonia sensor was tested without readout circuit. The sensor chip was set in the test chamber, and the LCR meter was used to measure its resistance variation at different NH3 concentrations. Figure 11 presents the measured results of the ammonia sensor without readout circuit at different ammonia concentrations. The initial resistance of the sensing resistor in the ammonia sensor was about 22.7 k (in air), and the resistance of the sensor varied to 25.5 k at 50 ppm NH3. The results showed that the resistance of the ammonia sensor increased as the concentration of ammonia increased. As shown in Figure 11, the ammonia sensor had a response time of about 60 sec at 50 ppm NH3 and a recovery time of 330 sec at 50 ppm NH3. Figure 10. Elements of polyaniline nanofiber film measured by energy dispersive spectrometer. 4. Results and Discussion A power supply, an oscilloscope, an LCR meter and a test chamber were utilized to measure the performance of the ammonia sensor chip. In order to characterize the variation of resistance in the sensing part, the ammonia sensor was tested without readout circuit. The sensor chip was set in the test chamber, and the LCR meter was used to measure its resistance variation at different NH3 concentrations. Figure 11 presents the measured results of the ammonia sensor without readout circuit at different ammonia concentrations. The initial resistance of the sensing resistor in the ammonia sensor was about 22.7 k (in air), and the resistance of the sensor varied to 25.5 k at 50 ppm NH3. The results showed that the resistance of the ammonia sensor increased as the concentration of ammonia increased. As shown in Figure 11, the ammonia sensor had a response time of about 60 sec at 50 ppm NH3 and a recovery time of 330 sec at 50 ppm NH3. Sensors 2009, 9 The ammonia sensor with readout circuit was set in the test chamber and was tested at room temperature with different ammonia concentrations. In order to understand the influence of the input voltage, the sensor was provided with different input voltages. The power supply provided a bias voltage of 3.3 V and different input voltages of 1, 2 and 3 V, respectively, to the readout circuit in the sensor. The oscilloscope was employed to record the output signal of the sensor to ammonia changes. Figure 12 displays the output voltage of the ammonia sensor with different input voltages of 1, 2 and 3 V. In this investigation, 1 to 50 ppm of ammonia gas was supplied. With an input voltage of 1 V, the output voltage of the ammonia sensor changed from 948 mV to 958 mV as the concentration of ammonia gas varied from 1 to 50 ppm. The variation of the output voltage was 10 mV over 1-50 ppm NH3, so the sensitivity of the ammonia sensor with an input voltage of 1 V was about 0.2 mV/ppm. As shown in Figure 12(b), when an input voltage of 2 V was supplied, the measured results showed that the output voltage of the ammonia sensor varied from 1.754 V to 1.772 V as the concentration of ammonia gas changed from 1 to 50 ppm. Thereby, the variation of the output voltage was 18 mV in 1- 50 ppm NH3, and the ammonia sensor with an input voltage of 2 V had a sensitivity of about 0.36 mV/ppm. When the input voltage was changed to 3 V, as depicted in Figure 12(c), the output voltage of the ammonia sensor changed from 2.491 V to 2.534 V as the concentration of ammonia gas varied from 1 to 50 ppm. The variation of the output voltage was 43 mV for 1-50 ppm NH3, so the sensitivity of the ammonia sensor with an input voltage of 3 V was approximately 0.88 mV/ppm. Therefore, the integrated ammonia senor had a sensitivity of 0.88 mV/ppm when providing a bias voltage of 3.3 V and an input voltage of 3V. The polyaniline nanofiber gas sensor, fabracted by Virji et al. [7], was used to detect a low concentration of 3 ppm ammonia gas and had a high sensitivity. Sutar et al. [19] proposed a polyaniline nanofiber ammonia sensor with the detection limit of 0.5 ppm NH3. Sensors 2009, 9 Crowley et al. [20] manufactured an ammonia gas sensor by using inkjet-printed polyaniline nanoparticles, and the sensor had a detection limit of 1 ppm NH3. In this study, the experiments showed that the detection limit of the ammonia sensor was about 1 ppm NH3. The resistive ammonia sensor with a sensitive film of polyaniline, presented by Lee et al. [8], had a sensitivity of about 40% at 50 ppm ammonia. Li et al. [11] proposed an ammonia sensor with a sensitive material of 11-mercaaptoundecanoic acid, and it had an output voltage of 7 V in 1 ppm ammonia gas. The sensitivity of 0.88 mV/ppm in this work exceeds that of Li et al. [11]. Sensors 2009, 9 877 Sensors 2009, 9 Sensors 2009, 9 Sensors 2009, 9 Figure 11. Relation between the resistance variation and NH3 concentration for the ammonia sens Figure 12. Measured results of the ammonia sensor with input voltage of (a) 1 V, (b) 2 V and (c) 3 V. Figure 12. Measured results of the ammonia sensor with input voltage of (a) 1 V, (b) 2 V and (c) 3 V Figure 12. Measured results of the ammonia sensor with input voltage of (a) 1 V, (b) 2 V and (c) 3 V. 878 Sensors 2009, 9 sensor was about 0.88 mV/ppm at room temperature when providing a bias voltage of 3.3 V and an input voltage of 3 V. sensor was about 0.88 mV/ppm at room temperature when providing a bias voltage of 3.3 V and an input voltage of 3 V. References 1. Timmer, B.; Olthuis, W.; Van Den Berg, A. Ammonia sensors and their application-A review. Sens. Actuat. B 2005, 107, 666-677. 1. Timmer, B.; Olthuis, W.; Van Den Berg, A. Ammonia sensors and their application-A review. Sens. Actuat. B 2005, 107, 666-677. 2. Christie, S.; Scorsone, E.; Persaud, K.; Kvasnik, F. Remote detection of gaseous ammonia using the near infarad transmission peroperties of polyaniline. Sens. Actuat. B 2003, 90, 163-169. 2. Christie, S.; Scorsone, E.; Persaud, K.; Kvasnik, F. Remote detection of gaseous ammonia using the near infarad transmission peroperties of polyaniline. Sens. Actuat. B 2003, 90, 163-169. 3. Boeker, P.; Horner, G.; Roesier, S. Monoliththic sensor array based on a quartz microbalance transducer with enhanced sensitivity for monitoring agricultural emissions. Sens. Actuat. B 2000, 70, 37-42, 3. Boeker, P.; Horner, G.; Roesier, S. Monoliththic sensor array based on a quartz microbalance transducer with enhanced sensitivity for monitoring agricultural emissions. Sens. Actuat. B 2000, 70, 37-42, 4. Moos, R.; Muller, R.; Plog, C.; Knezevic, A.; Leye, H.; Irion, E.; Braun, T.; Marquardt, K.J.; Binder, K. Selective ammonia exhaust gas sensor for automotive application. Sens. Actuat. B 2002, 83, 181-189. 4. Moos, R.; Muller, R.; Plog, C.; Knezevic, A.; Leye, H.; Irion, E.; Braun, T.; Marquardt, K.J.; Binder, K. Selective ammonia exhaust gas sensor for automotive application. Sens. Actuat. B 2002, 83, 181-189. 5. Pushkarsky, M.B.; Webber, M.E.; Baghdassarian, O.; Narasimhan, L.R.; Patel, C.K.N. Laser- based photoacoutic ammonia sensors for industrial applications. Appl. Phys. B 2002, 75, 391-396. 6. Huang, J.; Kaner, R.B. Nanofiber formation in the chemical polymerization of aniline: a mechanistic study. Angew. Chem. Int. Ed. 2004, 43, 5817-5821. 7. Virji, S.; Huang, J.; Kaner, R.B.; Weiller, B.H. Polyaniline nanofiber gas sensors: Examination of response mechanisms. Nano Lett. 2004, 4, 491-496. 8. Lee, Y.S.; Song, K.D.; Huh, J.S.; Chung, W.Y.; Lee, D.D. Fabrication of clinical gas sensor using MEMS process. Sens. Actuat. B 2005, 108, 292-297. 9. Mitzner, K.D.; Sternhagen, J.; Galipeau, D.W. Development of a micromachined hazardous gas sensor. Sens. Actuat. B 2003, 93, 92-99. 10. Llobet, E.; Molas ,G.; Molinas, P.; Calderer, J.; Vilanova, X.; Brezmes, J.; Sueriras, J.E.; Correig, X. Fabrication of Highly selective tungsten oxide ammonia sensors. J. Electrochem. Soc. 2000, 147, 776-779. 11. Li, P.; Li, X. A single-side micromachined piezoresistive SiO2 cantilever sensor for ultra- sensitive detection of gaseous chemicals. J. Micromech. Microeng. 2006, 12, 2539-2546. 12. Acknowledgements The authors would like to thank National Center for High-performance Computing (NCHC) for chip simulation, National Chip Implementation Center (CIC) for chip fabrication, Chunghwa Picture Tubes (CPT) Ltd for financial support, and the National Science Council of the Republic of China for financially supporting this research under Contract No NSC 97-2221-E-005-056. 5. Conclusions A polyaniline nanofiber ammonia sensor integrated with readout circuit was successfully implemented using the commercial 0.35 m CMOS process and the post-process. The ammonia sensor was comprised of a sensing film and a sensing resistor. The sensing film, which was prepared by a chemical polymerization method, was composed of polyaniline nanofibers that had a large surface area. The sensing resistor was formed by the polysilicon layer of the CMOS process. The post-process used etchants to etch the sacrificial layers to expose the sensing resistor, and then polyaniline was coated on the sensing resistor. The resistance of the resistive type ammonia sensor changed upon ammonia gas adsorption. A readout circuit was employed to convert the variation in resistance of the ammonia sensor into a voltage output. Experimental results showed that the sensitivity of the ammonia 879 References Connolly, E.J.; Timmer, B.; Pham, H.T.M.; Groeneweg, J.; Sarro, P.M.; Olthuis, W.; French, P.J. A porous SiC ammonia sensor. Sens. Actuat. B 2005, 109, 44-46. 13. Kim, J.W.; Takao, H.; Sawada K.; Ishida, M. Integrated inductors for RF transmitters in CMOS/MEMS smart microsensor systems. Sensors 2007, 7, 1387-1398. 14. Dai, C.L.; Chen, Y.L. Modeling and manufacturing of micromechanical RF switch © 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). Sensors 2009, 9 Sensors 2009, 9 880 Sensors 2007, 7, 2660-2670. 15. Dai, C.L.; Tai, Y.W.; Kao, P.H. Modeling and Fabrication of Micro FET Pressure Sensor with Circuits. Sensors 2007, 7, 3386-3398. 16. Neamen, D.A. Semiconductor Physics and Devices. Richard D. Irwin: Homewood, D.A. Semiconductor Physics and Devices. Richard D. Irwin: Homewood, IL, 1992. 17. Sengupta, P.P.; Barik, S.; Adhikari, B. Polyaniline as a gas sensor material. Mater. Manuf. Processes 2006, 21, 263-270. 18. He, Y.; Lu, J. Synthesis of polyaniline nanostructures with controlled morphology by a two-phase strategy. React. Funct. Polym. 2007, 67, 476-480. 19. Sutar, D.S.; Padma, N.; Aswal, D.K.; Deshpande, S.K.; Gupta, S.K.; Yakhmi, J.V. preparation of nanofiberous polyaniline filma and their application as ammonia gas sensor. Sens. Actuat. B 2007, 128, 286-292. 20. Crowley, K.; Morrin, A.; Hernandez, A.; O’Malley, E.; Whitten, P.G.; Wallace, G.G.; Smyth, M.R., Killard, A.J. Fabrication of an ammonia gas sensor using inkjet-printed polyaniline nanoparticles. Talanta 2008, 77, 710-717. © 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W2887132285	https://openresearch.surrey.ac.uk/view/delivery/44SUR_INST/12139758460002346/13140554550002346	English	null	Adaptation of the Ambulatory and Home Care Record for collecting palliative care service utilisation data from family carers in the UK: a pilot study	Pilot and feasibility studies	2,018	cc-by	6,999	RESEARCH Open Access © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Adaptation of the Ambulatory and Home Care Record for collecting palliative care service utilisation data from family carers in the UK: a pilot study Laura M. Holdsworth1* , Heather Gage2, Peter Williams3 and Claire Butler4 Abstract Background: Measuring service use and costs is an important aspect of service delivery evaluation. In end-of-life care, there is heavy reliance on care by family/friends (informal carers) and this should be reflected in the total cost of care alongside formal services. The Ambulatory and Home Care Record, developed in Canada, is both comprehensive in coverage and validated for collecting data on formal and informal caring. This study aimed to adapt and pilot the Ambulatory and Home Care Record questionnaire for use in the UK within a study evaluating a new palliative care service. The objectives were to test if family carers could be recruited and assess acceptability and usability of data gathered. Methods: Single cohort pilot study using a structured telephone questionnaire carried out every other week. Family carers of patients newly added to the palliative care register or referred to hospice services in the South East of England were invited to participate by mail. Volunteers remained in the study for a maximum of six interviews or until the patient died. Results: In total, 194 carers were invited by mail to participate in the study, of which 23 (11.8%) completed at least one interview and 16 (8.2%) completed all possible interviews. Recruitment to the study was lower than anticipated, but most participants seemed to find the interviews acceptable. The modified questionnaire produced usable and relevant data for an economic evaluation of formal and informal caring costs. Conclusions: Modifications are needed to the process of recruitment as a postal recruitment strategy did not have a high response rate. The Ambulatory and Home Care Record has proved a viable tool for use in the UK setting, with a few minor modifications, and will be used in a larger study comparing hospice models. ce utilisation, Palliative care, Questionnaire, Telephone interview, Pilot study, Informal care Keywords: Service utilisation, Palliative care, Questionnaire, Telephone interview, Pilot study, Inf * Correspondence: l.holdsworth@stanford.edu 1Primary Care and Population Health, Stanford University School of Medicine, 1265 Welch Road, MSOB, Stanford, CA 94305, USA Full list of author information is available at the end of the article Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 https://doi.org/10.1186/s40814-018-0332-2 Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 https://doi.org/10.1186/s40814-018-0332-2 Background more unpaid carer time and outpatient services for home death patients, and the resource implications of hospice and home become largely equivalent as places of death [5]. Early studies of palliative care have often not included all components of costs, particularly time and money pro- vided by family carers [6, 7]. Capture of these items is challenging, and issues arise around valuing caregiver time [8, 9]. Various approaches have been used to try and as- semble details of the formal and informal inputs in pallia- tive care settings, including record reviews, modelling, structured questionnaires and interviews with family carers before and after the death of the patient. Amongst Measuring the costs of palliative and end-of-life care is challenging but is important for the evaluation of different service delivery models [1, 2]. Studies typically report that hospital stays and residential care represent the major cost of care, depending on the cause of death [3, 4]. However, when care provided by family carers (informal care) is in- cluded in cost analyses, hospital costs are replaced by * Correspondence: l.holdsworth@stanford.edu 1Primary Care and Population Health, Stanford University School of Medicine, 1265 Welch Road, MSOB, Stanford, CA 94305, USA Full list of author information is available at the end of the article Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 Page 2 of 10 Page 2 of 10 the primary data collection methods, only one instrument, the Ambulatory and Home Care Record (AHCR), has been found comprehensive in coverage and validated [10, 11]. weekly basis. It collects information from an informal carer about the care received by “the patient”. The ques- tions are grouped around services received inside the home, outside the home (outpatient, emergency depart- ment), inpatient stays (hospital and hospice), telephone calls, medications, supplies and equipment and unpaid caregiving by family and friends (see Additional file 1). Background information about the patient and family carer is gathered in the first interview. Although the over- all structure of the AHCR remained the same, small changes were made to suit the particular features of the UK health and social care system (Table 1). p g The AHCR was first developed in Canada in 1997 to prospectively capture ambulatory and home services for economic evaluations and has been validated with cystic fibrosis patients [12]. It has subsequently been used in Canada for assessing the private costs of home care [13] and in studies of palliative care that include unpaid (in- formal) caring costs [14–16]. The AHCR involves inter- views with family carers of patients at two weekly intervals, up to the point of death, to collect information on the services that have been received. This paper re- ports the findings from a pilot study that sought to adapt and test the AHCR for gathering information on palliative care costs in the United Kingdom (UK) as cur- rently no suitable tool exists in the UK setting. Improve- ments to end-of-life care is a health policy priority in the UK which emphasises better coordination of services and enabling greater choice and control over the place of death for patients and carers [17]. A lack of informa- tion on end-of-life care costs and a need for more re- search on the resource implications of alternative models of care has been identified to help establish cost-effective service configurations and to inform ser- vice commissioning [17–23]. The service-use data that are captured provide a societal perspective on costs covering three categories: public, pri- vate and unpaid (informal) care costs [16]. Methods Design The design of the pilot was a single cohort with no com- parison group. In the UK, public costs relate to services financed by government from tax revenue (and some palliative care services pro- vided by the voluntary sector): healthcare appointments; emergency department visits; laboratory and diagnostic tests; treatments; prescription medicines, supplies, equip- ment; inpatient care (hospital, hospice, nursing home); and home care services. Prescription medicines and some supplies are exempt from charges for palliative care pa- tients. Private costs are all costs not publicly financed: out-of-pocket expenses on healthcare appointments, paid home carers, travel expenses, private insurance payments (if applicable), treatments bought privately, over-the-coun- ter medications and co-payments for means tested items. Unpaid (or informal) care costs include the time devoted by family and friends to caring (both time lost from paid employment and from leisure or household tasks). The AHCR was adapted and trialled as a pilot embed- ded in a study evaluating a new palliative care telephone navigation service offered by a major hospice provider in South East England. This was a pilot test of the AHCR’s acceptability to family carers and utility to researchers for gathering service-use data, in preparation for embarking on a large study comparing models of home palliative care [24]. The study aimed to explore whether a modified AHCR could be successfully used in the UK context for gathering information on formal and informal service use, from which costs of end-of-life care could be calculated. The specific objectives were to test: We also captured patient functional status using the Eastern Cooperative Oncology Group (ECOG) scale [25], carer burden [26] and views on service satisfaction as part of the telephone survey. These were not part of the AHCR but were warranted for other reasons; ECOG and carer burden because the scores may correlate with service use and informal caring; and service satisfaction because the provider of the new navigation service was interested in feedback. 1) Recruitment and retention of family carers to a study using the AHCR; 2) Acceptability of the adapted instrument and data collection process; and Sample and access Potential participants were identified by primary care practice managers in South East England when patients were added to their palliative care registers and by a hospice data manager when patients were referred to the hospice. Letters of invitation to participate in the re- search, an information sheet, consent form and pre-paid reply envelope were sent to carers by post by the pri- mary care practice and hospice. Where no carer was listed in the patient’s medical records, a letter was sent to the patient asking them to identify a carer who would then be sent an invitation by the researcher. Volunteers replied direct to an independent university researcher 3) Usability of the data for economic evaluation of palliative care models. Procedure Telephone surveys were carried out until: the patient died, the end of the study period, or when six surveys (approximately 10 weeks) had been completed with the carer. If the patient had survived, the maximum number of surveys was set at six to avoid overburdening carers. The usability of the data collected for the economic evaluation was assessed through descriptive analysis and inspection of data. The characteristics of participants were analysed descriptively, including ECOG performance sta- tus [25], carer burden scales [26] and service satisfaction. Survey interviews were recorded with carer permission for reference and responses were noted and transcribed into an Excel spreadsheet immediately following the interview. A narrative summary of the survey interview was added to the spreadsheet to aid analysis. Since participants were in the study for different pe- riods of time, service-use data were standardised by cal- culating the number of contacts, for each service, over a 28-day period. Unit costs obtained from validated na- tional sources [28] were applied to service use to calcu- late patient costs per 28 days and the distribution of expenditure on different categories of care. The number of hours per day reported as spent by the primary and secondary carers were converted to hours per 28 days and a replacement cost attributed based on a clinical support worker [28] (see Additional file 2). Costs were reported in British pounds using rates from 2016. Instrument adaptation Greater variability in local services in the UK; palliative care patients use the whole system of health and social care. People over 60 years and/or at a palliative stage (medical exemption) do not pay for items obtained on prescription in UK. Carer’s Allowance is a benefit available in UK. (LMH) who carried out the telephone survey. We aimed to recruit 30 carers which is considered a suitable sam- ple size for a pilot study [27]. to indicate retention of carers in the study. The reasons for withdrawal were explored, where possible. Acceptability of the instrument and the two-weekly data collection process was assessed subjectively through experiences noted by the interviewer and objectively by recruitment and retention rates. Instrument adaptation The AHCR was designed as a structured, paper-based in- strument for self-administration or interview, on a two- Page 3 of 10 Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 Table 1 Ambulatory and Home Care Record (AHCR) adaptations made for a UK population Original AHCR UK AHCR (adapted) Reason for change Home visits, health care only Home visits, health and social care Most healthcare visits are paid for by public funds; social care is usually free at end-of-life if deemed medically necessary (otherwise means tested). Length of home visits Removed Prior experience of research team indicated this would be poorly reported and because nationally validated data are available.a Payment for home visits and reimbursement received Removed Prior experience of research team indicated this would be poorly reported and unlikely to be relevant since palliative care costs are covered free of charge by the National Health Service and voluntary sector. Phone calls to palliative team only Phone calls to any health and social service, not just palliative care team Greater variability in local services in the UK; palliative care patients use the whole system of health and social care. Questions about medications and costs One generic question about whether exempt from prescription costs People over 60 years and/or at a palliative stage (medical exemption) do not pay for items obtained on prescription in UK. New question Whether carer received Carer’s Allowance Carer’s Allowance is a benefit available in UK. New question Whether services met (or fell short or exceeded expectations) was added The pilot was being conducted in the context of the roll out of a new telephone navigation service and satisfaction with services was of interest to the provider. Language and terminology Minor changes, e.g. use of A&E instead of ER To customise language and terminology to UK. aUnit Costs of Health and Social Care, Personal and Social Sciences Research Unit. http://www.pssru.ac.uk/project-pages/unit-costs/ Prior experience of research team indicated this would be poorly reported and unlikely to be relevant since palliative care costs are covered free of charge by the National Health Service and voluntary sector. Phone calls to palliative team only Questions about medications and costs New question New question Greater variability in local services in the UK; palliative care patients use the whole system of health and social care. Analysis The recruitment rate was assessed by monitoring the number of referrals from primary care or the hospice, in- vitations sent and consents received. The number of com- pleted surveys, relative to expected number (i.e. every 2 weeks between recruitment and patient death) was used Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 Page 4 of 10 Page 4 of 10 Results (107, 35.5%) were not sent a letter of invitation to the study for multiple reasons, most commonly because the hospice had not had recent contact with the patient (68), or no reason was given (27). In total, 194 carers/patients were invited to participate over the 15-month recruit- ment period and 28 (14.4%) carers provided written con- sent. Three of these carers could not be contacted and never had a first interview. Two patients had died prior to the first interview with the carer and so were ex- cluded. Twenty-three (11.9%) carers were included in the final analysis for each objective. Recruitment, retention and reasons for withdrawal , Recruitment and retention is shown in Fig. 1. Recruit- ment took place between August 2012 and February 2014 (with a break between September and December 2013 due to service changes), until sufficient data were collected to decide feasibility. Initially, six primary care practices had agreed to identify patients, but only one practice participated. Thirty-nine patients were added to the palliative care register from this practice, and 262 were newly referred to the hospice. More than one third Fig. 1 Recruitment and retention of carers Page 5 of 10 Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 Page 5 of 10 Page 5 of 10 Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 Sixteen carers (69.6%) completed the study (i.e. completed six interviews, or the patient died and the carer was interviewed after death). The carers who withdrew from the study (Table 2) either did not re- turn telephone messages after three attempts (4 carers), or notified the researcher that they wished to withdraw due to feeling overwhelmed with patient care (1 carer), or the patient had died and the carer did not wish to complete an after death interview (2 carers). Eleven (47.8%) patients died by the end of the study and seven (63.6%) of those carers com- pleted an interview following the patient’s death. Usability and relevance of data Usability and relevance of data Interviews yielded complete and usable data on the background characteristics of participants (Table 3) and service use (Table 4). Most carers lived with the patient and were spouses, a quarter were in paid em- ployment and one half were university educated. Carer burden increased closer to the death of the pa- tient. Results The average age of patients was 70 years, and most had a diagnosis of cancer. In most cases, carers reported that services met or exceeded expectations. The only services used by more than 50% of patients were primary care, district nursing and hospital out- patient consultations; a small number (30%) used home care intensely, and hospital or hospice stays were the costliest items. Reported out-of-pocket ex- penditure was relatively low (average of £6.40/month). Many respondents reported receiving supplies and equipment on loan from health, social and voluntary services (Table 4). Acceptability of the instrument and data collection procedure Survey interviews were carried out approximately every 2 weeks. Even though some carers requested to reschedule to suit their needs, the average time between surveys was 15 days. Though the AHCR questions are structured, responses frequently took on unstructured characteristics as carers volunteered unsolicited information about their experiences thus ac- counting for a wide variation in survey interview length, anywhere from 3 min to 40 min, with most taking be- tween 5 and 10 min. Several carers kept track of appoint- ments in their diaries and used these to aid with answering questions. Interviews had a conversational style to encourage participants to give details of service use as these stories often helped them to recall and clarify service utilisation. This was particularly useful as carers some- times forgot what services the patient had received. For example, a carer would say that they had not made any phone calls, but then while talking about a hospital admission, it would emerge that the carer had called urgent care and then emergency services for an am- bulance, which led to the hospital admission. During such lengthy discussions, the researcher would inter- pret and agree with the carer a structured response whilst noting their additional comments. Informal caring reported by the primary carer ranged from 1 to 24 h per day and was not significantly corre- lated with reported burden. Almost 40% of carers re- ported having a second person to assist with the caring responsibilities. Over a 28-day period, the mean (me- dian) total cost of the primary and supplementary (sec- ondary) informal carer hours, based on replacement cost method using the hourly rate of a clinical support worker, was £10,389 (£10,360). Discussion This pilot study explored potential for using the AHCR in the UK for capturing service use at end-of-life in preparation for a larger observational study to compare models of palliative care. Amongst a variety of different methods used to assess pallia- tive care service use, many of which are specific to individual projects and not comparable or compre- hensive, the AHCR has been identified as a validated framework [10]. In assessing the feasibility of using an adapted version of the AHCR as an instrument Table 2 Number of telephone survey interviews completed by carers and whether they completed all study surveys or withdrew, n = 23 Number of completed survey interviews Number of carers Carer completed study Patients alive at last survey Carer withdrew Reason withdrew 1 2 – – 2 1—did not return messages 1—requested withdrawal (too much going on) 2 3 2 – 1 Patient died, carer did not want follow-up 3 4 4 – – – 4 5 1 – 4 3—did not return messages 1—patient died, carer did not want follow-up 5 1 1 1 – – 6 8 8 8 – – Total 23 16 9 7 hone survey interviews completed by carers and whether they completed all study surveys or withdrew, n = 23 y Number of carers Carer completed study Patients alive at last survey Carer withdrew Reason withdrew Holdsworth et al. aBenefit available in the UK Discussion Pilot and Feasibility Studies (2018) 4:141 Page 6 of 10 Table 3 Characteristics of the carers and patients included in the analysis, n = 23 Carer (participant) Gender Female 16 (69.6) Age Median (range) 63 (44–81) Relationship to patient Spouse/partner 17 (73.9) Child 4 (17.4) Parent 1 (4.3) Other 1 (4.3) Lives with/staying with patient 21 (91.3) In full or part time work (vs retired) 6 (26.0) College or university educated 11 (47.8) Baseline; last recorded Carer burden during end-of-life care [26], range 0 (best)–100 (worst) Mean 34.78; 40.83 Standard deviation 25.16; 26.89 Median 29.17; 35.00 Range 0–90; 0–100 Best quartile, n % 11(47.8); 8(34.8) 2nd quartile, n % 7(30.5); 9(39.1) 3rd quartile, n % 3(13.0); 3(13.0) Worst quartile, n % 2(8.6); 3(13.0) Patient Gender Female 10 (43.5) Age Median (range) 70 (43–93) Lives alone (vs with spouse/other) 3 (13.0) College or university educated 9 (45.0) Receives attendance allowancea 11 (47.8) Diagnosis Cancer 20 (87.0) Non-cancer 3 (13.0) Place of death Hospice 4 (17.4) Hospital 4 (17.4) Home 2 (8.7) Unknown or alive at end of study 13 (56.5) Baseline; last recorded Functional status—ECOG [25] 0. Fully active 0; 0 1. Restricted in strenuous activity 1(4.3); 2(8.7) 2. Ambulatory, capable of self-care but not work 11(47.8); 5(21.7) 3. Limited self-care 7(30.4); 8(34.8) 4. Completely disabled 4(17.4); 3(13.0) 5. Dead 0; 5(21.7) Service satisfaction Baseline; last recorded Exceeds expectations 10(43.5); 10(43.5) Meets expectations 10(43.5); 12(52.2) Falls short of expectations 3(13.0); 1(4.3) aBenefit available in the UK Service satisfaction Holdsworth et al. Discussion Pilot and Feasibility Studies (2018) 4:141 Page 7 of 10 Table 4 Service use and costs reported by 23 carers, standardised to number of contacts/costs per 28 days Category Service Service use Costs (£, 2016) Number with zero % with zero Mean SD Median Range Mean cost (£) SD cost (£) Median cost (£) Cost range (£) In home GP visit 11 47.8 .705 1.16 .346 0–4.31 49.89 82.33 24.46 0 to 304.77 District nurse 10 43.5 3.61 6.44 .66 0–26.96 98.62 175.92 17.99 0 to 736.09 Hospice nurse 15 65.2 .372 .650 0 0–2.05 10.16 17.71 0 0 to 55.93 Hospice outreach 21 91.3 .110 .417 0 0–1.93 3.01 11.37 0 0 to 52.72 Home care 16 69.6 20.48 37.4 0 0–105.5 245.81 448.82 0 0 to 1266.46 Counsellor 22 95.7 .017 .080 0 0–.38 0.70 3.36 0 0 to 16.11 Physiotherapist 19 82.6 .373 1.35 0 0–6.41 8.21 29.70 0 0 to 141.01 Occupational therapist 20 87.0 .095 .302 0 0–1.37 2.09 6.64 0 0 to 30.05 Nutritionist 21 91.3 .045 .158 0 0–.69 0.99 3.47 0 0 to 15.21 Chiropodist 21 91.3 .002 .006 0 0–.03 0.07 0.25 0 0 to 1.10 Ambulance 18 78.3 .143 .344 0 0–1.47 14.16 34.05 0 0 to 145.89 Home enteral nutrition 22 95.7 .045 .216 0 0–1.04 .99 4.76 0 0 to 22.81 Out of home/hospitala GP office visit 18 78.3 .247 .543 0 0–1.75 10.88 23.87 0 0 to 77.00 Hospital clinic 7 30.4 2.699 4.474 .97 0–20 318.47 527.93 113.93 0 to 2360.00 Hospice clinic 15 65.2 .536 .809 0 0–2.90 63.27 105.04 0 0 to 341.79 Clinic (not specified) 20 87.0 .198 .643 0 0–2.95 23.33 75.88 0 0 to 347.79 Oncologist consultation 18 78.3 .220 .640 0 0–2.95 33.64 97.85 0 0 to 450.95 Hospice consultant 20 87.0 .205 .606 0 0–2.95 24.15 78.62 0 0 to 347.79 Hospice nurse 19 82.6 .258 .729 0 0–2.95 3.73 10.57 0 0 to 42.74 A&E 16 69.6 .273 .643 0 0–2.95 38.40 90.29 0 0 to 414.37 Hospital nights 14 60.9 1.323 .642 0 0–15.71 490.92 1349.31 0 0 to 5827.41 Hospice overnights 17 73.9 1.449 3.637 0 0–12.88 537.73 1275.23 0 0 to 4778.48 Medication, supplies (self-paid costs, £)b OTC, food supplement 12 52.2 n/a n/a n/a n/a 3.96 8.36 0 0 to 37.58 Continence, sheets 16 69.6 n/a n/a n/a n/a 2.41 6.90 0 0 to 32.01 Informal caring (h) Primary carer 0 0 391.2 228.7 368.7 18.7– 672.0 7824 4574 7373 373– 13,440 Secondary carer 14 60.9 122.9 104.5 71.9 0–366 2459 2090 1473 0–6720 aTests: carers reported that seven patients received tests, most frequently blood tests bMedication costs do not include prescription medications as all participants received these free (data on what medications were prescribed were not gathered). Strengths and limitations h h f h d Once recruited, the retention rate (70%) was reasonable, given the sensitive nature and timing of data collection, and provides an indirect endorsement of acceptability. Participants who were retained in the study indicated that they were comfortable with the questions asked, indicating good acceptability of the adapted AHCR. For some carers, the opportunity to talk about their experiences seemed to be therapeutic, though this required additional time to collect the data and not all commentary related to AHCR questions. Carers frequently contextualised their answers rather than just giving numerical responses. This quite often gave insight into how and why patients and carers used the services they did and also helped to enhance the completeness of the data. Though not the intention of the tool, the two-weekly telephone interview approach produced useful chronological qualitative data on the patient and carer experience of the services used. The strength of the study is in its conception as a pilot study to assess the acceptability of the AHCR method and usability of data generated in a palliative care patient group prior to a larger observational study and recognition of the need for cultural adaptation of the tool before wide use in the UK. In this regard, the study provided useful messages around recruitment and retention, and the costs and bene- fits of data gathering by telephone survey, to inform the de- sign of future studies in this area. This study achieved a smaller sample size than was planned (23 analysed cases vs 30 planned). However, the study yielded sufficient informa- tion to improve the overall process of using the AHCR for the larger, follow-up study. We did not check the accuracy of self-reported service use with records kept by the health system, and for some carers, we perceived that they gave in- complete responses to more complex events, like hospital admission. However, this seemed to be related to the structure of the questions rather than carer recall, as add- itional probing by an experienced researcher usually pro- vided the required information. We believe that having a qualitatively trained researcher with experience of the pal- liative care setting administering the questionnaire im- proved the quality of the data because she could ask appropriate clarifying questions. Discussion No cost was provided for many of the supplies that were reported because they were provided free of charge, e.g. mattresses, hospital bed, speech and language supplies, cushions. One person reported purchasing a disabled car for £4000—including this in the calculation raises the mean (SD) to £86.26 (402.2) OTC over the counter GP general practitioner Tests: carers reported that seven patients received tests, most frequently blood tests bMedication costs do not include prescription medications as all participants received these free (data on what medications were prescribed were not gathered). No cost was provided for many of the supplies that were reported because they were provided free of charge, e.g. mattresses, hospital bed, speech and language supplies, cushions. One person reported purchasing a disabled car for £4000—including this in the calculation raises the mean (SD) to £86.26 (402.2) OTC over the counter and another similar study in the UK (28.5%) [30]. Only one of the six primary care practices that agreed to recruit for the study sent out invitation letters to carers. This low follow-through rate calls into ques- tion the suitability of primary care practices as a set- ting for recruiting participants for palliative care studies. It should be noted that this project took for collecting formal and informal care costs at the end-of-life, we considered measures of recruitment and retention, acceptability and usability of the data. for collecting formal and informal care costs at the end-of-life, we considered measures of recruitment and retention, acceptability and usability of the data. Recruitment of family carers to this study was prob- lematic and we had a lower enrolment rate (11.9%) than has been reported in previous studies carried out in Canada which used the AHCR (41.8%, 70.4%) [14, 29] Recruitment of family carers to this study was prob- lematic and we had a lower enrolment rate (11.9%) than has been reported in previous studies carried out in Canada which used the AHCR (41.8%, 70.4%) [14, 29] Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 Page 8 of 10 place around the same time as the implementation of centrally imposed changes to primary care (Health and Social Care Act 2012) which may have absorbed primary care practice attention and resources. The one primary care practice that did participate had a research nurse funded by the National Institute of Health Research Clinical Research Network. Strengths and limitations h h f h d Alternatively, we could have tested the AHCR against a self-report diary to see which would provide greater recruitment, retention and completeness of data balanced against the research cost of implementing the method. The AHCR proved successful for data collection and yielded complete and usable data on most items. Missing information was minimal due to the survey interview format and the researcher having a good understanding of how ser- vices operate and investigating any discrepancies in carer ac- counts. As might be expected, informal caring costs, using the replacement cost method, were very high confirming the importance of including this element in evaluative stud- ies and the saving that informal carers afford the health care system. Debate exists, however, about applicability of using replacement methods; if carers gain utility from caring, the costs associated with this should be lower [8, 9]. Discussion The hos- pice had dedicated a staff member to identify patients as part of the parent project which was to evaluate its new telephone navigation service. Such a low re- cruitment rate raises concerns about research costs and study delays and creates doubts about the representative- ness of the samples, suggesting alternative approaches may need to be considered. Lessons may also be drawn from Canadian studies in which palliative care team staff recruited participants direct from their caseload by tele- phone, rather than by mail as was done here [14, 29]. Recruitment might therefore be more successful with a direct contact from the provider who should have ring-fenced resources to support the recruit- ment process. them to recall-selected, high cost or commonly used ser- vices. Whereas visits inside and outside the home were more easily recalled, phone calls were most difficult for carers to recall and so were not included in analysis due to lack of confidence in the data. We did not ask carers to report the number of prescription medications used as they did not pay out-of-pocket towards them but would choose to do so in the future study for under- standing the full cost to the system. Issues with recall may be reduced by distributing an aide memoire to carers to remind them of questions of interest and as a place to record service use, particularly as the carers who used a diary seemed to have an easier time report- ing their service use at the two-weekly survey interview. Additionally, we would propose changes to the AHCR tool to create a better structure for recording data re- garding equipment and supplies on loan as these were recorded as free text but could be structured to make for easier analysis following data collection. Conclusion Many service use items were not widely used, such as counselling and home enteral nutrition, so it might be possible to reduce the burden on carers by only asking The study confirms that the AHCR is a feasible instru- ment for collecting data on palliative care service use Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 Page 9 of 10 Page 9 of 10 Received: 19 April 2018 Accepted: 7 August 2018 Funding 11. Gardiner C, Ingleton C, Ryan T, et al. What cost components are relevant for economic evaluations of palliative care, and what approaches are used to measure these costs? A systematic review. Palliat Med. 2017;31(4):323–37. g This work was supported by Pilgrims Hospices in East Kent. References 1. May P, Morrison RS, Murtagh EM. Current state of the economics of palliative and end of life care: a clinical view. Palliat Med. 2017;31(4):293–5. 1. May P, Morrison RS, Murtagh EM. Current state of the economics of palliative and end of life care: a clinical view. Palliat Med. 2017;31(4):293–5. 2. Smith S, Brick A, O’Hara S, et al. Evidence on the cost and cost-effectiveness of palliative care: a literature review. Palliat Med. 2014;28(2):130–50. 3. Guest JF, Ruiz FJ, Greener MJ, et al. Palliative care treatment patterns and associated costs of healthcare resource use for specific advanced cancer patients in the UK. Eur J Cancer Care. 2006;15(1):65–73. 4. Van der Plas AG, Oosterveld-Vlug MG, Pasman HR, et al. Relating cause of death with place of care and health care costs in the last year of life for patients who died from cancer, chronic obstructive pulmonary disease, heart failure and dementia. A descriptive study using registry data. Palliat Med. 2017;31(4):338–45. 5. Yu M, Guerriere DN, Coyte PC. Societal costs of home and hospital end of life care in palliative care patients in Ontario, Canada. Health Soc Care Community. 2015;23(6):605–18. Received: 19 April 2018 Accepted: 7 August 2018 and informal caring costs, though refinement of the re- cruitment process is needed to make it practical for re- search purposes. An advantage is that data are gathered regularly thereby reducing the risk of recall errors rela- tive to approaches that contact the carer several months after the patient has died. This improvement in data quality needs to be balanced against the costs of multiple telephone surveys. The focus of the AHCR is on gather- ing service-use data and understanding the costs of pal- liative care from a system and societal perspective. Such data have historically been difficult to obtain but are im- portant elements for robust service delivery evaluation. Consent for publication Not applicable. 18. National Audit Office. End of life care. HC 1043. London: TSO, 2008. 19. Addicott R, Dewar S. Improving choice at end of life: a descriptive analysis of the impact and costs of the Marie Curie ‘Delivering Choice’ programme for Lincolnshire. London: King’s Fund; 2008. Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. 20. Hughes-Hallett T, Craft A, Davies C, et al. Funding the right care and support for everyone: creating a fair and transparent funding system. Report of the Palliative Care Funding Review, 2011. Abbreviations 8. Van den Berg B, Brouwer WB, Koopmanschap MA. Economic valuation of informal care. An overview of methods and applications. Eur J Health Econ. 2004;5(1):36–45. AHCR: Ambulatory and Home Care Record; GP: General practitioner Acknowledgements 9. Brouwer WB, van Exel NJ, van den Berg B, van den Bos GA, Koopmanschap MA. Process utility from providing informal care: the benefit of caring. Health Policy. 2005;74(1):85–99. We thank Hilary Pinnock, Helen Sansom, Helena Neal and all of the carers who participated in the study. Lawrence Matini assisted with data organisation. We also thank Denise Guerriere for permission to publish our adapted version of the Ambulatory and Home Care Record. 10. Gardiner C, Bereton L, Frey RA, et al. Approaches to capturing the financial costs of family care-giving within a palliative care context: a systematic review. Health Soc Care Community. 2016;24(5):519–31. Availability of data and materials 12. Guerriere DM, Unger WJ, Corey M, et al. Evaluation of the ambulatory and home care record: agreement between self-reports and administrative data. Int J Tech Assess Health Care. 2006;22(2):203–10. 12. Guerriere DM, Unger WJ, Corey M, et al. Evaluation of the ambulatory and home care record: agreement between self-reports and administrative data. Int J Tech Assess Health Care. 2006;22(2):203–10. The datasets generated and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request. 13. Guerriere DM, Wong AYM, Croxford R, Leong UW, McKeever P, Coyte P. Costs and determinants of privately financed home-based health care in Ontario. Canada. Health Soc Care Community. 2008;16(2):126–36. 14. Guerriere DN, Zagorski B, Fassbender K, et al. Cost variations in ambulatory and home-based palliative care. Palliat Med. 2010;24(5):523–32. 13. Guerriere DM, Wong AYM, Croxford R, Leong UW, McKeever P, Coyte P. Costs and determinants of privately financed home-based health care in Ontario. Canada. Health Soc Care Community. 2008;16(2):126–36. Authors’ contributions LMH and HG conceived the work and design and drafted the manuscript. LMH, HG and CB modified the AHCR. LMH collected the data. HG and PW conducted the analysis. All authors revised and approved the final manuscript. y 14. Guerriere DN, Zagorski B, Fassbender K, et al. Cost variations in ambulatory and home-based palliative care. Palliat Med. 2010;24(5):523–32. 14. Guerriere DN, Zagorski B, Fassbender K, et al. Cost variations in ambulatory and home-based palliative care. Palliat Med. 2010;24(5):523–32. 15. Chai H, Guerriere DN, Zagorski B, et al. The size, share, and predictors of publically financed healthcare costs in the home setting over the palliative care trajectory: a prospective study. J Palliat Care. 2013;29(3):154–62. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 21. NHS National End of Life Care Programme. Reviewing end of life care costing information to inform the QIPP End of Life Care Workstream. Report. Leicester: NHS National End of Life Care Programme, 2012. Additional files Additional file 1: UK adapted version of the Ambulatory and Home Care Record (AHCR). (DOCX 48 kb) 6. Guerriere DN, Coyte PC. The ambulatory and home care record: a methodological framework for economic analyses in end-of-life care. J Aging Res. 2011;2011:11. Article ID 374237. https://doi.org/10.4061/2011/374237. Additional file 2: Unit costs used in the costing analysis. (DOCX 14 kb) 7. Gardiner C, Bereton L, Frey RA, Wilkinson-Meyers L, Gott M. Exploring the financial impact of caring for family members receiving palliative and end of life care, a systematic review of the literature. Palliat Med. 2014;28(5):375–90. Ethics approval and consent to participate 16. Chai H, Guerriere DN, Zagorski B, et al. The magnitude, share and determinants of unpaid care costs for home-based palliative care service provision in Toronto, Canada. Health Soc Care Community. 2014;22(1):30–9. The study received NHS ethics approval from the NRES Committee South East Coast—Kent on 13 November 2012, reference 12/LO/1311. The study received NHS ethics approval from the NRES Committee South East Coast—Kent on 13 November 2012, reference 12/LO/1311. 17. Department of Health. End of life care strategy: promoting high quality care for all adults at the end of life. London: Department of Health; 2008. 26. Dumont S, Fillion L, Gagnon P, et al. A new tool to assess family caregivers’ burden during end-of-life care. J Palliat Care. 2008;24(3):151–61. 27. Lancaster GA, Dodd S, Williamson PR. Design and analysis of pilot studies: recommendations for good practice. J Eval Clin Pract. 2004;10(2):307–12. 28. Curtis L, Burns A. Unit costs of health and social care 2016. Report. Canterbury: Personal and Social Services Research Unit; 2016. https://www. pssru.ac.uk/pub/uc/uc2016/full.pdf?uc=2016-full. 29. Leong VW, Guerriere DN, Croxford R, et al. The magnitude, share and determinants of private costs incurred by clients (and their caregivers) of in-home publicly financed care. Health Policy. 2007;3(1):e141–59. 30. Rowland C, Hanratty B, Pilling M, van den Berg B, Grande G. The contributions of family care-givers at end of life: a national post-bereavement census survey of cancer carers’ hours of care and expenditures. Palliat Med. 2017;31(4):346–55. Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 29. Leong VW, Guerriere DN, Croxford R, et al. The magnitude, share and determinants of private costs incurred by clients (and their caregivers) of in-home publicly financed care. Health Policy. 2007;3(1):e141–59. 30. Rowland C, Hanratty B, Pilling M, van den Berg B, Grande G. The contributions of family care-givers at end of life: a national post-bereavement census survey of cancer carers’ hours of care and expenditures. Palliat Med. 2017;31(4):346–55. 26. Dumont S, Fillion L, Gagnon P, et al. A new tool to assess family caregivers’ burden during end-of-life care. J Palliat Care. 2008;24(3):151–61. 27. Lancaster GA, Dodd S, Williamson PR. Design and analysis of pilot studies: recommendations for good practice. J Eval Clin Pract. 2004;10(2):307–12. Author details 1 22. Georghiou T, Davies S, Davies A, et al. Understanding patterns of health and social care use at the end of life. Report. London: Nuffield Trust, 2012. 1Primary Care and Population Health, Stanford University School of Medicine, 1265 Welch Road, MSOB, Stanford, CA 94305, USA. 2Surrey Health Economics Centre, Department of Clinical and Experimental Medicine, University of Surrey, Guildford GU2 7XH, UK. 3Department of Mathematics, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford GU2 7XH, UK. 4Centre for Health Services Studies, University of Kent, Canterbury CT2 2NF, UK. 23. Georghiou T, Bardsley M. Exploring the cost of care at the end of life. Report. London: Nuffield Trust, 2014. 23. Georghiou T, Bardsley M. Exploring the cost of care at the end of life. Report. London: Nuffield Trust, 2014. 24. Thabane L, Ma J, Chu R, et al. A tutorial on pilot studies: the what, why and how. BMC Med Res Methodol. 2010;10:1. 24. Thabane L, Ma J, Chu R, et al. A tutorial on pilot studies: the what, why and how. BMC Med Res Methodol. 2010;10:1. 25. Oken MM, Creech RH, Tormey DC, et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982;5:649–55. Page 10 of 10 Holdsworth et al. Pilot and Feasibility Studies (2018) 4:141 28. Curtis L, Burns A. Unit costs of health and social care 2016. Report. Canterbury: Personal and Social Services Research Unit; 2016. https://www. pssru.ac.uk/pub/uc/uc2016/full.pdf?uc=2016-full.
https://openalex.org/W2031296888	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0113418&type=printable	English	null	Influence of Nonenzymatic Posttranslational Modifications on Constitution, Oligomerization and Receptor Binding of S100A12	PloS one	2,014	cc-by	10,541	Influence of Nonenzymatic Posttranslational Modifications on Constitution, Oligomerization and Receptor Binding of S100A12 Kerstin Augner1, Jutta Eichler2, Wolfgang Utz1, Monika Pischetsrieder1* 1. Department of Chemistry and Pharmacy, Food Chemistry Division, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg (FAU), Erlangen, Germany, 2. Department of Chemistry and Pharmacy, Medicinal Chemistry Division, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg (FAU), Erlangen, Germany monika.pischetsrieder@fau.de OPEN ACCESS Citation: Augner K, Eichler J, Utz W, Pischetsrieder M (2014) Influence of Nonenzymatic Posttranslational Modifications on Constitution, Oligomerization and Receptor Binding of S100A12. PLoS ONE 9(11): e113418. doi:10.1371/ journal.pone.0113418 Editor: Barry I. Hudson, University of Miami, United States of America Received: May 9, 2014 Accepted: October 28, 2014 Published: November 26, 2014 Copyright: 2014 Augner et al. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and repro- duction in any medium, provided the original author and source are credited. Editor: Barry I. Hudson, University of Miami, United States of America Received: May 9, 2014 Accepted: October 28, 2014 Published: November 26, 2014 Copyright: 2014 Augner et al. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and repro- duction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Abstract This study examined the effect of methylglyoxal (MGO)-derived nonenzymatic posttranslational modifications (nePTMs) on the binding affinity of S100A12 to its natural receptor for advanced glycation end-products (RAGE). Binding of MGO- modified S100A12 to RAGE decreased significantly with increasing MGO concentration and incubation time. Ca2+-induced S100A12 hexamerization was impaired only at higher MGO concentrations indicating that the loss of affinity is not predominantly caused by disturbance of ligand oligomerization. nePTM mapping showed carboxyethylation of lysine (CEL) and the N-terminus without preferential modification sites. Besides, hydroimidazolone, hemiaminals, argpyrimidine, and tetrahydropyrimidine rapidly formed at R21. Even at the highest modification rate, hexamerization of synthesized CEL-S100A12 was unaffected and RAGE-binding only slightly impaired. Thus, nePTMs at R21 seem to be the major cause of MGO- induced impairment of S100A12 oligomerization and RAGE binding. RESEARCH ARTICLE Influence of Nonenzymatic PTMs on S100A12-RAGE Binding carbonyl compounds such as glyoxal, methylglyoxal (MGO), or 3-deoxyglucosone [1, 2]. Increased levels of glycation products, AGEs and/or oxidation products have been associated with various diseases such as diabetes [3], uremia [4], or chronic liver disease [5]. nePTMs severely alter the proteins’ primary structure by diverse covalent modifications of amino acid side chains. Consequently, modifications of the protein structure may lead to a conformational change resulting in a change of biological function. Alternatively, nePTMs may be located directly in protein– protein-, enzyme–substrate-, or protein–DNA interaction sites. Thus it was shown, for example, that the glycation of aldehyde reductase impaired enzymatic activity, but it is not clear which glycation products and which sites were responsible for this loss of activity [6]. The reaction of human serum albumin with the AGE-precursor MGO led to a loss of esterase activity. Because R410, which was predominantly modified, is located in the active site, it was concluded that the AGE formation directly disrupted the enzyme–substrate interaction [7]. Finally, intermolecular cross-linking of proteins may drastically change protein properties and impair their biological functionality. Cross-linking of collagen, for example, increases the stiffness of the collagen network, thus decreasing the resistance of cartilage to fatigue [8]. The present study investigated the influence of nePTMs on the ligand–receptor interaction of the protein S100A12 and the receptor for advanced glycation end- products (RAGE). RAGE belongs to the immunoglobulin superfamily of cell surface receptors. Several endogenous ligands of RAGE have been described, such as amphoterin (HMGB1) or S100 proteins. RAGE is expressed at relatively low levels in most cell types and tissues, but its expression is upregulated at sites where its ligands accumulate. Because most of these ligands are related to various diseases, the engagement of RAGE triggers diverse signaling cascades and activates transcription factor nuclear factor-kB (NF-kB) leading to pro-inflammatory reactions. Thus, RAGE is associated with pathologies like chronic inflammation, atherosclerosis, neurodegenerative diseases, diabetic complications, and cancer [9]. The natural RAGE ligand S100A12 is involved in the inflammatory response by triggering cell activation and generation of pro-inflammatory mediators via RAGE [10]. S100A12 is a member of the S100 protein family, a family of small (10–12 kDa), acidic, Ca2+-binding EF hand proteins [11]. Like most S100 proteins, S100A12 forms homodimers, but hexamerizes at elevated Ca2+- concentration by the association of three dimers [12]. PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Introduction Nonenzymatic posttranslational modifications (nePTMs) of proteins are formed by spontaneous chemical reactions of amino acid side chains by diverse mechanisms such as oxidation or glycation. Protein glycation results from the reaction between reducing sugars and amino groups of proteins. Advanced glycation end-products (AGEs) are formed either by degradation of primary glycation products or by the reaction of amino acid side chains with reactive Funding: The ‘‘Deutsche Forschungsgemeinschaft’’ (DFG) contributed to the UHPLC-MS/MS unit used for experiments. Otherwise, the authors received no specific funding for this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 1 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding expressed at inflammation sites and nePTM formation is promoted under inflammatory conditions [17, 18]. Although the biological half-life of peptide ligands is relatively short, CML-modified S100A8 and S100A8/9 have been detected in inflamed gut tissue [19]. Consequently, it was postulated that CML modifications may enhance S100-induced inflammatory reactions. However, CML is not the most prevalent nePTM in vivo. In diabetic patients, for example, MGO-derived hydroimidazolone was detected in 20–40 times higher concentra- tions than CML. MGO reacts rapidly with amino acid side chains so that structural and functional effects can already be observed after a few hours of reaction time [2]. Thus, MGO may also severely modify protein ligands with a relatively short half-life. Because it is well established that nePTMs impair diverse protein functions, it can be expected that MGO-derived nePTMs of S100A12 may interfere with its RAGE-binding. The aim of the present study was to test how MGO-derived nePTMs modulate RAGE binding. Furthermore, we were interested in the chemical modifications and molecular mechanisms that are responsible for the observed inhibition of receptor binding. Therefore, nePTM mapping was performed to identify the major molecular structures and binding sites of the predominant nePTMs. Analysis of the oligomerization behavior and defined synthesis and analysis of CEL-modifications of S100A12 led to a molecular model how MGO-derived nePTMs may reduce binding of S100A12 to RAGE. The hexamer is the active form of S100A12, which binds to RAGE dependent on Ca2+- and Zn2+-ions [13]. It is well established that RAGE signaling is strongly modulated by the presence of nePTMs [14]. The introduction of AGEs, such as Ne-carboxymethyllysine (CML) or Ne-carboxyethyllysine (CEL), triggers the binding of non-natural ligands, e.g. human serum albumin, to RAGE [15]. It was proposed that AGE formation of non-ligands generates novel binding sites and that, for example, the negatively charged CEL fits inside a positively charged cavity of the V-domain of RAGE [16]. Apart from that, it can be expected that nePTM formation may also impair binding of RAGE to its endogenous ligands, such as S100A12. S100A12 is PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 2 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Construction of the expression plasmid The cDNA for S100A12 was cloned into the expression vector pTXB1 using the IMPACT Kit. The expression of a fusion protein containing the target protein, an intein and a chitin-binding domain allowed the purification in a single step including the cleavage of the target protein from the intein [20]. I.M.A.G.E cDNA clone [21] containing the coding sequence for S100A12 (I.M.A.G.E cloneID 30415232) was obtained from imaGenes (Berlin, Germany). S100A12 was amplified by polymerase chain reaction (PCR) using the following oligonucleo- tides (Eurofins MWG, Ebersberg, Germany): 59-GGTGGTCATATGACAAAAC- TTGAAGAG-39 and 59-GGTGGTACTAGTGCATCTCCCGTGATGCACTCTTT- GTGGGTGTGG-39. The restriction-digested PCR-product was ligated into the NdeI and SpeI site of pTXB1. The recombinant clones were confirmed by DNA sequencing. The resulting plasmid was transformed into Escherichia coli strain One Shot BL21 (DE3; Invitrogen, Darmstadt, Germany). Influence of Nonenzymatic PTMs on S100A12-RAGE Binding Chemicals IMPACT Kit, restriction enzymes, Crimson Taq DNA polymerase and T4 DNA ligase were purchased from New England Biolabs (Frankfurt am Main, Germany). The 2xYT-medium was obtained from Carl Roth (Karlsruhe, Germany). HBS- P+10x Buffer was supplied by GE Healthcare (Freiburg, Germany) and recombinant human RAGE Fc chimera from a mouse myeloma cell line by R&D Systems (Wiesbaden, Germany). Calibration standards for size exclusion chromatography, MGO (40% aqueous solution), sodium pyruvate, and liquid chromatography-mass spectrometry (LC-MS)-grade acetonitrile were purchased from Sigma-Aldrich (Taufkirchen, Germany). GluC and chymotrypsin were supplied by Roche (Mannheim, Germany). 2,5-Dihydroxyacetophenone (DHAP) and protein and peptide calibration standards for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS were obtained from Bruker Daltonik (Bremen, Germany). All other chemicals were, unless otherwise noted, purchased from Sigma-Aldrich or Acros (Geel, Belgium) and were at least of analytical grade. H2O was purified water from a Synergi-185 laboratory water system (Millipore, Schwalbach, Germany). 3 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Modification of S100A12 with MGO Aliquots of 900 mL each of a S100A12 solution (2.6 mg mL21 in 200 mM phosphate buffer, pH 7.4 with 0.04% sodium azide) were mixed with 100 mL each of 40 mM, 400 mM, 4 mM, and 40 mM MGO or 100 mL of buffer, respectively. The resulting solutions (final MGO concentrations were 0 mM, 4 mM, 40 mM, 400 mM, and 4 mM) were filter-sterilized (sterile syringe filter, 0.22 mm, PVDF, Carl Roth, Karlsruhe, Germany) and aliquots of 200 mL were incubated at 37 ˚C in a dry block shaker (350 rpm, Eppendorf, Hamburg, Germany) for 1, 3, and 7 days respectively. An unincubated sample without MGO served as negative control. Samples were stored at –20 ˚C until ultrafiltration. CEL-modification of S100A12 CEL-modified S100A12 was synthesized according to Koito et al. [23], with modifications. S100A12 was incubated with various concentrations of pyruvate and sodium cyanoborohydride to provide samples with different extents of modification. Briefly, 280 mL of a S100A12 solution (3.5 mg mL21 in 200 mM phosphate buffer, pH 7.8) was mixed with 35 mL of NaBH3CN solution (0.17 mM, 0.33 mM, 1.67 mM, 8.35 mM, and 16.70 mM) and 35 mL of pyruvate solution (3 mM, 6 mM, 30 mM, 150 mM, and 300 mM) or 70 mL of buffer, respectively, and filter-sterilized. Aliquots of 200 mL were incubated at 25 ˚C and 350 rpm for 16 h. An unincubated sample without pyruvate and NaBH3CN served as negative control. Samples were stored at –20 ˚C until ultrafiltration. Influence of Nonenzymatic PTMs on S100A12-RAGE Binding residue, however, did not impair receptor binding, oligomerization, or MS analysis. The present study investigated the influence of MGO-derived modifications on oligomerization and RAGE binding. Therefore, the functionality of the recombinant S100A12 was verified by size-exclusion chromatography and surface plasmon resonance spectroscopy (SPR; see below). Purification of recombinant S100A12 Escherichia coli cells were grown in 500 mL of 2xYT-medium containing 100 mg mL21 of ampicillin at 37 ˚C and 150 rpm until the optical density (OD)600 reached 0.4. Protein expression was induced by adding isopropyl-b-D-thioga- lactopyranoside (IPTG) to a final concentration of 400 mM. After incubation at 37 ˚C for 4 h, cells were harvested by centrifugation (4000 rpm, 4˚C, 20 min). The cell pellet was re-suspended in 50 mL of ice-cold column buffer (20 mM Tris- HCl, 500 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.5) and lysed with a BeadBeater (Biospec, Bartlesville, OK). After centrifugation (11000 rpm, 4 ˚C, 30 min), the supernatant was loaded onto a chitin column containing 5 mL of chitin beads equilibrated with column buffer. The column was washed with 100 mL of column buffer and on-column cleavage was induced by flushing with 15 mL of cleavage buffer (column buffer containing 50 mM dithiothreitol (DTT)). Then the column flow was stopped and the column was incubated for 18 h at room temperature. S100A12 was eluted with 10 mL of column buffer. The eluate was dialyzed three times for 2 h against H2O, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to confirm protein mass and purity and freeze-dried. S100A12 was dissolved in H2O and loaded onto an Illustra NAP-5 column (GE Healthcare, Solingen, Germany) equilibrated with H2O. After elution with H2O, S100A12 was freeze-dried. The amino acid sequence of S100A12 was confirmed with sequence coverage of 100% by MALDI-TOF-MS after digestion with GluC. The circular dichroism (CD) spectra of the protein showed proper folding of S100A12, the spectra being in accordance with literature (data not shown) [22]. Protein concentrations were determined with the Bio-Rad Protein Assay (Munich, Germany). During cleavage of the target protein from the intein, one methionine residue preceeding the N-terminus was only partially cleaved, so that the signal for M1S100A12 was predominant in the S100A12 mass spectra. The N-terminal M PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 4 / 21 Removal of excess glycating reagent Amicon Ultra-0.5 mL centrifugal filters were washed by centrifugation (14000 rpm, 30 min, 20 ˚C) of 200 mL of H2O. Samples were transferred to the filter, centrifuged and washed four times with 200 mL of water. The filter device was placed upside down in a new micro centrifuge tube and the protein solution was recovered by centrifugation (1000 rpm, 2 min, and 20 ˚C). The filter was washed once with 50 mL of H2O, which was combined with the recovered protein solution. Then the samples were freeze-dried and reconstituted with 200 mL of H2O. Determination of the oligomerization behavior with size-exclusion chromatography Aliquots of 30 mL each of the samples were mixed with 30 mL of H2O and 15 mL of buffer (50 mM Tris-HCl, 750 mM NaCl, 2.5 mM CaCl2, 50 mM ZnCl2, pH 7.4) and centrifuged at 20 ˚C and 100006g for 10 min. Fifty microliters of the supernatant was injected into the HPLC system for analysis. The Jasco HPLC system (series 1580, Jasco Deutschland, Groß-Umstadt, Germany) consisted of a PU-1580 pump, a DG-1580-53 degasser, a LG-1580-02 ternary gradient unit, an AS-1555 autosampler and a MD-1510 diode array detector. A Superdex 75 10/300 GL column was used with 10 mM Tris-HCl, 150 mM NaCl, 500 mM CaCl2, 10 mM ZnCl2, pH 7.4 as running buffer and a flow rate of 0.6 mL min21. The column was calibrated with bovine serum albumine, carbonic anhydrase, cytochrom C, and aprotinin for each test series. The chromatographic data were collected and processed using Borwin software. Influence of Nonenzymatic PTMs on S100A12-RAGE Binding measurements were performed on a Biacore X100 instrument (Biacore AB, GE Healthcare, Chalfont St. Giles, UK) at 25˚C at a flow rate of 30 mL min21. A quantity of 7200 response units (RU) of recombinant human RAGE Fc chimera was coupled to the measuring flow cell of a CM5 chip using amine coupling with N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide accord- ing to the manufacturer’s instructions. Binding analysis was performed with HBS- P buffer (10 mM HEPES, 150 mM NaCl, 0.05% surfactant 20, pH 7.4) containing 500 mM CaCl2 and 10 mM ZnCl2 as running buffer. Samples were injected with a 180 s association and a 180 s dissociation phase to both flow cells. The response of the reference cell was subtracted from the response of the measuring cell to compensate for non-specific binding of S100A12 to the sensor chip. The sensor chip was regenerated with a 60 s pulse of 10 mM glycine, pH 2.0. Investigation of the binding of S100A12 to RAGE by SPR Twenty microliters of the samples was mixed with 50 mL of H2O, 10 mL of 5 mM CaCl2, 10 mL of 100 mM ZnCl2, and 10 mL of HBS-P+10x buffer. SPR Investigation of the binding of S100A12 to RAGE by SPR Twenty microliters of the samples was mixed with 50 mL of H2O, 10 mL of 5 mM CaCl2, 10 mL of 100 mM ZnCl2, and 10 mL of HBS-P+10x buffer. SPR PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 5 / 21 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding MALDI-TOF-MS analysis was carried out on a Bruker Autoflex system (Bruker Daltonik, Bremen, Germany) equipped with a nitrogen laser (l5337 nm). Measurements of intact and digested proteins were performed using delayed extraction (330 ns and 150 ns, respectively). Laser-desorbed positive ions were analyzed after acceleration by 20 kV in the linear mode for the intact proteins and by 19 kV in the reflector mode for the partial hydrolyzed protein. External calibration was conducted with a mix of insulin, ubiquitin I, cytochrome C, and myoglobin for the intact proteins and with a mix of bradykinin 1–7, angiotensin I and II, substance P, bombesin, renin substrate, ACTH clip 1–17 and 18–39, and somastostatin 28 for the peptides. For each spectrum, 150 laser shots were averaged from several positions on a spot. Data evaluation was performed with mMass [25]. Analysis of nePTMs of S100A12 by UHPLC-ESI-MS/MS Analysis of nePTMs of S100A12 by UHPLC ESI MS/MS MGO-modified S100A12 was analyzed after digestion with GluC or chymotrypsin, while CEL-modified S100A12 was digested only with GluC. Aliquots of 12 mL of the samples were mixed with 3.3 mL of 125 mM ammonium bicarbonate buffer, pH 8.0 and 1.2 mL of GluC (0.5 mg mL21) or chymotrypsin (0.5 mg mL21), respectively. For digestion with GluC, the solution was incubated at 37 ˚C and 350 rpm for 16 h. For digestion with chymotrypsin, the solution was incubated at 25 ˚C and 350 rpm for 16 h. After addition of 63.5 mL of H2O, 10 mL of acetonitrile, and 10 mL of 5% formic acid, the samples were filtered and aliquots of 10 mL each were injected into the ultrahigh-performance liquid chromatography (UHPLC)-electrospray ioniza- tion (ESI)-MS/MS system. An Ultimate 3000 RS UHPLC (degasser, binary pump, autosampler, column oven; Dionex, Germering, Germany) was coupled to an API 4000 QTRAP mass spectrometer equipped with an ESI-source (AB Sciex, Foster City, CA). Analyst 1.5.1 with BioAnalyst extensions was applied for instrument control as well as data acquisition and processing. An ACQUITY UPLC BEH300 C18 column (100 mm62.1 mm, 1.7 mm particle size; Waters, Eschborn, Germany) was used at 30 ˚C with the following gradient: A, formic acid (0.1%); B, acetonitrile; flow rate, 0.3 mL min21; (time [min]/% B) 25.0/5, 0.0/5, 5.0/5, 55.0/50, 55.5/95, 60.0/95. All flow eluting before 2.0 or after 40.0 min was discarded by a two- position valve prior to mass analysis. The ESI-source was operated at 500 ˚C with a voltage of +5500 V and nitrogen as drying gas. Time periods were chosen to perform different product ion scans during one chromatographic run. Declustering potential, collision energy and scanned mass range were optimized for each peptide. MGO-modified S100A12 was analyzed after digestion with GluC or chymotrypsin, while CEL-modified S100A12 was digested only with GluC. Aliquots of 12 mL of the samples were mixed with 3.3 mL of 125 mM ammonium bicarbonate buffer, pH 8.0 and 1.2 mL of GluC (0.5 mg mL21) or chymotrypsin (0.5 mg mL21), respectively. For digestion with GluC, the solution was incubated at 37 ˚C and 350 rpm for 16 h. For digestion with chymotrypsin, the solution was incubated at 25 ˚C and 350 rpm for 16 h. After addition of 63.5 mL of H2O, 10 mL of acetonitrile, and 10 mL of 5% Analysis of nePTMs of S100A12 by MALDI-TOF-MS For analysis of the intact protein, 10 mL of the samples was purified with ZipTip pipette tips (Millipore, Schwalbach, Germany) as described by Meltretter et al. [24]. Three microliters of the purified protein solution was mixed with 3 mL of 2% trifluoro acetic acid and 3 mL of DHAP matrix. An aliquot of 1 mL was spotted onto a ground steel target and air-dried. For the DHAP matrix, 7.6 mg of DHAP were suspended in 375 mL of ethanol and 125 mL of diammonium hydrogen citrate (80 mM) was added to dissolve the DHAP. Peptides were analyzed after digestion with GluC. Therefore, 10 mL of the samples was mixed with 2.75 mL of 125 mM ammonium bicarbonate buffer, pH 8.0 and 1 mL of GluC (0.5 mg mL21). Subsequently the mixture was incubated at 25 ˚C and 350 rpm for 16 h. A quantity of 3 mL of the digested protein was mixed with 3 mL of 2% trifluoro acetic acid and 3 mL of DHAP matrix; an aliquot of 1 mL was spotted onto a ground steel target and air-dried. PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 6 / 21 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding different periods of time (1, 3, and 7 days) and binding of S100A12 to RAGE was analyzed by SPR spectroscopy. The physiologic running buffer (HBS-P) was supplemented with 500 mM Ca2+ and 10 mM Zn2+, because S100A12 interacts with RAGE dependent on these metal ions [13]. The applied concentrations of Ca2+ and Zn2+ were in the range of their serum levels. The recombinant RAGE was derived from a mouse myeloma cell line. The cell source may have major influence on the functionality of RAGE. Recently, it was shown that soluble RAGE (sRAGE) from mammalian cells exerts higher bioactivity than sRAGE from insect cells or bacterial cells [26]. Most likely, carboxylated N-glycans on sRAGE, which are formed only in mammalian cells, promote binding to S100 proteins [27]. (Figure 1) shows the results of the binding analysis with SPR. The maximum response was observed for the unincubated negative control with a value of 3047 RU. The negative control incubated without MGO showed a slight decrease in binding to 80.9¡5.1% (mean ¡ SD) after seven days compared to the unincubated control indicating that the incubation itself had an influence on the binding of S100A12 to RAGE. For this reason, samples were compared to the negative control incubated for the same period of time, but without MGO. The samples incubated with 4 mM and 40 mM MGO exhibited a similar binding behavior as the negative control. The incubation with 400 mM MGO led to a reduction of binding to 55.0¡9.6% after one day with further decrease during prolonged incubation. The reaction with 4 mM MGO reduced the binding to 10.7¡1.5% after one day with a further decline over the incubation time to 3.7¡1.8% after seven days. PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 MGO induces impaired binding of S100A12 to RAGE p g The present study investigated the effect of MGO-induced nePTM on the structure and functionality of S100A12. For this purpose, S100A12 was incubated with different concentrations of MGO (4 mM, 40 mM, 400 mM, and 4 mM) for PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 7 / 21 7 / 21 Influence of MGO on S100A12 oligomerization Because the active form of S100A12, which binds to RAGE, is a hexamer, the influence of MGO on the hexamerization of S100A12 was investigated by size- exclusion chromatography. A physiologic running buffer with 500 mM CaCl2 and 10 mM ZnCl2 was used, because the formation of S100A12 hexamers is also dependent on Ca2+ and Zn2+ [13]. The chromatograms of the negative controls (overlay in red) showed one peak with a retention time of 17.6 min (Figure 2). This retention time corresponds to the molecular weight of about 57 kDa, which is in the size range of the S100A12 hexamer indicating that the hexamer was actually formed under the applied conditions, and that the incubation itself had no influence on the hexamerization. The samples modified with 4 mM and 40 mM MGO showed the same elution profile (data not shown) indicating that the formation of the hexamer was not impaired under the reaction conditions. The peaks resulting from the samples modified with 400 mM MGO exhibited a small shoulder towards smaller molecular weight suggesting that a minor part of S100A12 was not present as hexamer. The effect, however, was independent from the incubation time suggesting a rapid reaction of MGO with the protein. The chromatograms of the samples incubated with 4 mM MGO, in contrast, displayed only a small part of the molecules present as hexamers, whereas the main peak had 8 / 21 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding Figure 1. Effects of methylglyoxal (MGO) on the binding of S100A12 to the receptor of advanced end- products (RAGE). S100A12 was incubated with different concentrations of MGO at 37 ˚C for 1, 3, and 7 days. Binding of the modified S100A12 to RAGE was investigated with surface plasmon resonance spectroscopy. The binding of the unincubated negative control was set as 100%. Values represent mean ¡ SD of three independent experiments with duplicate measurements; p,0.05, **p,0.001, significant differences are related to the control (0 mM MGO). d i 10 1371/j l 0113418 001 Figure 1. Effects of methylglyoxal (MGO) on the binding of S100A12 to the receptor of advanced end- products (RAGE). S100A12 was incubated with different concentrations of MGO at 37 ˚C for 1, 3, and 7 days. Binding of the modified S100A12 to RAGE was investigated with surface plasmon resonance spectroscopy. The binding of the unincubated negative control was set as 100%. Influence of MGO on S100A12 oligomerization Values represent mean ¡ SD of three independent experiments with duplicate measurements; p,0.05, *p,0.001, significant differences are related to the control (0 mM MGO). doi:10.1371/journal.pone.0113418.g001 a retention time of 19.9 min corresponding to a molecular weight of 28 kDa. This molecular weight is in the range of a trimer. Because no trimer of S100A12 has been reported before, the peak may also represent equilibrium between the a retention time of 19.9 min corresponding to a molecular weight of 28 kDa. This molecular weight is in the range of a trimer. Because no trimer of S100A12 has been reported before, the peak may also represent equilibrium between the Figure 2. Concentration-dependent effect of methylglyoxal (MGO) on the ability of S100A12 to form hexamers. S100A12 was incubated with different concentrations of MGO at 37 ˚C for 1, 3, and 7 days. Oligomerization behavior was analyzed with size-exclusion chromatography. The negative control incubated for the same time without MGO is shown in red. Chromatograms are representative for two independent experiments. doi:10.1371/journal.pone.0113418.g002 Figure 2. Concentration-dependent effect of methylglyoxal (MGO) on the ability of S100A12 to form hexamers. S100A12 was incubated with different concentrations of MGO at 37 ˚C for 1, 3, and 7 days. Oligomerization behavior was analyzed with size-exclusion chromatography. The negative control incubated for the same time without MGO is shown in red. Chromatograms are representative for two independent experiments. doi:10.1371/journal.pone.0113418.g002 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 9 / 21 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding Table 1. Overview of detected modifications of S100A12 after incubation with methylglyoxal at 37 ˚C. Table 1. Overview of detected modifications of S100A12 after incubation with methylglyoxal at 37 ˚C. Mass shift (Da) 4 mM MGO 40 mM MGO 400 mM MGO 4 mM MGO Interpretation +54 +54 Hydroimidazolone +72 CEL/MGO-hemiaminals/dihydroxyimidazolidine/CEA +80 Argpyrimidine +126 Dihydropyrimidine/hydroimidazolone + CEL (54+72 Da) +144 Tetrahydropyrimidine/CEL + hemiaminals (72+72 Da)/26CEL (72+72 Da) The intact protein was analyzed by MALDI-TOF-MS. All modifications were detected after one, three, and seven days of incubation. The table shows the results of three independent experiments; MGO, methylglyoxal; CEL, Ne-carboxyethyllysine; CEA, carboxyethylarginine. The intact protein was analyzed by MALDI-TOF-MS. All modifications were detected after one, three, and seven days of incubation. The table shows the results of three independent experiments; MGO, methylglyoxal; CEL, Ne-carboxyethyllysine; CEA, carboxyethylarginine. The intact protein was analyzed by MALDI-TOF-MS. All modifications were detected after one, three, and seven days of incubation. The table shows the results of three independent experiments; MGO, methylglyoxal; CEL, Ne-carboxyethyllysine; CEA, carboxyethylarginine. The intact protein was analyzed by MALDI-TOF-MS. All modifications were detected after one, three, and seven results of three independent experiments; MGO, methylglyoxal; CEL, Ne-carboxyethyllysine; CEA, carboxyethyla doi:10.1371/journal.pone.0113418.t001 S100A12 dimer and the tetramer. Although the slope of the calibration curve was relatively high (1 kDa 0.1 min21), it could also be possible that the signal represented the dimer, if the accuracy of the method was low or if the dimer showed inconsistent retention behavior. Thus, the high modification rate caused by 4 mM MGO resulted in a major impairment of S100A12 hexamerization. doi:10.1371/journal.pone.0113418.g003 Mass spectrometric nePTM mapping of MGO-modified S100A12 To investigate the molecular determinants of the reduced binding of MGO- modified S100A12 to RAGE and of the impaired hexamerization, the structures and binding sites of the resulting nePTMs were analyzed by MS. In the first step, untargeted analysis of nePTMs was performed by MALDI-TOF-MS of the intact S100A12 to gather a profile of all major structures that had been formed. In the second step targeted nePTM analysis was performed by UHPLC-ESI-MS/MS to identify the binding sites. MALDI-TOF-MS allows the untargeted analysis of the present nePTM modifications, because all major modifications appear as satellite signals of the native protein in the MALDI spectrum, independent from the structure of the modification [28]. The structures of the modifications were assigned by the mass difference between MGO-modified and the unmodified protein (Table 1) using data from literature [2, 29–31]. Two additional peaks were detected in the samples incubated with 400 mM MGO for one day. One peak showed a mass increase of +54 Da related to the native protein, which corresponds to the arginine modification hydroimidazolone (Figure 3). The other peak displayed a mass shift of +72 Da, which could be caused by several modifications on the arginine or lysine side chains. Arginine could react with MGO to hemiaminals (MGO-hemiaminals or dihydroxyimida- zolidine) or to carboxyethylarginine (CEA), which all have the same molecular weight. Alternatively, lysine could react to CEL, which causes the same mass increase. After the incubation with 4 mM MGO for one day, no signal of the native protein could be discerned suggesting that it was modified to a high extent. The hydroimidazolone was detected, but there was no peak indicating the +72 Da-modification. Instead, there were three additional peaks with a mass shift of +80 Da, +126 Da, and +144 Da, respectively. The mass increase of +80 Da can 10 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Figure 3. Structures of different advanced glycation end-products and their corresponding mass increase. Influence of Nonenzymatic PTMs on S100A12-RAGE Binding be explained by the arginine modification argpyrimidine, while the mass shift of +126 Da could be assigned to dihydropyrimidine formed on the arginine side chain or to a concurrent modification of the arginine residue to the hydroimidazolone (+54 Da) and of the lysine residue to CEL (+72 Da). There are also two possible explanations for the +144 Da-modification, which could be the arginine modification tetrahydropyrimidine or a twofold modification with CEL. Untargeted MALDI-TOF-MS analysis was also repeated after partial enzymatic hydrolysis with GluC to increase the sensitivity (data not shown). After hydrolysis, the modifications were observed already at lower MGO concentrations, but no additional MGO-derived nePTM structures were detected. For the identification of binding sites of the protein modifications in the amino acid sequence, the modified protein S100A12 was analyzed by targeted UHPLC- ESI-MS/MS after partial hydrolysis with the endoproteinase GluC. On the one hand, the localization of the binding site facilitates the prediction how a modification contributes to the observed loss of function. On the other hand, verification of ambiguous structure assignments could be achieved. Finally, Figure 3. Structures of different advanced glycation end-products and their corresponding mass increase. Figure 3. Structures of different advanced glycation end-products and their corresponding mass increase. Figure 3. Structures of different advanced glycation end-products and their corresponding mass increase. PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding targeted UHPLC-ESI-MS/MS analysis is much more sensitive than MALDI-TOF- MS, so that modifications of proteins incubated with lower MGO-concentrations could also be analyzed. For this purpose, product ion scans of all possible modified peptides were performed. Thus, the m/z-values of the peptides containing lysine and/or arginine residues with all possible modifications detected by untargeted MS analysis were calculated. Subsequently, product ion scans were performed using the calculated m/z-values as precursor ions. The modification sites were identified by fragment patterns showing the mass shift of the modification [32]. The peptide AA 10–32 fragmented very poorly, so that the amino acids of interest (R21, K22, and K30) were investigated in peptides AA 19–28 and AA 29–33 after digestion with chymotrypsin. Table 2 depicts the detected modifications and their assignment to the modified amino acid. After incubation with 4 mM MGO, only hydroimidazolone with a mass shift of +54 Da and a modification with a mass shift of +72 Da were detected, both at R21. Thus, it was confirmed that the +72 Da-modification was located on the arginine and was not caused by CEL. As mentioned above, a +72 Da-modification at an arginine side chain could be due to hemiaminals or CEA. However, the product ion spectra of the peptide R21+72 Da showed a loss of water at many fragments as well as a loss of the whole modification at some fragments. This fragmentation behavior strongly indicates that the peptide was modified by hemiaminals, which are relatively loosely bound early reaction products, and not by CEA [2]. This assignment is also supported by the time course of the formation of peptide R21+72 Da: The peak area of this modified peptide grew with increasing MGO concentration until 400 mM, but the peak areas of the samples modified with 4 mM MGO were about 10-fold smaller than the peak areas of the samples modified with 400 mM MGO and were similar to the peak areas of the samples modified with 40 mM MGO. This behavior indicates that the modification is a reaction product which undergoes further reactions with MGO, confirming the presence of a relatively labile intermediate such as a hemiaminal. However, the presence of CEA cannot be fully excluded. Incubation with 40 mM MGO additionally led to detectable levels of argpyrimidine and to tetrahydropyrimidine at R21. PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding Table 2. Overview of detected modifications of S100A12 after incubation with methylglyoxal at 37˚C for 1, 3, and 7 days. Mass shift (Da) Peptide Amino acid sequence 4 mM MGO 40 mM MGO 400 mM MGO 4 mM MGO Modification at Interpretation AA 1–6 MTKLEE +72a +72 +72 N-terminus Carboxyethylation +72a +72 +72 K3 CEL AA 2–6 TKLEE +72 +72 N-terminus Carboxyethylation +72 +72 K3 CEL AA 2–9 TKLEEHLE +72 N-terminus Carboxyethylation +72a K3 CEL AA 19–28 SVRKGHFDTL +54 +54 +54 +54 R21 Hydroimidazolone +72 +72 +72 +72 R21 MGO-hemiaminal/CEA +80 +80 +80 R21 Argpyrimidine +144 +144 +144 R21 Tetrahydropyrimidineb AA 29–33 SKGEL +72c +72 +72 K30 CEL AA 33–40 LKQLLTKE +72 +72 +72 K34 CEL +72 +72 +72 K39 CEL AA 41–50 LANTIKNIKD +72 +72 K46 CEL +72 +72 K49 CEL AA 51–56 KAVIDE +72a +72 K51 CEL AA 41–56 LANTIKNIKDKAVIDE +72 +72 K46 CEL +72 +72 +72 K49 CEL +72 +72 K51 CEL AA 74–92 FISLVAIALKAAHYHTHKE +72 +72 +72 K83 CEL +72 +72 +72 K91 CEL Peptides were analyzed with ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry after partial hydrolysis with GluC or chymotrypsin. Modifications were detected after one day of incubation with the exceptions as indicated in footnotes (a) and (c). The table shows the results of three independent experiments; a detected after three days, b five different peaks, c detected after seven days; MGO, methylglyoxal; CEL, Ne- carboxyethyllysine; CEA, carboxyethylarginine. verview of detected modifications of S100A12 after incubation with methylglyoxal at 37˚C for 1, 3, and 7 days. Table 2. Overview of detected modifications of S100A12 after incubation with methylglyoxal at 37˚C for 1, 3, Peptides were analyzed with ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry after partial hydrolysis with GluC or chymotrypsin. Modifications were detected after one day of incubation with the exceptions as indicated in footnotes (a) and (c). The table shows the results of three independent experiments; a detected after three days, b five different peaks, c detected after seven days; MGO, methylglyoxal; CEL, Ne- carboxyethyllysine; CEA, carboxyethylarginine. CEL was detected in samples with 40 mM MGO at K3, K30, K34, K39, K49, K83, and K91. After incubation with 400 mM MGO, CEL was also identified at K46 and K51. Additionally, carboxyethylation of the N-terminus was observed. Five peaks with the mass of the peptide containing tetrahydropyrimidine were most likely caused by different isomers [33]. The peak areas of the argpyrimidine-modified peptide increased with both MGO concentration and incubation time indicating that argpyrimidine represents a stable end-product of the reaction of R21 with MGO. This Incubation with 40 mM MGO additionally led to detectable levels of argpyrimidine and to tetrahydropyrimidine at R21. Five peaks with the mass of the peptide containing tetrahydropyrimidine were most likely caused by different isomers [33]. The peak areas of the argpyrimidine-modified peptide increased with both MGO concentration and incubation time indicating that argpyrimidine represents a stable end-product of the reaction of R21 with MGO. This observation is consistent with the study of Kloepfer et al. [29]. The modification with a mass increase of +126 Da, which was detected by MALDI-TOF-MS, could not be assigned to distinct amino acids by product ion spectra. Thus, it could not be elucidated if the mass increase represents the arginine modification dihydropyrimidine or a concurrent modification of the arginine residue to the hydroimidazolone and of the lysine residue to CEL. However, the assignment to dihydropyrimidine is more likely, because the chromatogram showed five peaks with the respective mass indicating a modification that can form different isomers. dihydropyrimidine or a concurrent modification of the arginine residue to the hydroimidazolone and of the lysine residue to CEL. However, the assignment to dihydropyrimidine is more likely, because the chromatogram showed five peaks with the respective mass indicating a modification that can form different isomers. 12 / 21 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding The existence of CEL and the absence of other modifications were confirmed by targeted UHPLC-ESI-MS/MS analysis of CEL-S100A12 samples after digestion with GluC. Analysis revealed that all lysine residues of S100A12 carried the CEL- modification and that the N-terminus was also carboxyethylated. The respective modifications were present already at the lowest concentration of pyruvate (300 mM). The peak areas of the modified peptides in the chromatograms grew with increasing concentration of pyruvate indicating a rising modification rate. Comparison of the peak areas of CEL-modified and MGO-modified peptides revealed that all MGO-modified S100A12 samples had equal or lower CEL- modification rates than the CEL-S100A12 sample prepared with 3 mM pyruvate. To analyze the effect of the CEL-modifications on the binding of S100A12 to RAGE, the binding was investigated with SPR (Figure 4). The incubated control without pyruvate and sodium cyanoborohydride exhibited a binding rate of 93.2¡1.8% compared to the unincubated control showing that the incubation had a small effect on the binding of S100A12 to RAGE. The binding of CEL- modified S100A12 to RAGE decreased with increasing modification rate. CEL- S100A12 prepared with 300 mM pyruvate showed a significantly reduced RAGE binding of 86.1¡2.7% of the control (p,0.001). Using 600 mM, 3 mM, 15 mM, and 30 mM pyruvate, the binding of the resulting CEL-S100A12 decreased to 84.4¡3.9%, 80.9¡3.3%, 43.1¡11.1%, and 34.0¡9.7%, respectively. The existence of CEL and the absence of other modifications were confirmed by targeted UHPLC-ESI-MS/MS analysis of CEL-S100A12 samples after digestion with GluC. Analysis revealed that all lysine residues of S100A12 carried the CEL- modification and that the N-terminus was also carboxyethylated. The respective modifications were present already at the lowest concentration of pyruvate (300 mM). The peak areas of the modified peptides in the chromatograms grew with increasing concentration of pyruvate indicating a rising modification rate. Comparison of the peak areas of CEL-modified and MGO-modified peptides revealed that all MGO-modified S100A12 samples had equal or lower CEL- modification rates than the CEL-S100A12 sample prepared with 3 mM pyruvate. To analyze the effect of the CEL-modifications on the binding of S100A12 to RAGE, the binding was investigated with SPR (Figure 4). The incubated control without pyruvate and sodium cyanoborohydride exhibited a binding rate of 93.2¡1.8% compared to the unincubated control showing that the incubation had a small effect on the binding of S100A12 to RAGE. Analysis of CEL-modified S100A12 nePTM mapping of MGO-modified S100A12 revealed the presence of CEL at all lysine side chains as well as the presence of hydroimidazolone, argpyrimidine, tetrahydropyrimidine, and hemiaminals/CEA at the arginine residue R21. In order to investigate the potential role of the different nePTMs on hexamerization and RAGE-binding of S100A12, an S100A12 derivative was synthesized, which contained only CEL-modifications at all lysine residues, but no arginine modifications. For this purpose, reductive amination was carried out using different concentrations of pyruvate (300 mM, 600 mM, 3 mM, 15 mM, and 30 mM) and NaBH3CN. 13 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 doi:10.1371/journal.pone.0113418.g004 The binding of CEL- modified S100A12 to RAGE decreased with increasing modification rate. CEL- S100A12 prepared with 300 mM pyruvate showed a significantly reduced RAGE binding of 86.1¡2.7% of the control (p,0.001). Using 600 mM, 3 mM, 15 mM, and 30 mM pyruvate, the binding of the resulting CEL-S100A12 decreased to 84.4¡3.9%, 80.9¡3.3%, 43.1¡11.1%, and 34.0¡9.7%, respectively. Figure 4. Effect of Ne-carboxyethyllysine (CEL)-modifications on the binding of S100A12 to the receptor for advanced glycation end-products (RAGE). S100A12 was incubated with the indicated concentrations of pyruvate and sodium cyanoborohydride at 25 ˚C for 16 h to achieve a similar CEL- modification as present in methylglyoxal (MGO)-modified S100A12. Binding of the CEL-modified S100A12 to RAGE was investigated with surface plasmon resonance spectroscopy. The binding of the unincubated negative control was set as 100%. #, in all MGO-modified proteins, CEL modification rates were # those of CEL-S100A12 at 3 mM. Values represent mean ¡ SD of three independent experiments with duplicate measurements; p,0.001, significant differences are related to the incubated control. The CEL- modifications did not inhibit the hexamerization of S100A12 because there was no elution behavior towards smaller molecular weight compared to the negative control (data not shown). Figure 4. Effect of Ne-carboxyethyllysine (CEL)-modifications on the binding of S100A12 to the receptor for advanced glycation end-products (RAGE). S100A12 was incubated with the indicated concentrations of pyruvate and sodium cyanoborohydride at 25 ˚C for 16 h to achieve a similar CEL- modification as present in methylglyoxal (MGO)-modified S100A12. Binding of the CEL-modified S100A12 to RAGE was investigated with surface plasmon resonance spectroscopy. The binding of the unincubated negative control was set as 100%. #, in all MGO-modified proteins, CEL modification rates were # those of CEL-S100A12 at 3 mM. Values represent mean ¡ SD of three independent experiments with duplicate measurements; **p,0.001, significant differences are related to the incubated control. The CEL- modifications did not inhibit the hexamerization of S100A12 because there was no elution behavior towards smaller molecular weight compared to the negative control (data not shown). doi:10.1371/journal.pone.0113418.g004 doi:10.1371/journal.pone.0113418.g004 14 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding Figure 5. Localization of resolved lysine (A) or arginine (B) side chains in the crystal structure of S100A12 (PDB-Code 2WCB). The two subunits of the S100A12 dimer are shown in red and green. The C- and N-atoms of lysine or arginine side chains are colored white and blue, respectively. Figure 5. Localization of resolved lysine (A) or arginine (B) side chains in the crystal structure of S100A12 (PDB-Code 2WCB). The two subunits of the S100A12 dimer are shown in red and green. The C- and N-atoms of lysine or arginine side chains are colored white and blue, respectively. doi:10.1371/journal.pone.0113418.g005 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Discussion The incubation of S100A12 with MGO led to different modifications dependent on the MGO concentration. At the lowest MGO concentration (4 mM), only hydroimidazolone and hemiaminals were detected at R21. Using 40 mM MGO, argpyrimidine and tetrahydropyrimidine were detected at R21 as well as CEL at four different lysine residues. At higher MGO concentrations, an additional CEL modification of most lysine residues was observed. These results confirm the higher reactivity of MGO towards arginine compared to lysine [34]. No other lysine modification besides CEL, a modification that is formed specifically between MGO and lysine [30], could be detected. The tandem mass spectrometric analysis of the MGO-modified S100A12 revealed that almost all lysines and the N-terminus were carboxyethylated after incubation with 40 mM MGO or 400 mM MGO. As an exception, no CEL was detected at K22. Thus, K22 may be shielded against reaction with MGO due to early and readily formed adducts at the neighboring R21. Alternatively, CEL at K22 may not be detected under the applied conditions, e.g. due to poor ionization properties. The CEL-modified S100A12 carried CEL on all lysine residues even after incubation with the lowest concentration of pyruvate (300 mM). Hence, there was no indication of a site-specific modification of the lysine residues in S100A12 either by reductive amination or by MGO. In S100A12, all lysine residues are located on the surface of the dimer (Figure 5A) indicating that the lysine residues are easily and equally accessible for the glycation reagents. Preferential glycation, however, is dependent on the differing accessibility of lysine side chains [35]. The arginine side chains are also located on the surface of the dimer (Figure 5B) providing easy access for MGO. The good accessibility of the lysine and arginine side chains on S100A12 apparently prevails over other effects that could lead to site-specific modification. In contrast, site-specific modifications were suggested for proteins like human serum albumin [7], hemoglobin [36], or RNase [37]. The CEL-modified S100A12 carried CEL on all lysine residues even after incubation with the lowest concentration of pyruvate (300 mM). Hence, there was no indication of a site-specific modification of the lysine residues in S100A12 either by reductive amination or by MGO. In S100A12, all lysine residues are located on the surface of the dimer (Figure 5A) indicating that the lysine residues are easily and equally accessible for the glycation reagents. PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Discussion Preferential glycation, however, is dependent on the differing accessibility of lysine side chains [35]. The arginine side chains are also located on the surface of the dimer (Figure 5B) providing easy access for MGO The good accessibility of the lysine and arginine 15 / 21 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding Figure 6. Localization of arginine side chains (orange) in the S100A12 hexamer (PDB-Code 1GQM). The three dimers of the S100A12 hexamer are shown in red, green, and blue, respectively. doi:10.1371/journal.pone.0113418.g006 Figure 6. Localization of arginine side chains (orange) in the S100A12 hexamer (PDB-Code 1GQM). The three dimers of the S100A12 hexamer are shown in red, green, and blue, respectively. Figure 6. Localization of arginine side chains (orange) in the S100A12 hexamer (PDB-Code 1GQM). The three dimers of the S100A12 hexamer are shown in red, green, and blue, respectively. doi:10 1371/journal pone 0113418 g006 doi:10.1371/journal.pone.0113418.g006 In contrast to the CEL-modification of S100A12, the incubation with MGO affected the hexamerization of S100A12. The hexamer was formed after incubation with 4, 40, and 400 mM MGO. By contrast, the incubation with 4 mM MGO did not lead to an oligomerization exceeding the tetramer. The peak areas of the CEL-modified peptides prepared with 3 mM pyruvate were equal to or higher than the peak areas obtained for all MGO-modified S100A12. This result suggests that the CEL-modifications of lysine residues do not influence the formation of the S100A12 hexamer. Since MGO-modified S100A12 impairs hexamerization, it can be concluded that these deteriorations are caused by the detected modifications at R21, which are the only structural difference between MGO-S100A12 and CEL-S100A12. The arginine side chains are located far from the hexamerization interface indicating that the inhibition of the hexamerization was not caused by a steric effect of the arginine modifications (Figure 6). One reason why R21 is important for hexamerization could be a stabilizing effect on the structure of S100A12. The guanidinium group of R21 is close enough to the amide group of Q35 to form a hydrogen bond (Figure 7). Q35 is located in an a-helix so that the hydrogen bond could stabilize the position of this a-helix relative to the loop containing the arginine. Modification of the arginine side chain could lead to a loss of this hydrogen bond causing an alteration of the tertiary structure of S100A12. This conformational change could then be the reason for impaired hexamerization. Discussion PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 16 / 21 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding Figure 7. Environment of R21 in the S100A12 hexamer (PDBCode 2WCE). The three dimers are shown in blue, red, and green. Nitrogen and oxygen atoms are shown in blue and red, respectively. Figure 7. Environment of R21 in the S100A12 hexamer (PDBCode 2WCE). The three dimers are shown in blue, red, and green. Nitrogen and oxygen atoms are shown in blue and red, respectively. doi:10.1371/journal.pone.0113418.g007 doi:10.1371/journal.pone.0113418.g007 SPR analysis revealed that the binding of S100A12 to RAGE was reduced after incubation with MGO in a concentration-dependent manner. The incubation of S100A12 with 4 mM and 40 mM MGO had no effect on its binding to RAGE consistent with the low modification degree detected by UHPLC-ESI-MS/MS. With increasing MGO concentration, the binding of S100A12 to RAGE declined consistently with the detection of more modifications. The specific introduction of CEL into S100A12 led to a concentration- dependent decrease of the binding of S100A12 to RAGE. This result suggests that the binding declines with increasing modification rate. However, the lysine modification rate of CEL-S100A12 prepared with 3 mM pyruvate was equal to or higher than the CEL-modification rate of S100A12 incubated with 4 mM MGO. Relative binding of the former CEL-S100A12 to RAGE, in contrast, reached 81%, whereas RAGE binding of the latter MGO-modified S100A12 was down to 4%. These results indicate that the modifications of R21 by MGO are major factors leading to the functional loss of S100A12, whereas CEL-modifications at the lysine residues play only a minor role herein. It has been shown before for other proteins, such as small heat shock proteins or human serum albumin, that specific arginine residues can be important determinants for protein biofunction [7, 38]. The structural reason for the impairment of S100A12-binding to RAGE by the modification of R21 is not clear. No major impairment of hexamerization was recorded when MGO concentrations lower than 4 mM were used, whereas even the incubation with 400 mM led to more than 50% suppression of RAGE binding. Therefore, it can be concluded that impaired S100A12 oligomerization is not the only mechanism leading to MGO-induced suppression of RAGE binding. Similar to the dimer, R21 is located on the surface of the S100A12 hexamer, which is the active form of the ligand (Figure 6). Influence of Nonenzymatic PTMs on S100A12-RAGE Binding Xie et al. suggested that the formation of a Ca2+-complex of S100A12 is a prerequisite of RAGE binding that leads to drastic conformational changes which allow interaction of Ca2+-S100A12 with RAGE [39]. Because calcium is coordinated, besides others, by K22, the modification of the neighboring R21 may impair calcium-coordination and subsequent conformational changes necessary for receptor binding. Furthermore, MGO-induced modification of R21 may additionally deteriorate the RAGE-binding site of S100A12. However, the structural basis of the RAGE–S100A12 interaction is not fully understood. Xie et al. described binding sites of the S100A12 hexamer that mediate binding to the C1C2 domain of tetramer RAGE [39]. In contrast, Dattilo et al. postulated that binding of S100 ligands is rather directed towards the V-domain of RAGE with secondary effects of the C1 domain [40]. Consequently, binding mechanisms of S100B to the VC1 domain of RAGE were described [41]. However, these results cannot be directly transferred to S100A12, because different S100 proteins are assumed to bind to different sites of RAGE [42]. Therefore, further studies on the structural basis of RAGE–S100A12 interaction are required to determine the mechanism how MGO-induced modifications of the ligand may directly interfere with receptor binding. The formation of nePTMs may modulate RAGE signaling by different mechanisms. It is well established that CML-modifications convert non-ligands into strong agonists of RAGE [15, 19]. Since CML-modified S100A8 und S100 A8/ 9 were detected in inflamed gut tissue, it can be speculated that CML- modifications further enhance the binding of these natural ligands of RAGE [19]. In the present study, MGO-induced nePTMS, mainly hydroimidazolone, inhibited RAGE binding. Because hydroimidazolone is present in vivo in higher concentrations than CML [3], it can be hypothesized that the inhibitory effect of hydroimidazolone overcompensates a possible advancing effect of CML. Geczy and coworkers found that the S100 proteins S100A8 and S100A9 are susceptible to oxidative modifications in vivo leading to alterations of the proinflammatory function [43]. Thus, they proposed that pronounced oxidation of S100A8 and S100A9, which is observed during inflammation, may act as a regulatory switch suppressing excessive inflammation. However, it remains speculation if the inactivating modification of S100A12 by MGO exerts a similar regulatory effect restoring homeostasis as the oxidation of S100A8 and S100A9. Discussion Thus, it is possible that the reduced binding as a consequence of R21 modification is due to steric interference at the S100A12–RAGE binding interface. 17 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding hexamerization at higher MGO concentrations. MS analysis revealed the presence of CEL-modifications at nine of the lysine residues and the N-terminus as well as at least four different modifications at R21. Synthetically prepared CEL-modified S100A12 also carried CEL-residues at all lysine residues and the N-terminus in concentrations as present in MGO-S100A12, but showed only minor impairment of RAGE-binding and no impairment of hexamerization. Thus, it can be concluded that mainly modifications at R21 are responsible for the functional consequences of S100A12-nePTMs. Most likely, reduced binding to RAGE is caused -to a minor extent- by allosteric inhibition of S100A12 hexamerization and mainly by allosteric or steric interference of the ligand-receptor interface. Author Contributions Conceived and designed the experiments: KA JE WU MP. Performed the experiments: KA WU. Analyzed the data: KA WU. Contributed reagents/ materials/analysis tools: JE MP. Contributed to the writing of the manuscript: KA JE WU MP. Conclusion It has been shown that S100 protein is subjected to nePTM in vivo at inflammation sites, which may influence its binding to the natural receptor RAGE [19]. In the present study, mechanisms were investigated how nePTMs may modulate S100A12–RAGE binding. Thus, nePTMs were introduced into S100A12 by MGO, which is a major nePTM precursor in vivo [44]. The formation of MGO-derived nePTMs led to a time- and concentration-dependent decrease of RAGE–S100A12 binding, which is accompanied by an interference of S100A12 18 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 18 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding 12. Moroz OV, Antson AA, Dodson EJ, Burrell HJ, Grist SJ, et al. (2002) The structure of S100A12 in a hexameric form and its proposed role in receptor signalling. Acta Crystallogr D Biol Crystallogr 58:407– 413. 13. Moroz OV, Burkitt W, Wittkowski H, He W, Ianoul A, et al. (2009) Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function. BMC Biochem 10:11. 14. Neeper M, Schmidt AM, Brett J, Yan SD, Wang F, et al. (1992) Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins. J Biol Chem 267:14998–15004. 15. Kislinger T, Fu C, Huber B, Qu W, Taguchi A, et al. (1999) N(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression. J Biol Chem 274:31740–31749. 16. Xue J, Rai V, Singer D, Chabierski S, Xie J, et al. (2011) Advanced glycation end product recognition by the receptor for AGEs. Structure 19:722–732. 17. Foell D, Kucharzik T, Kraft M, Vogl T, Sorg C, et al. (2003) Neutrophil derived human S100A12 (EN- RAGE) is strongly expressed during chronic active inflammatory bowel disease. Gut 52:847–853. 18. Anderson MM, Requena JR, Crowley JR, Thorpe SR, Heinecke JW (1999) The myeloperoxidase system of human phagocytes generates Nepsilon-(carboxymethyl)lysine on proteins: a mechanism for producing advanced glycation end products at sites of inflammation. J Clin Invest 104:103–113. 19. Andrassy M, Igwe J, Autschbach F, Volz C, Remppis A, et al. (2006) Posttranslationally modified proteins as mediators of sustained intestinal inflammation. Am J Pathol 169:1223–1237. 20. Chong S, Mersha FB, Comb DG, Scott ME, Landry D, et al. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192:271–281. 21. Lennon G, Auffray C, Polymeropoulos M, Soares MB (1996) The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics 33:151–152. 22. Dell’Angelica EC, Schleicher CH, Santome JA (1994) Primary structure and binding properties of calgranulin C, a novel S100-like calcium-binding protein from pig granulocytes. J Biol Chem 269:28929– 28936. 23. Koito W, Araki T, Horiuchi S, Nagai R (2004) Conventional antibody against Nepsilon- (carboxymethyl)lysine (CML) shows cross-reaction to Nepsilon-(carboxyethyl)lysine (CEL): immunochemical quantification of CML with a specific antibody. J Biochem 136:831–837. 24. References 1. Glomb MA, Monnier VM (1995) Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction. J Biol Chem 270:10017–10026. 1. Glomb MA, Monnier VM (1995) Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction. J Biol Chem 270:10017–10026. 2. Mittelmaier S, Pischetsrieder M (2011) Multistep ultrahigh performance liquid chromatography/tandem mass spectrometry analysis for untargeted quantification of glycating activity and identification of most relevant glycation products. Anal Chem 83:9660–9668. 2. Mittelmaier S, Pischetsrieder M (2011) Multistep ultrahigh performance liquid chromatography/tandem mass spectrometry analysis for untargeted quantification of glycating activity and identification of most relevant glycation products. Anal Chem 83:9660–9668. 3. Ahmed N, Babaei-Jadidi R, Howell SK, Beisswenger PJ, Thornalley PJ (2005) Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes. Diabetologia 48:1590–1603. 4. Thornalley PJ (2006) Advanced glycation end products in renal failure. J Ren Nutr 16:178–184. 5. Yagmur E, Tacke F, Weiss C, Lahme B, Manns MP, et al. (2006) Elevation of Nepsilon- (carboxymethyl)lysine-modified advanced glycation end products in chronic liver disease is an indicator of liver cirrhosis. Clin Biochem 39:39–45. 6. Takahashi M, Lu YB, Myint T, Fujii J, Wada Y, et al. (1995) In vivo glycation of aldehyde reductase, a major 3-deoxyglucosone reducing enzyme: identification of glycation sites. Biochemistry 34:1433–1438. 7. Ahmed N, Dobler D, Dean M, Thornalley PJ (2005) Peptide mapping identifies hotspot site of modification in human serum albumin by methylglyoxal involved in ligand binding and esterase activity. J Biol Chem 280:5724–5732. 8. Bank RA, Bayliss MT, Lafeber FP, Maroudas A, Tekoppele JM (1998) Ageing and zonal variation in post-translational modification of collagen in normal human articular cartilage. The age-related increase in non-enzymatic glycation affects biomechanical properties of cartilage. Biochem J 330 (Pt 1):345–351. 9. Fritz G (2011) RAGE: a single receptor fits multiple ligands. Trends Biochem Sci 36:625–632. 10. Hofmann MA, Drury S, Fu C, Qu W, Taguchi A, et al. (1999) RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 97:889–901. 11. Fritz G, Heizmann CW (2004) 3D Structures of the calcium and zinc binding S100 proteins. In: Messerschmidt A, Bode W, Cygler M, editors. Handbook of Metalloproteins Hoboken, N.J.: John Wiley & Sons Inc. pp. 529–540. 19 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Meltretter J, Becker CM, Pischetsrieder M (2008) Identification and site-specific relative quantification of beta-lactoglobulin modifications in heated milk and dairy products. J Agric Food Chem 56:5165–5171. 25. Strohalm M, Kavan D, Novak P, Volny M, Havlicek V (2010) mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data. Anal Chem 82:4648–4651. 26. Tae HJ, Kim JM, Park S, Tomiya N, Li G, et al. (2013) The N-glycoform of sRAGE is the key determinant for its therapeutic efficacy to attenuate injury-elicited arterial inflammation and neointimal growth. J Mol Med (Berl) 91:1369–1381. 27. Srikrishna G, Nayak J, Weigle B, Temme A, Foell D, et al. (2010) Carboxylated N-glycans on RAGE promote S100A12 binding and signaling. J Cell Biochem 110:645–659. 28. Meltretter J, Seeber S, Humeny A, Becker CM, Pischetsrieder M (2007) Site-specific formation of Maillard, oxidation, and condensation products from whey proteins during reaction with lactose. J Agric Food Chem 55:6096–6103. 29. Klopfer A, Spanneberg R, Glomb MA (2011) Formation of arginine modifications in a model system of Nalpha-tert-butoxycarbonyl (Boc)-arginine with methylglyoxal. J Agric Food Chem 59:394–401. 30. Ahmed MU, Brinkmann Frye E, Degenhardt TP, Thorpe SR, Baynes JW (1997) N-epsilon- (carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem J 324. (Pt 2):565–570. 31. Oya T, Hattori N, Mizuno Y, Miyata S, Maeda S, et al. (1999) Methylglyoxal modification of protein. Chemical and immunochemical characterization of methylglyoxal-arginine adducts. J Biol Chem 274:18492–18502. 20 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0113418 November 26, 2014 Influence of Nonenzymatic PTMs on S100A12-RAGE Binding 32. Meltretter J, Wust J, Pischetsrieder M (2013) Comprehensive analysis of nonenzymatic post- translational beta-lactoglobulin modifications in processed milk by ultrahigh-performance liquid chromatography-tandem mass spectrometry. J Agric Food Chem 61:6971–6981. 33. Ahmed N, Thornalley PJ (2002) Chromatographic assay of glycation adducts in human serum albumin glycated in vitro by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and intrinsic fluorescence. Biochem J 364:15–24. 34. Westwood ME, Thornalley PJ (1995) Molecular characteristics of methylglyoxal-modified bovine and human serum albumins. Comparison with glucose-derived advanced glycation endproduct-modified serum albumins. J Protein Chem 14:359–372. 35. Fogliano V, Monti SM, Visconti A, Randazzo G, Facchiano AM, et al. (1998) Identification of a beta- lactoglobulin lactosylation site. Biochim Biophys Acta 1388:295–304. 36. Gao Y, Wang Y (2006) Site-selective modifications of arginine residues in human hemoglobin induced by methylglyoxal. Biochemistry 45:15654–15660. 37. Brock JW, Hinton DJ, Cotham WE, Metz TO, Thorpe SR, et al. (2003) Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease. J Proteome Res 2:506–513. 38. Nagaraj RH, Panda AK, Shanthakumar S, Santhoshkumar P, Pasupuleti N, et al. (2012) Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics. PLoS One 7:e30257. 39. Xie J, Burz DS, He W, Bronstein IB, Lednev I, et al. (2007) Hexameric calgranulin C (S100A12) binds to the receptor for advanced glycated end products (RAGE) using symmetric hydrophobic target-binding patches. J Biol Chem 282:4218–4231. 40. Dattilo BM, Fritz G, Leclerc E, Kooi CW, Heizmann CW, et al. (2007) The extracellular region of the receptor for advanced glycation end products is composed of two independent structural units. Biochemistry 46:6957–6970. 41. Koch M, Chitayat S, Dattilo BM, Schiefner A, Diez J, et al. (2010) Structural basis for ligand recognition and activation of RAGE. Structure 18:1342–1352. 42. Leclerc E, Fritz G, Vetter SW, Heizmann CW (2009) Binding of S100 proteins to RAGE: an update. Biochim Biophys Acta 1793:993–1007. 43. Lim SY, Raftery MJ, Geczy CL (2011) Oxidative modifications of DAMPs suppress inflammation: the case for S100A8 and S100A9. Antioxid Redox Signal 15:2235–2248. 44. Thornalley PJ, Battah S, Ahmed N, Karachalias N, Agalou S, et al. (2003) Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry. Biochem J 375:581–592. 21 / 21
https://openalex.org/W2153332276	https://europepmc.org/articles/pmc4247940?pdf=render	English	null	Postoperative “Chimney” for Unintentional Renal Artery Occlusion after EVAR	Case reports in vascular medicine	2,014	cc-by	2,674	Hindawi Publishing Corporation Case Reports in Vascular Medicine Volume 2014, Article ID 170198, 5 pages http://dx.doi.org/10.1155/2014/170198 Hindawi Publishing Corporation Case Reports in Vascular Medicine Volume 2014, Article ID 170198, 5 pages http://dx.doi.org/10.1155/2014/170198 Hindawi Publishing Corporation Case Reports in Vascular Medicine Volume 2014, Article ID 170198, 5 pages http://dx.doi.org/10.1155/2014/170198 1. Introduction aortic aneurysm (AAA). Medical history was notable for obesity, hypertension, chronic depressed (<30%) left heart dysfunction, and chronic obstructive pulmonary disease. Preoperative computed tomography angiography (CT-A) highlighted the presence of a hostile proximal aortic neck characterized by a reversed tapered shape (Figures 1(a) and 1(b)) plus an acute 𝛽-angle > 60∘(Figure 1(c)). In the oper- ating room, a 28 mm transrenal bifurcated EG (Endurant- Medtronic; Santa Rosa-CA; USA) was implanted through conventional bilateral groin cut-down. Final angiography confirmed the complete exclusion of the AAA with no evi- dence of proximal or distal endoleak, as well as the visualiza- tion of the renal arteries (Figures 2(a) and 2(b)). The imme- diate postoperative serum creatinine level was 1.48 mg/dL (range, 0.6–1.3 mg/dL; preoperative level: 1.36 mg/dL). Twelve hours later, a progressive reduction of urine output was noted; at that time, serum creatinine level increased to 3.26 mg/dL, configuring a grade 4 AKI according to the Aneurysm Renal Injury Score (ARISe) [5]. Angiography was performed immediately thereafter: it showed the occlusion of the renal artery, bilaterally. During the same procedure, we attempted an endovascular revascularization of the renal Acute kidney injury (AKI) remains a known complica- tion after endovascular abdominal aortic aneurysm repair (EVAR): multiple factors may be involved in postoperative AKI including contrast-induced nephropathy, atheroem- bolism, occlusion of accessory renal arteries, or unintentional overstenting of the renal arteries [1, 2].t Generally, renal artery occlusion due to endograft (EG) overstenting is recognized rapidly and could be treated immediately during the procedure; in contrast, unexpected renal artery occlusion may lead to prolonged ischemic dam- age and potential permanent injury requiring hemodialysis [3, 4]. In this report, we describe the occurrence of an unin- tentional and unexpected bilateral renal artery overstenting successfully managed with a bilateral renal artery “chimney” stenting in the late postoperative course. Marco Franchin,1 Federico Fontana,2 Filippo Piacentino,2 Matteo Tozzi,1 and Gabriele Piffaretti1 1 Vascular Surgery, Department of Surgery and Morphological Sciences, Circolo University Hospital, University of Insubria School of Medicine, Via Guicciardini 9, 21100 Varese, Italy 2 Interventional Radiology, Department of Surgery and Morphological Sciences, Circolo University Hospital, University of Insubria School of Medicine, Via Guicciardini 9, 21100 Varese, Italy 1 Vascular Surgery, Department of Surgery and Morphological Sciences, Circolo University Hospital, University of Insubria School of Medicine, Via Guicciardini 9, 21100 Varese, Italy 2 Interventional Radiology, Department of Surgery and Morphological Sciences, Circolo University Hospital, University of Insubria School of Medicine, Via Guicciardini 9, 21100 Varese, Italy 1 Vascular Surgery, Department of Surgery and Morphological Sciences, Circolo University Hospital, University of Insubria School of Medicine, Via Guicciardini 9, 21100 Varese, Italy 2 Interventional Radiology, Department of Surgery and Morphological Sciences, Circolo University Hospital, University of Insubria School of Medicine, Via Guicciardini 9, 21100 Varese, Italy Correspondence should be addressed to Gabriele Piffaretti; gabriele.piffaretti@uninsubria.it Received 11 August 2014; Accepted 27 October 2014; Published 16 November 2014 Academic Editor: Atila Iyisoy Copyright © 2014 Marco Franchin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Renal artery obstruction during endovascular repair of abdominal aortic aneurysm using standard device is a rare but life- threatening complication and should be recognized and repaired rapidly in order to maintain renal function. Both conventional surgery and endovascular stenting have been reported. We report a case of late postoperative bilateral “chimney” to resolve a bilateral thrombosis of the renal artery following an uncomplicated endovascular aortic repair. 2. Case Report A 73-year-old man was admitted to our department with a diagnosis of an asymptomatic 57 mm fusiform abdominal 2 2 Case Reports in Vascular Medicine (b) (c) (a) 𝛽> 60∘ Figure 1: Preliminary CT-A showing (a, b) the reversed tapered shape of the proximal neck (arrows: renal arteries origin) and the severe 𝛽-angle (c). (c) 𝛽> 60∘ (a) (a) (b) (b) (c) Figure 1: Preliminary CT-A showing (a, b) the reversed tapered shape of the proximal neck (arrows: renal arteries origin) and the severe 𝛽-angle (c). RT renal (a) RT renal SMA LT renal (b) Figure 2: Predeployment preliminary angiography (a). Final con- trol (b) showed the patency of both the renal arteries (arrows). RT renal (a) RT renal SMA LT renal (b) arteries, which failed because of the acute onset of a high- response atrial fibrillation causing hemodynamic instability and acute respiratory distress. The patient was transferred in the intensive care unit and started a temporary renal replace- ment therapy and amiodarone (Cordarone-Sanofi-Aventis; Milano-Italy) intravenously. Four days later, hemodynamic stability was recovered; at that time, a percutaneous left transbrachial approach was used to catheterize selectively the renal arteries (Figure 3(a)) with a 0.014󸀠󸀠guidewire (Stabilizer-Cordis, Miami Lakes, FL, USA) coupled with a 4F vertebral catheter (Cordis; Miami Lakes-FL; USA). A 5 × 15 mm bare metal stent (Genesis-Cordis; Miami Lakes- FL; USA) was used bilaterally with the complete restora- tion of renal flow into the parenchyma (Figure 3(b)). The subsequent postoperative course was uneventful: the urine output improved progressively. He was discharged on day 6 postoperatively on acetylsalicylic acid (Cardioaspirin-Bayer; Milano-Italy) 100 mg/die ad infinitum. He was last seen eighteen months later; the patient is still alive, asymptomatic, and the follow-up CT-A confirmed the complete exclusion of the aneurysm without endoleaks and the patency of the renal stents with preservation of visceral flow (Figures 3(c) and 3(d)). At that time serum creatinine level was 1.38 mg/ dL. SMA (a) (b) Figure 2: Predeployment preliminary angiography (a). Final con- trol (b) showed the patency of both the renal arteries (arrows). 3 Case Reports in Vascular Medicine (a) (d ) (a) (b) (c) Figure 3: Selective angiography at the proximal extremity of the EG (a) showed the overstenting (arrows) of the origin of both the re arteries. Complete revascularization after bilateral stenting (b). 2. Case Report Follow-up CT-A: complete reperfusion of the parenchyma (c) as well persistent exclusion of the aneurysm and absence of endoleak (d). (c) (c) (d ) ( ) (a) ( ) (b) (d ) (b) Figure 3: Selective angiography at the proximal extremity of the EG (a) showed the overstenting (arrows) of the origin of both the renal arteries. Complete revascularization after bilateral stenting (b). Follow-up CT-A: complete reperfusion of the parenchyma (c) as well as persistent exclusion of the aneurysm and absence of endoleak (d). Figure 3: Selective angiography at the proximal extremity of the EG (a) showed the overstenting (arrows) of the origin of both the renal arteries. Complete revascularization after bilateral stenting (b). Follow-up CT-A: complete reperfusion of the parenchyma (c) as well as persistent exclusion of the aneurysm and absence of endoleak (d). 3. Discussion cause was a proximal migration of a bifurcated EG, an event that is anecdotal and mainly due to an iatrogenic upward thrust of the device during contralateral cannulation. When suprarenal stents were first introduced, there was concern that they would induce hyperplasia and narrow the renal orifices; up to date, insufficient high-level evidence has decreed for or against proximal transrenal fixation on EVAR- related renal occlusion [13–15]. In our case, as probably occurs in most of the cases, the mechanism that caused the renal artery thrombosis was an underhanded partial obstruction that can be difficult to identify on intraoperative angiograms. We had an uncomplicated deployment with visualization of the renal arteries at completion angiography: the reason that may explain the delayed onset of the thrombosis of the renal arteries can be ascribed to the imperceptible filling defect in the renal artery profile even when the EG covers much of the orifice so that the renal artery lumen often fills with contrast-enhanced blood. Furthermore, the partial obstructions of renal arteries by the graft material or the struts of the suprarenal hooks were potentially masked by intraoperative anticoagulation. The starting points for discussion of our case are either tech- nical or clinical: they concern the underhanded pathogenesis of the renal artery occlusion, the feasibility of a postoperative “chimney” technique to overcome the renal artery ostia overstenting, and the recovery of the renal function 96 hours after the onset of the acute kidney injury. t As all the innovative techniques, even EVAR has brought new complications such as endoleaks and migration; some others are shared with conventional aortic repair such as renal artery occlusion [6]. Acute kidney injury is a serious complication and harbinger of poor prognosis after EVAR [7, 8]: despite renal artery occlusion during EVAR remaining an uncommon complication, as EVAR gains popularity, the incidence of EVAR-associated renal artery obstruction may increase. EVAR-related renal artery occlusion is generally found intraoperatively following an EG maldeployment; however, it has been suggested that also those occlusions detected during the follow-up could be considered occluded since the initial operation [9–11]. Inan et al. [12] described a case of intraoperative bilateral renal artery occlusion: the Currently, there is no consensus about the treatment strategy of operative treatment and outcomes after prolonged Case Reports in Vascular Medicine 4 renal artery occlusion. 3. Discussion We believe that an attempt to revascu- larize the overstented renal arteries should have been manda- tory in our case. Twine and Boyle [5] reported the occlusion of both renal arteries in the early postoperative period of an ordinary infrarenal EVAR resulting in dialysis-dependent kidney injury. Our case shows that urgent revascularization could be effective for this type of complication. Maybe the favorable outcome was facilitated by the early development of a rich collateral circulation that has been studied to come up most commonly from the periureteral, peripelvic, and adrenal vessels and that can maintain viability of nephrons at sub-filtration arterial pressures; furthermore, it provides the rationale for rescue intervention after total renal artery occlusion, even when the diagnosis has been delayed [16, 17]. renal function compared with open repair,” Journal of Vascular Surgery, vol. 58, no. 4, pp. 886–893, 2013. renal function compared with open repair,” Journal of Vascular Surgery, vol. 58, no. 4, pp. 886–893, 2013. [3] [3] P. Cao, F. Verzini, S. Zannetti et al., “Device migration after endoluminal abdominal aortic aneurysm repair: analysis of 113 cases with a minimum follow-up period of 2 years,” Journal of Vascular Surgery, vol. 35, no. 2, pp. 229–235, 2002. [4] B. T. Katzen, A. A. MacLean, and H. E. Katzman, “Retrograde migration of an abdominal aortic aneurysm endograft leading to postoperative renal failure,” Journal of Vascular Surgery, vol. 42, no. 4, pp. 784–787, 2005. [5] C. P. Twine and J. R. Boyle, “Renal dysfunction after EVAR: time for a standard definition,” Journal of Endovascular Therapy, vol. 20, no. 3, pp. 331–333, 2013. [6] W. Grande and S. W. Stavropoulos, “Treatment of complications following endovascular repair of abdominal aortic aneurysms,” Seminars in Interventional Radiology, vol. 23, no. 2, pp. 156–164, 2006. g y [ ] Both open and endovascular techniques may be used as procedures to treat this uncommon, but important, especially if we consider the rapid and steady increase of EVARs either to treat conventional anatomies or complex aortic necks [18]. Open conversion still remains an option: when they covered the renal arteries for a number of weeks Adu et al. [18] proposed a flowchart with specific vascular bypasses depend- ing on the patency status of the celiac trunk. In contrast, endovascular revascularization is primarily determined by accessibility of the renal orifices [4, 10, 12]. Similar to our case, Hedayati et al. 3. Discussion [11] reported two cases of renal artery occlusion treated one week after a transrenal EVAR with renal artery stenting using a transfemoral approach which led to symptom resolution and recovery of renal function. In the present case the femoral approach has not been successful; in contrast, the second attempt using a brachial approach has led to an easier and more rapid catheterization of both renal arteries, perhaps more so for the presence of the transrenal bare fixation and despite the greater length of the shaft affecting its maneuverability. [7] B. Wisniowski, M. Barnes, J. Jenkins, N. Boyne, A. Kruger, and P. J. Walker, “Predictors of outcome after elective endovascular abdominal aortic aneurysm repair and external validation of a risk prediction model,” Journal of Vascular Surgery, vol. 54, no. 3, pp. 644–653, 2011. [8] G. T. Pisimisis, C. F. Bechara, N. R. Barshes, P. H. Lin, W. S. Lai, and P. Kougias, “Risk factors and impact of proximal fixation on acute and chronic renal dysfunction after endovascular aortic aneurysm repair using glomerular filtration rate criteria,” Annals of Vascular Surgery, vol. 27, no. 1, pp. 16–22, 2013. [9] B. Thomas and L. Sanchez, “Proximal migration and endoleak: impact of endograft design and deployment techniques,” Semi- nars in Vascular Surgery, vol. 22, no. 3, pp. 201–206, 2009. [10] P. H. Lin, R. L. Bush, and A. B. Lumsden, “Endovascular rescue of a maldeployed aortic stent-graft causing renal artery occlu- sion: technical considerations,” Vascular and Endovascular Sur- gery, vol. 38, no. 1, pp. 69–73, 2004. [11] N. Hedayati, P. H. Lin, A. B. Lumsden, and W. Zhou, “Prolonged renal artery occlusion after endovascular aneurysm repair: endovascular rescue and renal function salvage,” Journal of Vascular Surgery, vol. 47, no. 2, pp. 446–449, 2008. Conflict of Interests [14] A. Saratzis, P. Sarafidis, N. Melas et al., “Suprarenal graft fixation in endovascular abdominal aortic aneurysm repair is associated with a decrease in renal function,” Journal of Vascular Surgery, vol. 56, no. 3, pp. 594–600, 2012. The authors declare that there is no conflict of interests regarding the publication of this paper. [15] G. N. Kouvelos, I. Boletis, N. Papa, A. Kallinteri, M. Peroulis, and M. I. Matsagkas, “Analysis of effects of fixation type on renal function after endovascular aneurysm repair,” Journal of Endovascular Therapy, vol. 20, no. 3, pp. 334–344, 2013. 4. Conclusion Lessons learned from our case are never settled on the contrast effect into renal arteries at the time of completion angiography, and revascularization is still an option even when EVAR-related occlusion of the renal arteries has a delayed onset. Postoperative “chimney” might be considered a viable alternative if the renal artery overstenting is not completely occlusive also because it does not preclude a subsequent conventional bypass attempt. [12] K. Inan, A. Ucak, B. Onan, V. Temizkan, M. Ugur, and A. T. Yilmaz, “Bilateral renal artery occlusion due to intraoperative retrograde migration of an abdominal aortic aneurysm endo- graft,” Journal of Vascular Surgery, vol. 51, no. 3, pp. 720–724, 2010. [13] S. K. Subedi, A. M. Lee, and G. S. Landis, “Suprarenal fixation barbs can induce renal artery occlusion in endovascular aortic aneurysm repair,” Annals of Vascular Surgery, vol. 24, no. 1, pp. 113.e7–113.e10, 2010. References [1] R. Wald, S. S. Waikar, O. Liangos, B. J. G. Pereira, G. M. Chertow, and B. L. Jaber, “Acute renal failure after endovascular vs open repair of abdominal aortic aneurysm,” Journal of Vascular Surgery, vol. 43, no. 3, pp. 460–466, 2006. [16] M. Hamish, G. Geroulakos, D. A. Hughes, S. Moser, A. Shep- herd, and A. D. Salama, “Delayed hepato-spleno-renal bypass for renal salvage following malposition of an infrarenal aortic stent-graft,” Journal of Endovascular Therapy, vol. 17, no. 3, pp. 326–331, 2010. [2] M. Antonello, M. Menegolo, M. Piazza, L. Bonfante, F. Grego, and P. Frigatti, “Outcomes of endovascular aneurysm repair on [2] M. Antonello, M. Menegolo, M. Piazza, L. Bonfante, F. Grego, and P. Frigatti, “Outcomes of endovascular aneurysm repair on Case Reports in Vascular Medicine 5 [17] B. Williams, J. Feehally, A. R. Attard, and P. R. F. Bell, “Recovery of renal function after delayed revascularisation of acute occlu- sion of the renal artery,” The British Medical Journal, vol. 296, no. 6636, pp. 1591–1592, 1988. [18] J. Adu, N. J. Cheshire, C. V. Riga, M. Hamady, and C. D. Bicknell, “Strategies to tackle unrecognized bilateral renal artery occlu- sion after endovascular aneurysm repair,” Annals of Vascular Surgery, vol. 26, no. 8, pp. 1127.e1–1127.e7, 2012.
https://openalex.org/W2035394457	https://www.revistas.usp.br/anaismp/article/download/5373/6903	Portuguese	null	Influência da radiação de luz sobre acervos museológicos	Anais do Museu Paulista	2,001	cc-by	4,774	Anais do Museu Paulista. São Paulo. N. Sér. v. 8/9. p. 177-192 (2000-2001). Editado em 2003. A natureza da luz e seu comportamento Para abordamos este tema, sobre a natureza da luz e como ela age sobre os materiais expostos a ela, precisaremos da Óptica para as informações básicas e indispensáveis à compreensão de seu mecanismo, destinadas aos especialistas em conservação/ restauração. p Se fôssemos definir a luz diríamos que ela é uma forma de energia eletromagnética chamada radiação, e que tem origem a partir de uma fonte energética, tal como a luz natural e a luz artificial oriunda de lâmpadas. Chamamos de radiação ao processo de propagação de calor no qual a energia, denominada radiante, apresenta-se sob forma de ondas eletromagnéticas. A radiação é o único processo de propagação de calor que ocorre no vácuo. d d d d l d d l p p p g q A concepção moderna aceita a natureza da radiação da luz de duplo caráter: caráter de onda e de partícula devido a seu comportamento diferente conforme as condições do fenômeno que experimenta. Por exemplo: essa energia, quando atinge a matéria, pode se comportar de diferentes maneiras: pode ser refletida, absorvida, refratada, transmitida ou dispersada, dependendo das cores dos ambientes ou objetos nos quais incide. O ramo da Física que estuda estes fenômenos é a Óptica. Sabemos que as cores escuras absorvem mais a energia emitida, e as cores claras têm a propriedade de refletir. l d d f d f p p Esse comportamento é resultado da transformação que as radiações sofrem, uma vez que energia não se perde mas se transforma. Esta transformação pode ser, por exemplo, em calor, cor, som, etc. A Teoria Quântica explica o fenômeno da emissão e absorção de energia radiante entre a fonte de origem da radiação e a matéria. A Teoria Ondulatória explica o meio de transferência da energia de um lugar a outro (da fonte de energia para a matéria), isto é, seu movimento de propagação, que acontece através de movimentos ondulatórios, portanto comportando-se como uma onda. P l f d l Para simplificar, vamos considerar que a luz se propague como conseqüência de uma vibração de partículas (fótons). Nem todos os fótons têm a mesma capacidade de causar danos, uma vez que a energia está relacionada com a freqüência da radiação. As ondas eletromagnéticas têm características que ajudam a entender as radiações eletromagnéticas, objeto deste estudo. Influência da radiação de luz sobre acervos museológicos Norma Ciaflone Cassares Library Services Yara Lígia Mello Moreira Petrella Museu Paulista da USP Uma das causas mais comuns de degradação de materiais de acervos museológicos, bibliográficos e arquivísticos é a ação das radiações de luz, tanto natural quanto artificial presentes nos ambientes das instituições. Essa iluminação, natural e artificial, é um dos relevantes objetivos de estudos da área da Conservação Preventiva. As pesquisas buscam de forma incessante a compreensão da natureza da luz, os mecanismos de seu comportamento e seus efeitos sobre os diversos materiais que constituem as obras dos acervos. q Alguns indícios de degradação causada pela radiação de luz são identificados visualmente com certa facilidade, como é o caso do desbotamento ou mudança de cor dos materiais. Porém este sintoma é apenas uma indicação superficial de deterioração. Na realidade, ela atinge a estrutura química e física da matéria, e apesar de sua ação ser silenciosa, as conseqüências são desastrosas na conservação das obras. ç O caso do desbotamento causado pela ação da luz sobre o material colorido das obras é considerado um dos fatores dos mais graves de degradação em acervos, porque desfigura a obra, altera a sua leitura de forma irreversível. Por ser uma ação que se desenvolve fora do alcance das nossas vistas até que o dano seja detectado, é muito importante que se alcance o conhecimento amplo da natureza e comportamento dessas radiações para adotarmos procedimentos de conservação preventiva na proteção do acervo. Algumas características da radiação da luz que nos obrigam a não menosprezar seus poderes de degradação estão no fato de sua ação ser cumulativa e seus danos irreversíveis. 177 A natureza da luz e seu comportamento As mais importantes dizem respeito à freqüência e comprimento de onda Comprimento de Onda é a distância entre uma partícula em uma onda e a partícula correspondente na onda seguinte, ou a distância entre duas cristas, e é de vital importância para entendermos cada tipo de onda. As variações de comprimento de ondas entre as radiações eletromagnéticas são grandes. Há radiações com comprimento de onda de milionésimos de metro e de vários quilômetros. Já F üê i di i ú d ib õ i l id d Já a Freqüência diz respeito ao número de vibrações ou ciclos por unidade de tempo, que também é uma característica de diferencial das ondas. A medida é dada por ciclo/segundo (FIGURA 1). Enormes faixas de freqüência de radiações compõem o espectro eletromagnético. Este espectro é representado graficamente pelos diferentes comprimentos de ondas originados por uma fonte de emissão e pode ser dividido em oito regiões principais, dependendo do caráter geral das radiações: ondas elétricas, ondas de rádio, infravermelho, luz visível, ultravioleta, raio x, raios gama, raios cósmicos secundários. Esses nomes indicam áreas do espectro divididas com fins didáticos e práticos, pois o espectro é contínuo e não há diferenças abruptas entre as formas de radiação: todas constituem basicamente o mesmo fenômeno físico. Referindo-se à luz visível, esta afirmativa também é válida. No limite mais baixo de 400 nm ela se aproxima do intervalo do espectro da 178 radiação ultravioleta, e no outro limite, de 700 nm, ela se aproxima da infravermelha. Todas se irradiam pelo espaço com a mesma velocidade, que é de 300 mil km/s ou 3x108m/s. As diferenças estão no comprimento de onda e na freqüência da radiação, que fazem com que tenham diferentes características, como o poder de penetração dos raios x ou o aquecimento do infravermelho. As correspondências entre as unidades Angstron ( ) e nanômetros: 1 =10-8cm e 1nm=10 . A A A GURA 1 – Espectro eletro magnético. Fonte: MENDES, Marylka. Restauração Ciência e Arte. o de Janeiro: UFRJ-IPHAN, 1998,p. 288,289. ntre as unidades Angstron ( ) e nanômetros: 1 10 cm e 1nm 10 . A A A FIGURA 1 – Espectro eletro magnético. Fonte: MENDES, Marylka. Restauração Ciência e Arte. Rio de Janeiro: UFRJ-IPHAN, 1998,p. 288,289. FIGURA 1 – Espectro eletro magnético. Fonte: MENDES, Marylka. Restauração Ciência e Arte. Rio de Janeiro: UFRJ-IPHAN, 1998,p. 288,289. A natureza da luz e seu comportamento 179 Uma fonte de radiação, como o Sol, pode emitir luz de um espectro variado. Por exemplo, decompondo-se a luz solar com um prisma é possível ver o espectro de cores, como as do arco-íris. Outras são invisíveis ao olho humano, mas detectáveis por instrumentos (FIGURA 2). O termo luz, por convenção, indica a região de ondas eletromagnéticas que se encontram entre as frequências de ondas de 400 a 770 nm (nanômetros), indo da cor ultravioleta à infravermelha, passando pela azul, verde, amarela e roxa. Esta é a região do espectro óptico ou luz visível, capaz de reproduzir a sensação visual. Estas cores (cor luz), quando somadas em quantidades iguais, definem o aspecto da luz branca ou luz do Sol. Cada fonte de luz tem um espectro de radiação próprio, com comprimentos de onda distintos e com características e qualidades específicas. As radiações de infravermelho e de ultravioleta não são perceptíveis ao olho humano, mas podem ser detectadas por meio de película fotográfica ou equipamentos. Analisando as frequências de ondas do espectro, concluímos que partindo do ultravioleta para o infravermelho, o comprimento de onda vai do mais curto, que é a radiação mais energética, para o mais longo, menos energética. De onde concluímos que a radiação ultravioleta é a mais nociva dentro do espectro óptico, mas que não significa que é a única. Uma fonte de radiação, como o Sol, pode emitir luz de um espectro variado. Por exemplo, decompondo-se a luz solar com um prisma é possível ver o espectro de cores, como as do arco-íris. Outras são invisíveis ao olho humano, mas detectáveis por instrumentos (FIGURA 2). O termo luz, por convenção, indica a região de ondas eletromagnéticas que se encontram entre as frequências de ondas de 400 a 770 nm (nanômetros), indo da cor ultravioleta à infravermelha, passando pela azul, verde, amarela e roxa. Esta é a região do espectro óptico ou luz visível, capaz de reproduzir a sensação visual. Estas cores (cor luz), quando somadas em quantidades iguais, definem o aspecto da luz branca ou luz do Sol. C d f t d l t t d di ã ó i i t Cada fonte de luz tem um espectro de radiação próprio, com comprimentos de onda distintos e com características e qualidades específicas. A natureza da luz e seu comportamento As radiações de infravermelho e de ultravioleta não são perceptíveis ao olho humano, mas podem ser detectadas por meio de película fotográfica ou equipamentos. Analisando as frequências de ondas do espectro, concluímos que partindo do ultravioleta para o infravermelho, o comprimento de onda vai do mais curto, que é a radiação mais energética, para o mais longo, menos energética. De onde concluímos que a radiação ultravioleta é a mais nociva dentro do espectro óptico, mas que não significa que é a única. A influência da radiação de luz sobre os materiais de acervos museológicos e as conseqüências na degradação das coleções A influência da radiação de luz sobre os materiais de acervos museológicos e as conseqüências na degradação das coleções A leitura do espectro da radiação eletromagnética indica como interpretar a ação das radiações, com seus respectivos comprimentos de onda, sejam elas visíveis ou não. As radiações ultravioleta são a forma de luz mais destrutiva e energética devido ao comprimento de onda mais curto e com maior frequência e mais energia. Atingindo um objeto com maior energia e num tempo mais curto, apresenta um grande potencial de dano. A luz ultravioleta, não sendo visível ao olho humano, não faz falta ao ambiente de uma sala de exposição, e tudo deve ser feito para eliminá-la ou para que atinja o nível mínimo aceitável. p q j O processo fotoquímico é gerado principalmente pela ação da ultavioleta, por ser uma fonte poderosa de energia. A ação fotoquímica é o processo no qual uma molécula sofre uma mudança na sua estrutura química, por exemplo, por meio de energia de ativação provocada pela absorção de um fóton de energia da luz. Os fótons emitidos chocam-se com as moléculas dos objetos expostos, e se a energia tiver potência suficiente, produzirá danos químicos irreversíveis. Como o próprio termo diz – “reação fotoquímica” é aquela que ocorre com a absorção de luz. O processo de absorção de luz é melhor explicado nos termos da Teoria dos Orbitais Moleculares. Os elétrons dos átomos de uma molécula estão distribuídos nos orbitais atômicos em estados energéticos definidos. Cada orbital pode conter dois elétrons que ocupam uma região definida e uma energia também definida. Quando a molécula está no seu estado normal, esse sistema energético é o mais baixo possível e se mantém estável. Para que determinada molécula de uma matéria possa dar início a uma reação química com outras moléculas, ela necessita de uma certa quantidade de energia chamada energia de ativação. Os diferentes tipos de moléculas necessitam de diferentes energias de ativação. 180 RA 2 – Planta: é visível a ação da luz sobre a obra . O acondicionamento não cumpriu a o de proteger toda área da obra e as áreas que ficaram expostas tornaram-se escurecidas. rafia de Norma Cianflone Cassares. Acervo Biblioteca FAU/USP. URA 2 – Planta: é visível a ação da luz sobre a obra . O acondicionamento não cumpriu a ão de proteger toda área da obra e as áreas que ficaram expostas tornaram-se escurecidas. grafia de Norma Cianflone Cassares. Acervo Biblioteca FAU/USP. A influência da radiação de luz sobre os materiais de acervos museológicos e as conseqüências na degradação das coleções FIGURA 2 – Planta: é visível a ação da luz sobre a obra . O acondicionamento não cumpriu a função de proteger toda área da obra e as áreas que ficaram expostas tornaram-se escurecidas. Fotografia de Norma Cianflone Cassares. Acervo Biblioteca FAU/USP. 181 Diz-se que agora a molécula está em estado excitado. A energia de excitação pode ser transferida de uma molécula para outra. Quando os elétrons excitados são transferidos para outras moléculas, ou quando outro elétron ocupa posição deixada pelo elétron excitado, ocorrem mudanças químicas. A molécula se torna um íon ou um radical, ou ambos, de natureza muito reativa. A molécula resultante pode se combinar com ela mesma, ou provocar reações com as moléculas próximas, iniciando uma reação em cadeia. p ç p ç Portanto, quando uma molécula absorve fóton de energia de luz, natural ou artificial, seus elétrons são lançados para níveis mais altos de energia. Quando os elétrons voltam para seus níveis mais baixos de energia, eles liberam a mesma quantidade de energia que haviam absorvido. Esta liberação de energia pode romper as ligações e iniciar um processo de degradação. Conforme os comprimentos de onda ficam maiores na seqüência do espectro, elas tem menor energia, menor freqüência e menor capacidade de “excitar” as moléculas. q p Uma das reações fotoquímicas primárias é a oxidação, muito freqüente e nociva para todos os materiais. Nela a molécula “excitada” transfere sua energia para um oxigênio que reage com outras moléculas, iniciando assim um complexo processo de degradação. A luz ultravioleta, no caso de obras em suporte-papel, não ataca diretamente a molécula da celulose. A absorção de luz não rompe diretamente as ligações químicas de cadeia de celulose. Porém as impurezas, partículas metálicas e certos aditivos existentes no papel irão absorver a energia emitida pela luz. Essa energia absorvida irá projetar seus elétrons para órbitas mais energéticas da eletrosfera dos seus átomos. Quando esta energia é liberada e incide na celulose, pode ocorrer o rompimento das ligações que favorece o processo oxidativo. p g q p Os suportes como telas, tecidos, papel, como outras substâncias orgânicas, se oxidam sob a ação da radiação, e quando conjugados à ação da umidade se hidrolisam ou despolimerizam. O rompimento da cadeia polimérica se identifica pelo rompimento de fibras de papel, tecido, por exemplo. Outros materiais suscetíveis à ação da luz contidos numa pintura se danificam de formas diferenciadas. Os pigmentos e colorantes desbotam. A influência da radiação de luz sobre os materiais de acervos museológicos e as conseqüências na degradação das coleções Os vernizes tornam-se amarelecidos ou esbranquiçados, enquanto os aglutinantes das colas utilizadas na base de preparação podem se ressecar e contrair. A camada pictórica sobre esta base também é igualmente sensível à ação do infravermelho, que provoca a elevação de temperatura que, somada à umidade relativa acabam por produzir dilatação e contração desses materiais, surgindo assim os craquelados. ç ç g q Os danos causados pelos efeitos da radiação são tão mais graves quando: o tempo de exposição for mais longo, a intensidade da radiação de luz incidente no objeto for maior, quando a luz contém mais radiações azuis, violeta e ultravioleta, e quando a temperatura e a umidade relativa são mais altas. A elevação dos níveis de iluminação numa exposição para pôr em maior evidência o valor de uma obra, ou pela justificativa da estética da exposição, é totalmente equivocada e tem conseqüências nefastas para a conservação das pinturas. d f d d l d l d f d f q q p p A identificação de danos pela ação da luz pode ser feita de forma prática através de observação da aparência na celulose de papel, tintas, couros, têxteis utilizados em revestimentos das encadernações, jornais etc. (FIGURAS 3-9). Devemos lembrar que a ação das radiações, quando associadas às condições ambientais inadequadas, como temperatura e umidade relativa em índices elevados, assim como a presença de partículas de gases poluentes do ar, entre outras, potencializam as reações de degradação instaladas no material. 182 FIGURA 3 – O mesmo processo químico foi desencadeado no exemplar do jornal, com o agravante da existência de substâncias químicas residuais do processo de fabricação do papel. Fotografia de Norma Cianflone Cassares. Acervo particular. FIGURA 3 – O mesmo processo químico foi desencadeado no exemplar do jornal, com o agravante da existência de substâncias químicas residuais do processo de fabricação do papel. Fotografia de Norma Cianflone Cassares. Acervo particular. 183 184 FIGURA 4 – Obra sofreu danos devido ao longo tempo de exposição e iluminação em níveis elevados. O papel escureceu e as laterais estão mais claras porque estavam protegidas da luz. Fotografia de Norma Cianflone Cassares. Acervo particular. FIGURA 4 – Obra sofreu danos devido ao longo tempo de exposição e iluminação em níveis elevados. O papel escureceu e as laterais estão mais claras porque estavam protegidas da luz. Fotografia de Norma Cianflone Cassares. Acervo particular. 185 FIGURA 5 – Um exemplo de danos da radiação combinados com as impurezas de fabricação, entre elas partículas de metal (Fe) e o resultado é a degradação por oxidação e acidez. Fotografia de Norma Cianflone Cassares. Acervo particular. A influência da radiação de luz sobre os materiais de acervos museológicos e as conseqüências na degradação das coleções 184 FIGURA 5 – Um exemplo de danos da radiação combinados com as impurezas de fabricação, entre elas partículas de metal (Fe) e o resultado é a degradação por oxidação e acidez. Fotografia de Norma Cianflone Cassares. Acervo particular. 185 186 FIGURA 6 – A luz causou desbotamento na cor do couro, na área exposta à radiação de luz. Fotografia de Norma Cianflone Cassares. Acervo particular. FIGURA 6 – A luz causou desbotamento na cor do couro, na área exposta à radiação de luz. Fotografia de Norma Cianflone Cassares. Acervo particular. 186 187 FIGURA 7 – Danos causados pela ultravioleta e infravermelha no pergaminho utilizado na encadernação: ressecamento, distorção e fragilização. Fotografia de Norma Ciasnflone Cassares. Acervo particular. FIGURA 7 – Danos causados pela ultravioleta e infravermelha no pergaminho utilizado na encadernação: ressecamento, distorção e fragilização. Fotografia de Norma Ciasnflone Cassares. Acervo particular. 187 88 FIGURA 8 – Desbotamento do papel pela ação da luz ultravioleta nos cortes da guarda. Fotografia DE Norma Cianflone Cassares. Acervo Instituto de Biociências da USP. FIGURA 8 – Desbotamento do papel pela ação da luz ultravioleta nos cortes da guarda. Fotografia DE Norma Cianflone Cassares. Acervo Instituto de Biociências da USP. 188 FIGURA 9 – Galeria anteriormente iluminada com lâmpada mista (Azulada) criando tons esverdeados, alterando a cor das paredes. Fotografia de José Rosael, edifício Museu Paulista. FIGURA 9 – Galeria anteriormente iluminada com lâmpada mista (Azulada) criando tons esverdeados, alterando a cor das paredes. Fotografia de José Rosael, edifício Museu Paulista. 189 Princípio da Reciprocidade e as unidades de medidas da intensidade da luz A intensidade da iluminação de um ambiente se dá através da unidade de medida chamada lux. É definida como a iluminância que recebe uma superfície de 1m2 sobre a qual incide um fluxo luminoso de um lúmen. O Princípio da Reciprocidade considera o tempo de exposição dos objetos à radiação-luz e a intensidade da iluminação. Existe uma concordância entre os especialistas da área em que a meta de 200.000 lux/h por ano é aceitável para materiais sensíveis à luz. As opções para a exposição podem variar, por exemplo, em 50 dias com 100 lux por dia ou 50 lux por 100 dias. Enfim, o somatório de lux por ano não deve ultrapassar os índices recomendados. A influência da radiação de luz sobre os materiais de acervos museológicos e as conseqüências na degradação das coleções Portanto, devemos ressaltar que é de suma importância o planejamento de tempo de exposição de um objeto, e não só pela aplicação do nível de intensidade de luz recomendada em tabelas consideradas adequadas. q Materiais em processo de degradação têm que ser avaliados individualmente, se devem ou não ser expostos, considerando o conjunto das condições ambientais que se somam às ações da luz. Os processos de danos não ocorrem de forma isolada, mas sempre causados pelas complexas condições ambientais. Numa montagem de exposição devemos ter em consideração um planejamento de incidência de iluminação nas obras, sem esquecer a segurança e o conforto visual do visitante. Além deste estudo dos processos de danos, há ainda outros parâmetros a ser considerados. Quanto à qualidadedas lâmpadas atualmente oferecidas no mercado, tanto os fabricantes como os usuários estão preocupados com o consumo e custo de energia. Então, o uso racional e econômico de energia passou a ser mais uma referência na escolha das lâmpadas utilizadas num projeto de iluminação. p j Com o uso indiscriminado de iluminação artificial com níveis exagerados de irradiação nos locais de exposição, os danos estão se tornando ainda mais intensos e irreversíveis aos elementos componentes das obras expostas. Algumas considerações acrescentadas ao tema da Luz Temperatura de cor – é a grandeza que expressa a aparência de cor da luz e sua unidade é Kelvin (K). Quanto mais alta a temperatura de cor, mais branca é a cor da luz. A luz quente é a que tem aparência amarelada e temperatura de cor baixa: 3.000 K. A luz fria, ao contrário, tem aparência azul-violeta, com temperatura de cor elevada: 6.000 K ou mais. A luz branca natural é a emitida pelo Sol em céu aberto ao meio-dia, e tem a temperatura de cor de 5.800. A lâmpada mista em tom azulado, como mostra a Figura 9, cria tons esverdeados no ambiente em reação com as cores das paredes. Na Figura 10, utilizando-se as lâmpadas fluorescentes compactas amareladas, produz-se um ambiente mais agradável que se harmoniza com as cores das paredes. Embora seja um conceito subjetivo, a luz amarelada, para a maioria das pessoas, é considerada mais agradável aos nossos olhos. 190 FIGURA 10 – Galeria iluminada com lâmpada fluorescente compacta (amarelada) mantendo a d d F f d J R l d f M P l Recomendações importantes para amenizar ou mesmo excluir a incidência de radiação em acervos A instituição deve implementar uma política de controle ambiental, em que esteja incluído o controle de incidência de radiação luminosa tanto nos acervos, como nas reservas técnicas e exposições; buscar sempre especialistas que orientem quanto ao uso de tipos mais adequados de lâmpadas para a conservação do acervo; manter na política de conservação uma metodologia de controle e monitoramento das áreas onde incide a luz natural, e também a artificial e buscar a instalação de filtros adequados que sirvam de barreira para as radiações em áreas onde haja a incidência comprovada principalmente de ultravioleta e infravermelho. FIGURA 10 – Galeria iluminada com lâmpada fluorescente compacta (amarelada) mantendo a cor das paredes. Fotografia de José Rosael, edifício Museu Paulista. FIGURA 10 – Galeria iluminada com lâmpada fluorescente compacta (amarelada) mantendo a cor das paredes. Fotografia de José Rosael, edifício Museu Paulista. 191 Índice de Reprodução de Cor: Ra ou IRC – é a medida de correspondência entre a cor real de um objeto ou superfície e sua aparência diante da fonte de luz. A luz artificial, como regra, deve permitir ao olho humano perceber as cores corretamente ou o mais próximo possível da luz natural. Lâmpadas com Ra de 100% apresentam as cores com total fidelidade e precisão. Quanto mais baixo o índice, mais deficiente é a reprodução de cores. A lâmpada incandescente tem 100%, a fluorescente compacta 89, e a mista 50. Recomendações importantes para amenizar ou mesmo excluir a incidência de radiação em acervos Conservação de coleções em ambientes tropicais: coletando e comunicando dados do Museu Paulista/USP, Brasil (1997-2000) Conservação de coleções em ambientes tropicais: coletando e comunicando dados do Mus Paulista/USP, Brasil (1997-2000) g KEYWORDS: Climate in a Museum. Tropical Climate. Brazilian Climate. Brazilian Museums. Environmental Monitoring. Anais do Museu Paulista. São Paulo. N. Sér. v. 8/9. p.193-278 (2000-2001). Editado em 2003. Norma Ciaflone Cassares, Yara Lígia Mello Moreira Norma Ciaflone Cassares, Yara Lígia Mello Moreira O texto analisa a ação e os efeitos da radiação de luz sobre acervo museológico. Considera-se a luz como fator de dano produzindo alterações físicas e químicas sobre a estrutura molecular dos materiais dos objetos, que ocorrem sempre em conjunto com as condições ambientais e não de forma isolada. Aponta critérios técnicos, referências, relativos aos níveis de iluminação e ao tempo de exposição dos objetos. p j PALAVRAS-CHAVE: Conservação. Luz. Museus. Acervos. ç Anais do Museu Paulista. São Paulo. N. Sér. v. 8/9. p.177-192 (2000-2001). Editado em 2003. Anais do Museu Paulista. São Paulo. N. Sér. v. 8/9. p.177-19 REFERÊNCIAS CUTTLE, Christopher. Damage to Museum Objects Due to Light Exposure. International Journal of Lightning Research and Technology , v. 28, n. 1, p. 1,2,12 e 13. MICHALSKI, Stefan. Time’s Effects on Paintings. In: CONFERENCE SHARED RESPONSIBILITY: A SEMINAR FOR CURATORS AND CONSERVATORS, 1990, Ottawa. Shared Responsability. Ottawa: National Gallery of Canada. 1990. p. 39-53, reprinted. MICHALSKI Stefan . Towards Specific Lighting Guidelines. Preprints of THE 9TH TRIENNIAL MEETING, DRESDEN , GERMANY ( LOS ANGELES ICOM COMMITTEE FOR CONSERVATION 1990 ) : vol II Working Group #17 Lighting and Climate Control , p. 583-588. Reprinted by permission of the author. RESNICH, Robert. Física. [s. l.]: Ed. Livros Técnicos e Científicos Editora, 1974. v. 1, p. 154. WILLIAMS, John E. ; METCALFE , H. Clark; TRINKLEIN, Frederic E.; LEFLER, Ralph W. ; MELLO, L.J. Silva. Física moderna. Rio de Janeiro: Ed. Renes, 1970. v. 2, p. 259-304. Artigo apresentado em 10/2003. Aprovado em 11/2003. 192 Influência da radiação de luz sobre acervos museológicos Teresa Cristina Toledo de Paula As atividades de monitoramento e controle ambiental nas regiões temperadas originaram os parâmetros e práticas hoje estabelecidos mundialmente para a conservação de acervos; tais parâmetros e práticas, entretanto, podem não ser adequados à conservação de acervos em regiões tropicais. Este trabalho apresenta uma pesquisa sobre as condições ambientais em museu de região tropical, o Museu Paulista da Universidade de São Paulo. Trinta e três salas, halls e corredores nos quatro pavimentos do Museu Paulista têm sido monitorados por termo-higrógrafos desde 1997. Grandes variações na UR (30-98%) e temperatura (12-35 graus C) foram registradas. O efeito nocivo sobre as coleções, esperável em situações climáticas tão inconstantes, não foi encontrado onde há ventilação apropriada. O monitoramento possibilitou, também, a identificação de áreas de alto risco, onde ações localizadas podem ser introduzidas de forma econômica. Desenvolver um modo efetivo de comunicar essas informações ambientais à equipe do museu mostrou-se vital à implementação de medidas sustentáveis de monitoramento e controle climáticos. PALAVRAS-CHAVE: Clima em Museu. Clima Tropical. Clima Brasileiro. Museus Brasileiros. Monitoramento Ambiental. Anais do Museu Paulista. São Paulo. N. Sér. v. 8/9. p.193-278 (2000-2001). Editado em 2003. Caring for Collections in Tropical Environments: Collecting and Communicating Data at Muse Paulista/USP, Brasil (1997-2000) Caring for Collections in Tropical Environments: Collecting and Communicating Data at Museu Paulista/USP, Brasil (1997-2000) Norma Ciaflone Cassares, Yara Lígia Mello Moreira Norma Ciaflone Cassares, Yara Lígia Mello Moreira The text analyses the actions and effects of light on the museum’s collections. Light is considered of harmful effects leading to physical and chemical changes on the molecular structure of the materials of the objects, which always happen together with the environmental conditions rather than alone. It points out towards technical criteria, references, related to the levels of light and the exposure time of the objects. YWORDS: Conservation. Light. Museums. Collections. g Anais do Museu Paulista. São Paulo. N. Sér. v. 8/9. p.177-192 (2000-2001). Editado em 2003. Teresa Cristina Toledo de Paula The monitoring and environment control activities in the temperate regions have originated the parameters and the actions now established, world widely, for the conservation of the collections; such parameters and the actions, nevertheless, may not be adequate for the conservation of the collections in tropical regions. This work presents a research about the environmental conditions in a museum in a tropical region, the Museum of the University of São Paulo (Museu Paulista da Universidade de São Paulo). Thirty-three rooms, halls and corridors in the four of the museums floors have been monitored with thermo hygrographs since 1997. Huge variations in the UR (30-98%) and temperature (12-35 degrees C) have been registered. The harmful effect on the collections, expected in so changeable climatic situations, has not been found where there is proper ventilation. The monitoring has also enabled the identification of the high-risk areas, where local actions can be introduced in an economical way. Developing an effective way of passing ahead this environment information to the museum’s staff has been proved vital for the implementation of sustainable measures of monitoring and climatic control. 327 g KEYWORDS: Climate in a Museum. Tropical Climate. Brazilian Climate. Brazilian Museums. Environmental Monitoring. Anais do Museu Paulista. São Paulo. N. Sér. v. 8/9. p.193-278 (2000-2001). Editado em 2003.
https://openalex.org/W1979844929	https://zenodo.org/records/2317921/files/article.pdf	Dutch	null	Ballads, tunes and dances in Nash's works	Neophilologus	1,920	public-domain	4,229	40 40 Van Stockum. Artistieke mensch. Wel voelt ook deze zich als door eene kloof gescheiden van de menschen, maar niet uitgestooten, alleen hopeloos verwijderd, terwijl van ethische motieven, van een bewustzijn van minderwaardigheid nauwelijks lets blijkt. Is her te vermetel, deze verschillen te reduceeren tot een onderscheid in den duur van de nawerking van verschillende bewustzijnstoestanden, in casu aandoeningen? Claudio maakt zeer sterk den indruk van lichtzinnig, ver- anderlijk, ,,flatterhaft" te zijn. Men denke slechts aan de wijze, waarop hij zijne geliefde verlaat: ,,Und dab du mich dann ,,Und dab du mich dann Fortwarfest, achtlos, grausam wie ein Kind, Fortwarfest, achtlos, grausam wie ein Kind, Des Spielens miJd, die Blumen fallen 1/iBt" en aan het verloop van zijne vriendschap: ,,Du, schnellbefreundet, fertig schnell mit jedem". Daartegenover geeft ons de ,,Bajazzo", afgezien van eenige buien van uitgelatenheid, duidelijk her beeld van eene duurzame, op de nawerking en het onderling zich versterken van droeve ervaringen berustende melancholic. g g Zoo komt her mij voor, dat, terwijl beide gevallen eene zeer fundamenteele overeenkomst vertoonen, de tragiek in ,,Der Bajazzo" aanmerkelijk uitge- sprokener is, die van Claudio echter door het ontbreken van den socialen factor en misschien ook juist door de hem eigene lichtzinnigheid meer karakteristiek is voor de tragische ontwikkeling van den aesthetisch aan- gelegden mensch als zoodanig. Dr. Trt. C. VAN STOCKUM. Hilversara. BALLADS, TUNES AND DANCES IN NASH'S WORKS. As could only be expected of a man like Nash, whose writings were so actual at the time when they appeared, frequent reference is made in his works to ballads, songs, tunes and dances. Mr. Me Kerrow in his excellent edition of the works of Thomas Nash(e), has in most cases been able to elucidate the references in his ample notes, but a few allusions have eluded his efforts to identify the ballads and dances or the tunes to which they were composed. The benefit I have derived from Mr. Mc Kerrow's edition, makes it a pleasure to offer the following additional notes. p g In Have witk you to Saffron-Walden (III 30, 31) we read: As newfangled and idle, and prostituting my pen like a Curtizan, is the next ltera that you taxe me with; well it may and it may not bee so, for neither will I deny it nor will I grant it; onely thus farre lle goe with you, that twise or thrise in a month, when res est angusta dorai, the bottome of my purse is turned downeward, & my conduit of incke will no longer flowe for want of reparations, I am faine to let my plow stand still in the midst of a furrow, and follow some of these newfangled Oaliardos and Senior Fantasticos, to whose amorous Villa- hellas and Quipassas I prostitute my pen in hope of gaine. Q p p y p p g In his note to this passage (IV, 317) the editor says: =Quipassas]. The only other example of the word known to me is in Stanyhurst's account of Ireland, Swaen. 41 Nash's works. Swaen. in Holinshed's Chron., ed. 1807-8, VI. 13 mid., 'And trulie they [i. e. jests] beeset a divine as well, as for an asse to twang quipassa on a harpe or gitterne, or for an ape to friske trenchmoore in a paire of buskins and a doublet.' It probably represents the opening words of some well-known song, but I cannot identify it." g y The word also occurs in The Two Noble Kinsmen, III, 5, 98: Quipassa o'th bels and bones. Prof. Herford in his note (The Temple Dramatists) says: ,Qui passa: 'here passes'; but the point is unexplained." Mr. Tucker Brooke (The Shake- speare Apocrypha) passes the word in silence. BALLADS, TUNES AND DANCES IN NASH'S WORKS. Swaen 4 42 42 Swaen. Nash's works. For a description of the galliard I refer to Chappell, Popular Music of the olden Time, pp. 155, 6. , pp , There can be no doubt that Qui passa stands for the Italian Chi pussa, the first words of the galliard 'Chi passa per questa strada" mentioned in Thesaurus musicus (1574), in a German lutebookl) and in Met Luilboek van ThyMus. The German lutebook is in the library of 'de Vereeniging vo0r Noord-Nederlands Muziekgeschiedenis', at Amsterdam. It was written in Saxony about 1580, and is known as ,,Manuscript in deutscher Lauten- Tabulatur. Ende 16. Jahrh. V B 13." On p. 47 v. we find: 'Chi passa' in red characters, and under it 'gagliarda XXVII', which means that is the twenty-seventh of the galliards in the MS. In I-let Luitboek van ThyMus (Land's edition, p. 136, No. 131) the fuller name is given: 'Chi passa per questa strada' (Gaillarde). Qui for chi is merely the result of replacing the unfamiliar ch = 'k' by the familiar qu, 'qui passa' being probably taken for French. Why, he hath made a Prologue longer then his Play: nay, 'tis no Play neyther, but a shewe. Ile be sworne, the Iigge of Rowlands God-sonne is a Gyant in comparison of it. Summers Last Will and Testament. The Prologue. III, 235. In a note Mr. Mc Kerrow says: ',A first and second part of 'Rowlandes godson moralized' were entered in the Stationers' Register to John Wolf on April 18 and 29, 1592. The 'ligge' itself was presumably earlier, but nothing seems to be known of it." IV, 421. In tlerrigJs Archly, Vol. CXIV, pp. 326-357, Wilhelm Bolle has published "Das Liederbuch Ms. Rawlinson Poet. 185," a collection of Elizabethan bal- lads and songs preserved in the Bodleian Library. The ballads and songs were composed before 1590. Nr. XIV of Bolle's edition is entitled: ~A proper new ballett, intituled Rowlands god sonne. To the tune of Loth to departe." It is a lively dialogue in stanzas, based on the sixty-seventh tale in Boecaccio's Decamerone. The interlocutors are Besse (the wife), Jhon (the lover) and the husband. Two stanzas will give some idea of the form and tone of this spirited dialogue. Besse: Tell me, Jhon, why art thow soe sade? tell me Jhon, tell me Jhon, what isle will make thee glade? 0 These two references I owe to the kindness of Dr. D. $eheurleer, who also informs me that there is no mention of a galliard 'Qui passa' in Caroso's II Ballarino (1581) or in Tabourot's Orchisographie 0589). BALLADS, TUNES AND DANCES IN NASH'S WORKS. In Joseph Lilly's A collection of seventy-nine Black.Letter Ballads and Broadsides, printed in the reign of Queen Elizabeth, between the years 1559 and 1597 there is on pp. 138--40 ,,A proper newe Ballad sheweing that philosophers learnynges are full of good warnynges. And songe to the tune of my Lorde Marques Galyarde, or the first traces of Que passa". The accompanying note says: ,,A dance, properly called Qai passa, but sometimes spelt quipascie or kypaseie. There is a song 'to the tune of Kypascie' in the Handeful of Pleasant Delites, 1584" (p. 297). The song in A Handful of Pleasant Delights (Arber's Reprint, pp. 46, 47) is entitled: ',A Sonet of two faithfull Louers, exhorting one another to be constant. To the tune of Kypascie." Sonet in this case simply means song. From the title of the ballad in Lilly's collection it is quite evident that Quipassa is a dance. 'Trace' is 'a series of steps in dancing; a measure; a dance' (N. E. D.). Then the ballad could also be sung to 'my Lorde Marques Galyarde', which settles the character of the dance. And finally it is mentioned in the passage from Stanyhurst together with the Trenchmore, which is also a galliard. Evidently the movement of the Quipassa was not always the same, as appears from a comparison of the opening stanzas of the ballad and the 'sonet'. Philosophers learnings are ful of good warnings, In memorye yet left to schoole vs, So be ther contayned, in poietries fained, Great documents to rate and rule vs; As well fGr continuance of life, helth and substance, Whose vanities the world requireth, As for the derection of life by correction From lyberties that lust desireth. As well fGr continuance of life, helth and substance, As for the derection of life by correction From lyberties that lust desireth. The famous Prince of Macedon, The famous Prince of Macedon, Whose wars increst his worthy name Triumphed not so, when he had won By conquest great, immortall fame, As I rejoice, reioice, For thee, my choice, with heart and voice, Since thou art mine, Whom, long to loue, the Gods assigne. Whose wars increst his worthy name Triumphed not so, when he had won By conquest great, immortall fame, As I rejoice, reioice, For thee, my choice, with heart and voice, Since thou art mine, Whom, long to loue, the Gods assigne. BALLADS, TUNES AND DANCES IN NASH'S WORKS. thow knowest thy misteries loves thee well, soe dearely as I shune to tell. Tell me, I praye thee, lett nothing dismaye thee, but let me inioye thy love, thy love. y y y Jhon: 0 misteries myne, I cannot be merrye. Bes: Tell me, Jhon, tell me, Jhon, why lookes thow soe heavylye? Jhon: my master carries a jealous eie, and warnes me ffrom your companie. Jhon: my master carries a jealous eie, and warnes me ffrom your companie. y p Bess: heavens forfend it l - Jo: you maist amende it, or ells fare well to our love, our love. Bess: heavens forfend it l - Jo: you maist amende it, or ells fare well to our love, our love. 0 These two references I owe to the kindness of Dr. D. $eheurleer, who also informs me that there is no mention of a galliard 'Qui passa' in Caroso's II Ballarino (1581) or in Tabourot's Orchisographie 0589). $waen 4 43 Nash's works. $waen. Nash calls Rowlands Oodson a ,jigge", and an acquaintance with the ballad gives point to the above quoted passage. Prof. C. H. Firth in the chapter on Ballads and Broadsides in the second Volume of Shakespeare's Eagland, gives the following qualification of a jig. ,,At times more than one minstrel took part, and a ballad developed into a duet, or a semi- dramatic performance, in which several performers united. To these the name of 'jig' was usually given, which has been defined as 'a dramatic ballad or a ballad drama written to dance music, and capable of presentation by dance action on the stage.' In one example, 'Mr. Attowell's jigge', the characters are 'Francis, a gentleman; Richard, a farmer; and their wives'. In 'Rowlands Godson' there are three characters, Bess, Bess's husband, and John, Bess's lover. In 'Clod's Carrol' there are two characters only: it is described as 'a proper new jigg to be sung between a man and a woman that would need be married'. Some jigs extend to the length of twenty-six or twenty-eight verses" (pp. 515, 516). y g (pp ) This explains the meaning of the opening lines of 'The Prologue' of Tambarlaiae : From iygging vaines of riming mother wits, And such conceits as clownags keepes in pay, Weele lead you to the stately tent of war l). In a note on p. i) Mommsen's translation (vom Leierton gereimten Unverstands), quoted in Wagner's edition (p. V), entirely misses the point. BALLADS, TUNES AND DANCES IN NASH'S WORKS. 357 Herr Bolle says: ~In meiner Vorlage (i. e. the trans- cript made by Dr. S. Schayer) findet sich eine Notiz, in der bezweifelt wird, ob Titel und Lied zusammen geh6ren. Balladen in Dialog'form finden sich aber sehr h~iufig; die Melodie wurde eben yon den beiden Personen ab- wechselnd aufgenommen. Auch ist ja hier eine vollkommen strophische Gliederung vorhanden, es ist keineswegs ein fortlaufend dramatisiertes Ge- dicht .... Zwei verschiedene Melodien, benannt Loth to depart, sind bei Chappell I 102 abgedruckt; jedoch paBt keine yon ihne~. Der text eines schottischen Loath to depart steht in den Bagford ballads, II, 481." In an 'Appendix of supplementary Ballads', Mr. A. Clark in his edition of The Shirburn Ballads (1904) prints 'Rowlands godson', from the same MS., arranged as a 'four-act ballad drama', with stage directions (pp. 354- 360). In his prefatory note he remarks: The title 'Rowland's godson' is peculiar. When taken in connexion with No. LXX it seems as though Rowland in Elizabethan folklore were a generic name for a libertine or Don Juan." No. LXX of the Shirburn Ballads is 'A new song intituled: To wappe with a widdow', also called, from its first line, 'In Christmas time, as it befell.' The hero, a sower of wild oats, is named Rowland. At a later period 'jig' was only the name of a dance. There were numerous jigs, mostly named after some person, for instance: The King's, The Duke of Monmouth's, Johnson's, The Queen's, The Bishop of Chester's Jig, etc. In his note Mr. Me Kerrow refers to 'Rowlandes godson moralized'; bal- lads were often 'moralized', a good example being 'Greene Sleves moralised to the Scripture' (Stationers' Registers Sept. 15, 1580; Nash, III, 104). Swaen 4 44 Swaen. Nash's works. 'Peruse but the Ballet In Sandon soyle as late befell, and you will be more soundly edified by sixe parts.' III, 36. Mr. Mc Kerrow says the ballad is unknown to him. So is it to me, but there is a religious ballad in the Roxburghe Collection (Ballad Society's edition, Vol. I, pp. 401--400) entitled 'Glad tydings from Heaven. To the tune of The Dolefull Shepherd, or Sandy Soyle. BALLADS, TUNES AND DANCES IN NASH'S WORKS. But rather I deeme that from the harsh grating in his eares 8: continuall crashing of sextens spades against dead mens bones (more dismall musique to him than the Voyce or Ghosts Hearse) he came so to be incenst & to inueigh against the dead. 1-1ave with you to Saffron-Walden, III, 88. crashing of sextens spades against dead mens bones (more dismall musique to him than the Voyce or Ghosts Hearse) he came so to be incenst & to inueigh against the dead. 1-1ave with you to Saffron-Walden, III, 88. In a note the editor refers to The Shirburn Ballads, ed. A. Clark, p. 337, ,,where we find a ballad 'to the tune of The gosl~s hearse alias The voice of the earth'," adding 'So far as I am aware the original has perished.' The ballad is not printed in the Shirburn Ballads, only referred to with the addition of the three opening lines. The whole has been published in the publication of MS Rawlinson Poet. 185, referred to above (l-lerriKs Archiv, CXIV, pp. 354, 5). The title is: A sonnge (Shirb. Ball. 'sounge') in praise of the single life. To the tune of the gostes hearse, alius (Shirb. Ball. 'alias') the voice of the earth.' This ballad is identical with No. 10 of Deloney's The Oarland of Kood Will (The Works of Thomas Deloney, edited by F. O. Mann, pp. 328-30). I can find no trace of the original ballad or song. g g y ff , , In a note the editor refers to The Shirburn Ballads, ed. A. Clark, p. 337, ,,where we find a ballad 'to the tune of The gosl~s hearse alias The voice of the earth'," adding 'So far as I am aware the original has perished.' The ballad is not printed in the Shirburn Ballads, only referred to with the addition of the three opening lines. The whole has been published in the publication of MS Rawlinson Poet. 185, referred to above (l-lerriKs Archiv, CXIV, pp. 354, 5). The title is: A sonnge (Shirb. Ball. 'sounge') in praise of the single life. To the tune of the gostes hearse, alius (Shirb. Ball. 'alias') the voice of the earth.' This ballad is identical with No. 10 of Deloney's The Oarland of Kood Will (The Works of Thomas Deloney, edited by F. O. Mann, pp. 328-30). i) There is an 'Irish Boree', in the Dancing Master (1721, 200). BALLADS, TUNES AND DANCES IN NASH'S WORKS. I can find no trace of the original ballad or song. In stead of all these (I say) here is the tufft or labell of a rime or two, the trick or habit of which I got by looking on a red nose Ballet-maker that resorted to our Printing-house. They are to the tune of Labore Dolore, or the Parlement tune of a pot of ale and nutmegs and ginger, or Eldertons ancient note of meetin K the divell in conjure house lane. If you hit it right, it will go maruellous sweetly. y Gabriel l-laraey, fames dacklinK, g y Gabriel l-laraey, fames dacklinK, hey noddie, noddie, noddie: Is made a KoslinK and a sucklinK, hey noddie, noddie, noddie. -- Or that's not it, I haue a better. Dilla, my Doctor deare, Sin K dilla, diUa, dilla : Nashe hath spoyled thee cleare With hH quilla, quilla, quiUa. Have with you to Saffron Walden III 133 g y Gabriel l-laraey, fames dacklinK, hey noddie, noddie, noddie: Is made a KoslinK and a sucklinK, hey noddie, noddie, noddie. -- Or that's not it, I haue a better. Dilla, my Doctor deare, Sin K dilla, diUa, dilla : Nashe hath spoyled thee cleare With hH quilla, quilla, quiUa. Have with you to Saffron Walden III 133 hey noddie, noddie, noddie: Is made a KoslinK and a sucklinK, hey noddie, noddie, noddie. -- Sin K dilla, diUa, dilla : Nashe hath spoyled thee cleare With hH quilla, quilla, quiUa. Have with you to Saffron-Walden, III, 133. y This passage teems with difficulties. I believe Labore Dolore must be explained in the following manner. 'Labore' stands for La Boree, that is la bourr6e, a rustic dance belonging to Auvergne, first danced in Paris in 1587, and originally perhaps introduced from Spain. On p. 49 r. of the above- mentioned German Lutebook it is called El Burato. In Starter's Friesche Lusthof the song beginning 'Stil, stil een reys' (van VIoten's edition p. 42) is written to the tune of 'De Nieuwe Labor~'. Cf. Luitboek van Thysius, pp. 380, 395, 396 and Oud-l-lolland, I, p. 109 1). We have similar cases in 45 Nash's works. Swaen. Swaen. lacaranto, lagoranto for the coranto, courante, a favourite dance in Elizabethan days (v. l-lerr~ffs Archiv, CXIV, 332, 351); and in lava#o, lavolto, lavolt, lovalto, levalto, levolto for the Italian dance 'la volta' (cf. my article on 'Das Liederbuch MS. BALLADS, TUNES AND DANCES IN NASH'S WORKS. Rawlinson Poet. 185' in Herrigs Archiv, CXVI, pp. 374, 5). Perhaps the 'Margott laborez' of the Fitzwilliam VirgCnalbook (I, 321) also stands for 'la bor6e'. As regards the dolores I can only suggest that it is on a par with the dolorosa of the 'Gagliarda dolorosa' and the 'Pavana dolorosa' of the Fitzwilliam Virginalbook (ed. J. A. Fuller Maitland and W. Barclay Squire, 1894, I, p. 327 and p. 321); cf. the 'Gaillarde Bedroevet harteken' of Her Luitboek van ThyMus, p. 33. y , p 'Nutmegs and ginger' must be separated by a comma from 'a pot of ale' with which it has no connection. Readers of Beaumont and Fletcher will remember the following passage in the first act of The Knight of the Burning Pestle: y p 'Nutmegs and ginger' must be separated by a comma from 'a pot of ale' with which it has no connection. Readers of Beaumont and Fletcher will remember the following passage in the first act of The Knight of the Burning Pestle: g Old Mer. within. Nose, Nose, jolly red Nose, and who gave thee this jolly red Nose ? g Old Mer. within. Nose, Nose, jolly red Nose, and who gave thee this jolly red Nose ? j y Mist. Met. Hark my Husband he's singing and hoiting, And I'm fain to cark and care, and all little enough. j y Mist. Met. Hark my Husband he's singing and hoiting, And I'm fain to cark and care, and all little enough. g Husband, Charles, Charles Merry-thought. g Husband, Charles, Charles Merry-thought. Husband, Charles, Charles Merry-thought. y g Old Mer. Nutmegs and Ginger, Cinnamon and Cloves, And they gave me this jolly red Nose. Old Mer. Nutmegs and Ginger, Cinnamon and Cloves, And they gave me this jolly red Nose. Old Mer. Nutmegs and Ginger, Cinnamon and Cloves, And they gave me this jolly red Nose. j y Beaumont and Fletcher, ed. by A. R. Waller, Vol. VI, p. 176. This is one of the King Henry's Mirth or Freemen's Songs in Deutero- melia; or, the Second Part of Musick's Melodic, 1609, p. 7. Chappell (p. 75) gives the music. It seems still to have been sung in the nineteenth century. For further details I refer to my paper 'Notes on Ballads and Tunes in W. Sampsons Vow-Breaker a in Neophilologus, III (p. 152). BALLADS, TUNES AND DANCES IN NASH'S WORKS. p p g p 'Hey noddie, noddie, noddie' is an amusing variant of 'Hey honey (nonny, nony), honey', enabling Nash to call his antagonist a noddy. 'Hey honey honey, occurs in Res publica, III, 6: p p g p 'Hey noddie, noddie, noddie' is an amusing variant of 'Hey honey (nonny, nony), honey', enabling Nash to call his antagonist a noddy. 'Hey honey honey, occurs in Res publica, III, 6: Cantent: Hey, honey, nbny, houghe for money, etc.; in Much Ado About Nothing, II. 3: Cantent: Hey, honey, nbny, houghe for money, etc.; in Much Ado About Nothing, II. 3: Converting all your sounds of woe Into Hey nonny, nonny; Converting all your sounds of woe Into Hey nonny, nonny; g y Into Hey nonny, nonny; y y, y; in Hamlet IV, 5: They bore him barefaced on the bier: y y y in Hamlet IV, 5: They bore him barefaced on the bier: Hey non nonny, nonny, bey nonny; Hey non nonny, nonny, bey nonny; and in various songs and ballads. and in various songs and ballads. 'Dilla' afforded Nash another hit at Harvey, whom he accused of not being properly a doctor. 'Dilly' is used in the refrains of songs and ballads, for instance: and in various songs and ballads. 'Dilla' afforded Nash another hit at Harvey, whom he accused of not being properly a doctor. 'Dilly' is used in the refrains of songs and ballads, for instance: From thee lie never depart: Thou art my Ciperlillie: And I thy Trangdidowne dilly, And sing hey ding a ding ding. g Wily Beguiled, 2450- 3, Swaen 4 Swaen. Swaen. 46 Nash's works, Nash's works, and fa la la Langtidowne, Langtidowne dilly, the burden of Joane's song in the first act of Heywood's The Witches of LancasMre. the burden of Joane's song in the first act of Heywood's The Witches of LancasMre. 'Dill' is the name of an umbelliferous plant (Anetham graveolens) from which a carminative draught, called dill.water, is prepared. The 'a' of 'dilla' is the well-known postfixed ' a(h)', still common in songs t). What could be more apposite than the refrain 'sing dilla, dilla, dilla', which every reader would at once connect with 'dill' and with 'dilly', in a song meant to ridicule a doctor? In the last stanza 'quilla' stands similarly for 'quill-a; and the meaning is 'Nashe hath spoyled thee clcare with his quill', i. BALLADS, TUNES AND DANCES IN NASH'S WORKS. e. with his pamphlets. These allusions would appeal to a reader of that time and enhance his enjoyment of Nash's merciless onslaught on Harvey. A.E.H. SWAEN. Amsterdam. l) For instance: There was a London gentlewoman That lov'd a country-man-a; And she did desire his company A little now and than-a. Fa la~ &c. Th EEN JAPANSCH PORTRET VAN MILTON. De verschiUende afbeeldingen van Milton hebben aanleiding gegeven tot heel wat geschrijf. Dit is het gevolg hiervan dat er afbeeldingen bestaan wier echtheid onomstootelijk vaststaat en andere omtrent wier echtheid be- denken wordt gekoesterd. Een korte opsomming der voornaamste portretten zal niet ondienstig zijn. g j I. Het ,,Disney portrait", Milton voorsteUende als knaap van tien jaar en misschien geschilderd door Cornelis Janssen. g j I. Het ,,Disney portrait", Milton voorsteUende als knaap van tien jaar en misschien geschilderd door Cornelis Janssen. 2. Een geschilderd portret van Milton als jonkman van twintig jaar, afkomstig uit de nalatenschap van's dichters weduwe. In 1828 werd het verkocht, doch men weet niet waar het is gebleven. Her is welbekend door de gravure van Vertue. g 3. De teekening van Faithorne, gemaakt in 1640, {Den Milton twee en zestig jaar oud was. Dit portret, dat in 1760 nog bestond, is verdwenen, maar een gravure ervan werd door Faithorne zelf gemaakt voor Milton's History of Britain en het werd ge~tst door Cipriani. Het is het bekende konterfeitsel met de stroeve trekken. 4. Een portret dat reel op het vorige gelijkt, in het bezit is der familie Baker te Bayfordbury, en in 1861 gephotografeerd werd voor een boek van S. L. Sotheby, Ramblings in the elucidation of the autograph of Milton. 4. Een portret dat reel op het vorige gelijkt, in het bezit is der familie Baker te Bayfordbury, en in 1861 gephotografeerd werd voor een boek van S. L. Sotheby, Ramblings in the elucidation of the autograph of Milton. y g f g p f Deze portretten worden als authentiek beschouwd. Deze portretten worden als authentiek beschouwd. 5. Het borstbeeld in de boekerij van Christ's College te Cambridge, dat nu vrijwel algemeen als echt word{ aangenomen en dat omtrent 1654 ge- maakt moet zijn naar een pleisterafgietsel.
https://openalex.org/W4385247546	https://cp.copernicus.org/articles/19/2127/2023/cp-19-2127-2023.pdf	English	null	Reply on RC2	null	2,023	cc-by	22,031	Reconstructing 15 000 years of southern France temperatures from coupled pollen and molecular (branched glycerol dialkyl glycerol tetraether) markers (Canroute, Massif Central) Reconstructing 15 000 years of southern France temperatures from coupled pollen and molecular (branched glycerol dialkyl glycerol tetraether) markers (Canroute, Massif Central) Léa d’Oliveira1, Lucas Dugerdil1,2, Guillemette Ménot2, Allowen Evin1, Serge D. Muller1, Salomé Ansanay-Alex2, Julien Azuara3, Colline Bonnet1, Laurent Bremond1, Mehmet Shah4, and Odile Peyron1 1ISEM UMR 5554, CNRS, IRD, EPHE, Université de Montpellier, 34090 Montpellier, France 2LGL-TPE UMR 5276, CNRS, ENS de Lyon, Université Lyon 1, 69364 Lyon, France 3Chrono-environnement UMR 6565, CNRS, Université de Franche-Comté, 25030 Besançon, France 4ASM UMR 5140 U i ité d M t lli 3 34199 M t lli F Correspondence: Léa d’Oliveira (lea.d-oliveira@umontpellier.fr) Correspondence: Léa d’Oliveira (lea.d-oliveira@umontpellier.fr) Received: 28 March 2023 – Discussion started: 4 April 2023 Revised: 4 September 2023 – Accepted: 18 September 2023 – Published: 1 November 2023 Received: 28 March 2023 – Discussion started: 4 April 2023 Revised: 4 September 2023 – Accepted: 18 September 2023 – Published: 1 November 2023 ent modern databases highlights the importance of consid- ering environmental and ecological constraints when using transfer functions on pollen sequences. Pollen- and brGDGT- inferred climate trends are consistent, notably for the Late Glacial and the Early and Late Holocene. However, the re- constructions notably differ concerning the presence of a Holocene thermal maximum with the MAT pollen-based method, but no difference is apparent with the BRT pollen method nor brGDGT. The temperature reconstructions es- timated from the two independent pollen and lipid proxies are then compared to regional climate signals (chironomids, pollen, molecular biomarkers) to better understand global regional climatic patterns in southern Europe. Altogether, our results from the Canroute sequence and those already available in southern Europe reveal that for the Late Glacial and Early Holocene, the regional climate trends are consis- tent between sites and proxies, supporting the reliability of their reconstructions despite some discrepancies. During the Holocene, the temperature signal of Canroute does not in- dicate the clear presence of a pronounced HTM, but rather stable temperatures. Abstract. Climatic changes in southern Europe during the Holocene are characterized by a strong spatial and temporal heterogeneity whose patterns are still poorly understood, no- tably the presence or not of a Holocene thermal maximum (HTM; 10 000–6000 cal BP). The climatic patterns also dif- fer according to the proxies used (e.g. pollen, chironomid) and the latitude of the record. Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. Reconstructing 15 000 years of southern France temperatures from coupled pollen and molecular (branched glycerol dialkyl glycerol tetraether) markers (Canroute, Massif Central) Léa d’Oliveira1, Lucas Dugerdil1,2, Guillemette Ménot2, Allowen Evin1, Serge D. Muller1, Salomé Ansanay-Alex2, Julien Azuara3, Colline Bonnet1, Laurent Bremond1, Mehmet Shah4, and Odile Peyron1 1ISEM UMR 5554, CNRS, IRD, EPHE, Université de Montpellier, 34090 Montpellier, France 2LGL-TPE UMR 5276, CNRS, ENS de Lyon, Université Lyon 1, 69364 Lyon, France 3Chrono-environnement UMR 6565, CNRS, Université de Franche-Comté, 25030 Besançon, France 4ASM UMR 5140, Université de Montpellier 3, 34199 Montpellier, France Correspondence: Léa d’Oliveira (lea.d-oliveira@umontpellier.fr) Received: 28 March 2023 – Discussion started: 4 April 2023 Revised: 4 September 2023 – Accepted: 18 September 2023 – Published: 1 November 2023 Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction This highlights the need for further palaeoclimatic studies in this region based on independent proxies, particu- larly in southern Europe, for which the past climate remains poorly understood (Peyron et al., 2013, 2017; Samartin et al., 2017). In recent years, quantitative reconstructions of tempera- ture based on branched GDGTs (brGDGTs) were proposed in both marine and continental ecosystems (e.g. Schouten et al., 2013; Zheng et al., 2015, 2018; Ardenghi et al., 2019; Rodrigo-Gámiz et al., 2022; Dugerdil et al., 2021a, b). Transmembrane lipids, synthesized by Archaea or Bac- teria (Weijers et al., 2009; Pearson and Ingalls, 2013), have been identiﬁed in a wide range of environments including soils, peat, and lake and marine sediments (Hopmans et al., 2004; Weijers et al., 2006; Huguet et al., 2010; Pearson et al., 2011; Peterse et al., 2012; De Jonge et al., 2014b; Li et al., 2016; Naafs et al., 2017a, b). Two main fam- ilies of GDGTs can be distinguished: isoprenoid GDGTs (isoGDGTs) and brGDGTs. Although the archaeal (Thau- marchaeota) origin of isoGDGTs has been validated, the brGDGTs’ source remains a subject of debate and investi- gation (Sahonero-Canavesi et al., 2022; Zeng et al., 2022). However, recent studies have shown GDGT production in bacterial culture (Chen et al., 2022; Halamka et al., 2023), supporting a bacterial origin for brGDGTs. A relationship between the structure of biomolecules synthesized by organ- isms and environmental conditions to maintain cell viabil- ity has been demonstrated (Weijers et al., 2004; Naafs et al., 2021). More speciﬁcally, relationships between the degree of methylation and temperature (MBT) as well as between the degree of cyclization (CBT) and pH have been documented (Weijers et al., 2004, 2007). Furthermore, studies showed the presence of methyl isomers at the C6 position (6-methyl iso- mer), which when excluded from the MBT index resulted in the better-temperature-correlated MBT’5Me index (Naafs et al., 2017b), and a novel C7 position (7-methyl isomer) that co-elute with the 5- and 6-methyl brGDGTs (Ding et al., 2016). De Jonge et al. (2019) revealed that brGDGTs had a varied relationship with temperature and pH in “warm” and “cold” bacterial communities, demonstrating that those correlations are also reliant on the bacterial population. A “community index” (CI ratio; De Jonge et al., 2019) has been deﬁned to assess whether there is a change between the bacterial community and temperature or pH. De Jonge et al. 1 Introduction dialkyl glycerol tetraethers (GDGTs) (Naafs et al., 2019), whose distribution and abundance are partly governed by en- vironmental (e.g. pH) and climatic (e.g. temperature) factors, making them ideal proxies for palaeoclimatic reconstructions in the continental domain (Raberg et al., 2022). However, lit- tle research based on GDGTs has been conducted on the con- tinental realm thus far, with the majority focused on lacus- trine environments (c.f., Sun et al., 2011; Sinninghe Damsté, 2016; Russell et al., 2018). The Holocene epoch (last 11 700 cal BP) is considered to be a stable climatic period compared to the previous Late Glacial period, which corresponds to the deglaciation be- tween ca. 15 000 and 11 700 cal BP and alternating phases of rapid warming and cooling (Mayewski et al., 2004). How- ever, the Holocene demonstrates regional climate oscillations at millennial and centennial timescales (Smith et al., 2016). In Europe, at a millennial scale, palaeoclimatological studies have indicated the occurrence of a Middle Holocene thermic optimum called the “Holocene thermal maximum” (HTM) (Liu et al., 2014) dated between 10 000 and 6000 cal BP (e.g. Renssen et al., 2012; Marcott et al., 2013; Kaufman et al., 2020; Cartapanis et al., 2022). The temperature trends dur- ing this optimum show strong latitudinal patterns which dif- fer between regions (Herzschuh et al., 2023). Marine prox- ies record an optimum in southern Europe and the Mediter- ranean region (Kaufman et al., 2020; Marriner et al., 2022), but this optimum is not clear for terrestrial proxies. Several studies suggest major differences according to region and latitude, as well as the proxy or seasonal parameter stud- ied (Samartin et al., 2017; Erb et al., 2022). Pollen-based palaeoclimate studies have highlighted the Holocene climate heterogeneity in southern Europe and suggest for the Mid- dle Holocene similar or cooler conditions than the current ones (Cheddadi et al., 1997; Davis et al., 2003; Mauri et al., 2015; Marsicek et al., 2018; Erb et al., 2022; Herzschuh et al., 2023). However, these climate patterns are not sup- ported by atmospheric climate model outputs, which indi- cate a clear warming of northern and southern Europe during the Holocene (Mauri et al., 2014; Liu et al., 2014; Erb et al., 2022). Reconstructing 15 000 years of southern France temperatures from coupled pollen and molecular (branched glycerol dialkyl glycerol tetraether) markers (Canroute, Massif Central) Here, a multi-proxy approach combining pollen and lipid biomarkers (branched glycerol dialkyl glycerol tetraethers, brGDGTs) is applied to the Can- route sedimentological sequence (Massif Central, France) to reconstruct the climatic variation over the last 15 000 years in southern Europe. This area is poorly documented in terms of vegetation and climate change. To provide reliable climate reconstructions, we have (1) performed a multi-method ap- proach applied to pollen (modern analogue technique, MAT; weighted averaging partial least squares regression, WA- PLS; boosted regression trees, BRT; and random forest, RF) and molecular biomarkers brGDGTs (ﬁve calibrations) and (2) investigated the role of modern databases and calibra- tions in climate reconstructions. Three different databases were tested for pollen data: one global database based on a Eurasian pollen database and two regional databases corre- sponding to Mediterranean–Temperate Europe and Temper- ate Europe–Scandinavian databases respectively. Five global calibrations were tested for lipid biomarkers including four for soil and one for peat. Results show that the use of differ- Published by Copernicus Publications on behalf of the European Geosciences Union. L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2128 1 Introduction Because crenarchaeol and GDGT-0 can be derived from Group I Crenarchaeota, the GDGT-0 / crenarchaeol ratio can be used to investigate the presence of methanogenic Archaea that thrive in anoxic con- ditions in sediments, whereas methanogenic Archaea synthe- size GDGT-0, but no crenarchaeol (Blaga et al., 2009). The lower the ratio, the lower the anoxic conditions. Indices and calibrations have been developed to allow quantitative recon- struction of palaeotemperatures based on archive type and re- gion (Weijers et al., 2007; Peterse et al., 2012; De Jonge et al., 2014b; Naafs et al., 2017a, b; Dearing Crampton-Flood et al., 2020). These calibrations were developed from global databases that group together surface samples, for which the current climatic conditions are known, from distinct types of substrates: soils (Peterse et al., 2012; De Jonge et al., 2014b; Naafs et al., 2017b; Dearing Crampton-Flood et al., 2020), lake sediments (Russell et al., 2018; Martinez-Sosa et al., 2021; Raberg et al., 2021; Zhao et al., 2023), and peat (Naafs et al., 2017a). The aim of this study is (1) to address Holocene regional climate variability within the context of the Holocene ther- mal maximum in southern Europe from a peat sequence ex- tracted in southern Massif Central (Canroute peatland) us- ing a pollen–brGDGTs multi-proxy approach; (2) to con- tribute to palaeoclimatic reconstructions based on brGDGTs extracted from peat, which are still very little addressed in the ﬁeld of biogeochemistry; and (3) to improve the reliabil- ity of the climate signal obtained from pollen data thanks to a multi-method approach (i.e. using four different methods in- cluding recent machine learning such as boosted regression trees) and the use of three modern pollen databases (regional to global). The use of brGDGTs to reconstruct annual temperatures in Europe during the Late Glacial and/or the Holocene is still rare (e.g. Martin et al., 2020; Robles et al., 2023; Rodrigo-Gámiz et al., 2022; Ramos-Roman et al., 2022). Due to the complexity and number of interactions through- out each ecosystem (Birks and Birks, 2006), it is advisable to use several independent proxies to obtain reliable tem- perature reconstructions (e.g. Ponel et al., 2022). 1 Introduction (2019) determined a threshold value of 0.64 to sepa- rate the two groups of bacterial communities. If the CI ra- tio threshold is exceeded, a shift in the bacterial commu- nities might be predicted, perhaps affecting the relationship between brGDGTs and temperature or pH (De Jonge et al., 2021). Furthermore, edaphic factors such as anoxic or oxic conditions have an impact on GDGT production and bacte- Terrestrial records provide especially useful information on climate change. However, terrestrial records can be in- ﬂuenced by environmental factors (e.g. erosion on detrital activity, elevation), which makes the responses of continen- tal ecosystems to climate change difﬁcult to interpret (Mar- tin et al., 2020). Erosion has an impact on the detrital con- tribution to terrestrial archives; its dynamics are linked to, but not solely to, climate changes. Land clearing, for exam- ple, can increase detrital activity and so impact the terrestrial record (van Andel et al., 1990). Peatlands are powerful envi- ronmental archives, used in palaeoecology for their capacity to conserve palaeoclimatic markers (Moore, 1989). The ac- cumulation and preservation of pollen and other proxies in peatlands make it possible to reconstruct quantitatively vari- ations in climatic parameters, such as mean annual and/or seasonal temperatures and/or precipitation (Salonen et al., 2019). Peatlands are particularly rich in organic matter, con- ducive to the presence of lipid biomarkers such as glycerol https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 2129 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures tiﬁcation should be interpreted with caution for periods with small temperature changes (< 2 ◦C), as is the case for the Late Holocene (last 2000 years) (Naafs et al., 2019). In this sense, multi-proxy approaches are one of the best ways to estimate the reliability of quantitative climate reconstruc- tions, as it is easier to disentangle local from regional events (Ponel et al., 2022). Although some studies compare palaeo- climatic reconstructions based on pollen and brGDGTs, al- lowing complementary and more robust interpretations to be reached (Martin et al., 2020; Dugerdil et al., 2021a, b; Rob- les et al., 2022, 2023; Ramos-Roman et al., 2022; Watson et al., 2018), very few have been carried out on peat se- quences, with none of them concerning south-western Eu- rope and the Mediterranean region nor covering the entire last 15 000 years. rial communities (Weber et al., 2018). 1 Introduction The gen- eral assumption is that all proxies used to reconstruct climate changes are to some extent dependent on climatic parame- ters, but other factors, including human activities, biologi- cal processes, edaphic conditions, pH shift, and so on, can inﬂuence their distribution and/or abundance (Sugita et al., 2006; Huguet et al., 2010; Martin et al., 2020; De Jonge et al., 2021). In peat, this local environmental context can be understood through geochemical and sedimentary analyses based on X-ray ﬂuorescence analysis (XRF) or loss on igni- tion (LOI) to investigate mineral inputs and organic matter content. However, the pollen record can also be inﬂuenced by anthropogenic, biological, and environmental processes which can alter pollen production, dispersal, and preserva- tion (Sugita et al., 2006). Production of molecular biomark- ers, such as brGDGTs, may also differ depending on the source, edaphic parameters (e.g. anoxic or oxic conditions), soil type, and vegetation (Weber et al., 2018; De Jonge et al., 2021; Robles et al., 2022). Human activities, like defor- estation and agriculture, can disturb the natural record of the vegetation–climate interaction, resulting in a biased quanti- tative reconstruction of climatic parameters from pollen data (Seppä and Bennett, 2003; Birks and Seppä, 2004). Fur- thermore, several studies document anthropogenic impacts on bacterial communities, demonstrating that reconstructions based on brGDGTs might be disrupted by human interven- tion in speciﬁc contexts, such as watersheds (Martin et al., 2019). In addition, due to the still signiﬁcant calibration er- rors (±3.8 to ±5.5 ◦C), brGDGT-based palaeoclimate quan- 2.1 Study area The Canroute peatland (43◦38′48′′ N, 02◦34′35′′ E; 790 m al- titude; Fig. 1a) is located in the south of the Massif Cen- tral (France), in the Monts de Lacaune. This soligenous Sphagnum peatland is supplied by several streams and small springs (Muller et al., 2018; Fig. 1b) and harbours a diversi- ﬁed vegetation including several species associated with the western Atlantic Ocean inﬂuence. The peatland is located at the conﬂuence of three distinct climatic regimes: Mediter- ranean inﬂuence from the south; the inﬂuence of the Atlantic Ocean from the west due to Atlantic air masses arriving from the country’s west coast, which are not prevented by any to- pographical obstacles in the Aquitaine Basin; and a moun- tainous regime from the north (Fig. 1a). These inﬂuences result in an average annual temperature of 9.5 ◦C, a tem- perature seasonality (TS; standard deviation of the monthly mean temperatures) of 0.5 ◦C (WorldClim 2.0; Fick and Hi- jmans, 2017), higher summer temperatures, and an average annual rainfall of 895 mm with a slightly drier summer pe- riod (Fig. 1c and Table A1; CRU TS version 4.06; Harris et al., 2020) and a precipitation seasonality (SoP; standard de- viation of the monthly precipitation) of 21 (WorldClim 2.0; Fick and Hijmans, 2017). Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2130 Figure 1. (a) Location and altitude of the Canroute peatland and modern surface samples (NASA JPL, 2013). Elevations of the sample departments (Aveyron, Gard, Hérault, Haute-Loire, Lozère, and Tarn). The palaeoclimate records discussed in the text are represented by black dots in the inset map on the right (1: Swiss Alps, Heiri et al., 2003; 2: Lake St Front (Massif Central), Martin et al., 2020; 3: lakes Gemini and Verdarolo (northern Apennines), Samartin et al., 2017; 4: Lake Matese (Italy), Robles, 2022; 5: Gulf of Lion, Jalali et al., 2016). The south-western Europe climate reconstruction provided by Davis et al. (2003) is based on numerous pollen records (extracted from the European Pollen Database, EPD), which are not shown for clarity. (b) Aerial view of Canroute peatland. (c) Current annual conditions (precipitation and temperature) of Canroute (Monts de Lacaune). Figure 1. (a) Location and altitude of the Canroute peatland and modern surface samples (NASA JPL, 2013). Elevations of the sample departments (Aveyron, Gard, Hérault, Haute-Loire, Lozère, and Tarn). 2.4 Sedimentological analysis The CAN02 sequence was analysed by energy-dispersive X-ray ﬂuorescence (ED-XRF) spectrometry using a Delta InnovX DP4000 portable spectrometer. The analyses were conducted on bulk sediments to preserve as much material as possible for other palaeoenvironmental studies. A three- beam “soil” analytical method was used to measure the con- tents of trace elements (Pb, Zn, Rb, Sr, Zr, Ba) and of Ti. The 2.1 Study area The palaeoclimate records discussed in the text are represented by black dots in the inset map on the right (1: Swiss Alps, Heiri et al., 2003; 2: Lake St Front (Massif Central), Martin et al., 2020; 3: lakes Gemini and Verdarolo (northern Apennines), Samartin et al., 2017; 4: Lake Matese (Italy), Robles, 2022; 5: Gulf of Lion, Jalali et al., 2016). The south-western Europe climate reconstruction provided by Davis et al. (2003) is based on numerous pollen records (extracted from the European Pollen Database, EPD), which are not shown for clarity. (b) Aerial view of Canroute peatland. (c) Current annual conditions (precipitation and temperature) of Canroute (Monts de Lacaune). 2.2 Coring and sampling team, 2023) and the R Studio software (Posit team, 2023) with the Clam package (Blaauw et al., 2022) using the Int- Cal20 calibration (Reimer et al., 2020). No reservoir effect corrections were performed on the 14C measurements. The Canroute core (CAN02) extraction was carried out in 2019 using a 100 cm long Russian corer. Two 100 cm sec- tions were taken, spaced 20 cm apart, to cover a total depth of 169 cm. Six peat surface samples from the Massif Central were taken to reﬁne the selection of calibrations for the reconstruc- tion based on brGDGTs (Fig. 1a and Table A1). 2.5.2 Indexes calculations Figure 2. Age–depth model from the CAN02 sequence, built with Clam (Blaauw et al., 2022) in R (R Core Team, 2022). The shaded period corresponds to the interval of accumulation rate increase be- tween 110 and 60 cm depth (6500–5000 cal BP). Different indices such as MBT’5Me, CBT5Me, CBT’, In- dex1, GDGT-0 / crenarchaeol, IR (isomer ratio), and CI ra- tio (community index) were calculated for the CAN02 se- quence and surface samples (Table 1). The mean annual temperature (MAAT) was reconstructed from three types of calibrations: the linear relationship between methylation indices (MBT’5Me) (De Jonge et al., 2014a; Naafs et al., 2017b) and Index1 (De Jonge et al., 2014a), multiple re- gression (mr) between MAAT and fractional abundances of selected brGDGTs (De Jonge et al., 2014a), and Bayesian calibration (Dearing Crampton-Flood et al., 2020; RMSE = 3.8 ◦C). Due to the removal of the pH-dependent 6-methyl brGDGTs, MBT’5Me- and Index1-based calibrations allow the substantial correlation between MBT and soil pH to be overcome (De Jonge et al., 2014a). Multiple regression con- nects the MAAT with the fractional abundance of tetra- and penta-methylated brGDGTs and shows a little accuracy im- provement over MBT’5Me-based calibration (De Jonge et al., 2014a). Bayesian-based calibration allows the intuitive rea- soning of the relationship between MBT’5Me and MAAT to be respected (i.e. brGDGT-producing Bacteria respond to temperature changes, not the other way around) (Dearing Crampton-Flood et al., 2020). The Bayesian calibration em- ployed in this study refers to the threshold-based calibration of Dearing Crampton-Flood et al. (2020), which calibrates the MBT’5Me index to the average temperature of all months with an average temperature above freezing. element contents of Si, K, Ca, and Fe were measured using a two-beam “minerai +” analytical mode. element contents of Si, K, Ca, and Fe were measured using a two-beam “minerai +” analytical mode. The organic matter content (OMC) was measured by loss on ignition (LOI) at 550 ◦C (Ball, 1964). For this study, 16 samples of 1 cm3 were taken every 10 cm. Each sample was weighed after drying for 12 h at 150 ◦C. Then, a calcination at 550 ◦C for 5 h was performed to estimate the OMC (percent of dry mass) (Decorsiere et al., 2019). The organic matter content (OMC) was measured by loss on ignition (LOI) at 550 ◦C (Ball, 1964). For this study, 16 samples of 1 cm3 were taken every 10 cm. 2.3 Age–depth model Radiocarbon dating of the CAN02 sequence was carried out by the Pozna´n Radiocarbon Laboratory (Poland) on 16 peat samples (bulk). The calibration and the age–depth model (Ta- ble 2 and Fig. 2) were performed with the R language (Posit Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2131 Figure 2. Age–depth model from the CAN02 sequence, built with Clam (Blaauw et al., 2022) in R (R Core Team, 2022). The shaded period corresponds to the interval of accumulation rate increase be- tween 110 and 60 cm depth (6500–5000 cal BP). samples were then analysed in hexane–isopropanol (99.8 : 0.2) by high-performance liquid chromatography with mass spectrometry (HPLC-MS; Agilent 1200). Ions in selected ion monitoring (SIM) are detected for mass-to-charge ratios (m/z): 744 for the internal standard C46, 1302, 1300, 1298, 1296, 1294, and 1292 for isoGDGTs and then 1050, 1048, 1046, 1036, 1034, 1032, 1022, 1020, and 1018 for brGDGTs (Hopmans et al., 2016; Davtian et al., 2021). The high- performance liquid chromatography allowed for the separa- tion of the 5-, 6-, and 7-methyl brGDGTs isomers (Ding et al., 2016; Naafs et al., 2017b). Concentrations are expressed in milligrammes per gramme of sediment (mgg−1 [sed]). The rel- ative abundances of each GDGT (iso and br) are determined by the ratio of the proportion of the compound to the sum of all iso- or brGDGTs (n = 6 and 19 respectively). Four sam- ples with contrasting GDGT compositions were measured and integrated ﬁve times to establish the reproducibility of the analytical set-up. 2.5.2 Indexes calculations Each sample was weighed after drying for 12 h at 150 ◦C. Then, a calcination at 550 ◦C for 5 h was performed to estimate the OMC (percent of dry mass) (Decorsiere et al., 2019). 2.6.1 Pollen analysis The MAT method, developed by Guiot (1990), is a method often used due to its simplicity of use, performance, and sen- sitivity. The MAT is based on measuring the degree of dis- similarity between a fossil pollen assemblage and modern pollen assemblages with known environmental characteris- tics to draw inferences about the temporal sequences of fossil samples with unknown environmental characteristics. A total of 65 samples have been analysed (sampling interval of 1 cm for 159–138 cm, 2 cm for 138–128 cm, and 4 cm for 128–0 cm), and the pollen was identiﬁed and counted under an optical microscope under a light microscope at a standard magniﬁcation of ×400. Pollen sums are at least 500 grains per sample and exclude spores; 148 taxa were identiﬁed, but here, a simpliﬁed pollen diagram was made to represent the vegetation variability over the last 15 000 years. The pollen data are presented as a function of age in calibrated years before present (Fig. 6). The WA-PLS method (ter Braak and Juggins, 1993) is a transfer function and is also a method often used; it assumes that the relationship between pollen proportion and climate is unimodal, where the abundance of a plant species is di- rectly related to its environmental tolerance. WA-PLS esti- mates the climatic optimum of a species from calibration data by calculating the average climatological conditions in which a species occurs, weighted by the abundance of that species (Chevalier et al., 2020). L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2132 computation of brGDGT indices, MAAT, and pH calibrations and RMSE (in degrees Celsius for MAAT). Table 1. Formulae used for computation of brGDGT indices, MAAT, and pH calibrations and RMSE (in degrees Celsius for MAAT). Table 1. Formulae used for computation of brGDGT indices, MAAT, and pH calibrations and RMSE (in deg Table 1. Formulae used for computation of brGDGT indices, MAAT, and pH calibrations and RMSE (in degrees Celsius for MAAT). Table 1. Formulae used for computation of brGDGT indices, MAAT, and pH calibrations and RMSE (in degrees Celsius for MAAT Index Formula RMSE Reference CBT5Me −log10 (Ib+IIb5Me) (Ia+IIa5Me) – De Jonge et al., 2014b CBT’ log10 (Ic+IIa6Me+IIc6Me+IIIa6Me+IIIa6Me) (Ia+IIa5–7Me+IIIa5–7Me) – De Jonge et al., 2014a MBT’5Me (Ia+Ib+Ic) (Ia+Ib+Ic+IIa5Me+IIb5Me+IIc5Me+IIIa5Me) – De Jonge et al., 2014b Index1 log10 (Ia+Ib+Ic+IIa6–7Me+IIIa6–7Me) (Ic+IIa5Me+IIc5Me+IIIa5–7Me) – De Jonge et al., 2014a IIIa/IIa IIIa5Me IIa5Me – Schouten et al., 2012 GDGT −0/crenarchaeol GDGT−0 Crenarchaeol – Blaga et al., 2009 IR IIab6Me+IIIab6Me IIab5Me+IIIab5Me+IIab6Me+IIIab6Me – De Jonge et al., 2021 CI ratio Ia Ia+IIa5Me+IIIa5Me – De Jonge et al., 2021 MAATSoil MBT’5Me −0.8571 + 31.45 × MBT’5Me 4.8 De Jonge et al., 2014a MAATBog MBT’5Me 52.18 × MBT’5Me −23.05 4.7 Naafs et al., 2017b MAATIndex1 5.05 + (14.86 × Index1) 4.7 De Jonge et al., 2014a MAATmr 5.58 + 17.91 × [Ia] + 25.9 × [Ib] −18.77 × [IIa5Me] 5.0 De Jonge et al., 2014a pHCBT’ 7.15 + 1.59 × CBT’ 0.52 De Jonge et al., 2014a 2.5.1 GDGT analysis GDGT analysis was carried out in the LGL-TPE ENS (Labo- ratoire de Géologie de Lyon: Terre, Planète, Environnement, Ecole Normale Supérieure de Lyon) laboratory in Lyon on 75 samples of the CAN02 core with a sampling step of 2 to 4 cm and on the six surface samples (Fig. 1a). Sam- pling was carried out using a 1 cm3 brass cutter (about 1 g), and then samples were freeze-dried for 24 to 72 h. After grinding and homogenization, the total lipid fraction was extracted twice by microwave (MARS 6 CEM) at 70 ◦C with 10 mL of a dichloromethane (DCM)–methanol (MeOH) mixture (3 : 1, v/v) and then ﬁltered on a solid-phase ex- traction (SPE) cartridge; 1000 ng of C46 GDGT (99 % n- hexane : 1 % isopropanol) was then added to the total liq- uid extract (TLE) to serve as an internal standard (Huguet et al., 2006). The TLE was then separated into polar and ap- olar fractions on a silica column with 5 mL of hexane–DCM (1 : 1) and 10 mL of DCM–MeOH (1 : 1) respectively. The Changes in peat pH can have a signiﬁcant impact on brGDGT-based temperature; hence pH reconstruction based on brGDGTs has been examined (De Jonge et al., 2021). The CBT’-based calibration was utilized (De Jonge et al., 2014a). Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 2.6 Pollen analysis and reconstruction of climate parameters from pollen assemblages MAT), and (3) recent machine learning techniques with re- gression trees (random forest, RF, and boosted regression trees, BRT) to quantify the climate parameters on the CAN02 sequence. L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures Table 2. Dating of the 16 samples of the Canroute core with men- tion of their depth in centimetres. AMS (accelerator mass spec- troscopy) radiocarbon dating and calibrated 2σ age interval of the CAN02 core. for BRT the under-represented samples in the previous tree have a higher probability of being selected. This approach is called “boosting” and increases the model’s performance concerning the least well-predicted elements (Salonen et al., 2019). Due to the regression tree signal variation, the BRT method’s ﬁnal signal is an average of 15 independent runs of the BRT algorithm. Laboratory code Depth AMS 14C Age (cm) (BP) (cal BP) Poz-129948 19–20 345 ± 30 480–310 Poz-109171 39–40 3150 ± 35 3450–3260 Poz-129949 59–60 4380 ± 35 5040–4860 Poz-141932 75–76 5120 ± 40 5980–5750 Poz-109172 79–80 5200 ± 35 6160–5900 Poz-142282 85–86 5640 ± 40 6490–6310 Poz-142283 95–96 5690 ± 40 6620–6360 Poz-129950 109–110 5710 ± 35 6620–6400 Poz-129951 119–120 5940 ± 40 6880–6670 Poz-109174 129–130 6710 ± 30 7660–7510 Poz-129995 139–140 7830 ± 50 8970–8450 Poz-129996 149–150 9790 ± 50 11 310–11 110 Poz-148568 154–155 9900 ± 50 11 600–11 200 Poz-129997 159–160 11620 ± 60 13 590–13 340 Poz-148570 164–165 11720 ± 60 13 750–13 460 Poz-109175 168–169 11070 ± 50 13 100–12 840 For these four methods, local taxa and hydrophytes were not used in the reconstruction of climate parameters as they could not be strictly related to the regional climate. The role of the modern pollen database in the reliabil- ity of climate reconstructions is also investigated here. The Eurasian Pollen Database (EAPDB; Fig. B1a), compiled by Peyron et al. (2013, 2017) and updated by Dugerdil et al. (2021a), was used as the ﬁrst modern pollen database. In addition, two other modern pollen databases were used, originating from a sub-sampling of the EAPDB database, grouping sites from the Mediterranean–Temperate Europe (MEDTEMP; Fig. B1c) database and sites originating from the Temperate Europe–Scandinavian (TEMPSCAND; Fig. B1b) database. The temperatures reconstructed from those two sub-sampled databases are compared to the tem- perature reconstructed from the EAPDB (Fig. 7). Here, we reconstruct the mean annual temperature (MAAT) from the Canroute pollen record as the MAAT is the only comparable parameter between pollen and brGDGT re- constructions. We also reconstruct the precipitations (MAP) to better discuss the links with the pH variations. dard deviation estimated from observed values (Josse and Husson, 2016). L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures Additionally, element counts have been nor- malized over the Ti element (Davies et al., 2015). Climate reconstructions and reliability tests (R2 and RMSE criterion) were performed with the rioja (Juggins, 2023), randomForest (Liaw and Wiener, 2002), and dismo (Hijmans et al., 2022) packages. All analyses were per- formed in R Studio (Posit team, 2023), using the ggplot2 (Wickham, 2016) package for plot creation and the ri- oja package (Juggins, 2023) for the pollen diagram with a CONISS (constrained incremental sum-of-squares agglom- erative clustering) hierarchy classiﬁcation method described by Grimm (1987). The reliability of pollen-inferred climate reconstruction methods was estimated by bootstrapping cross-validation by calculating the correlation coefﬁcient values between the variables (R2) and those of the root mean square error (RMSE) criterion. 2.7 Statistical treatments A principal component analysis (PCA) of the correlation ma- trix has been conducted on XRF and brGDGTs with the Fac- toMineR package (Lê et al., 2008). The PCA aims to illus- trate the components that explain the most variations across samples and thus allows the exploration of relationships be- tween the variables. For both PCA, a k-means clustering al- lowed the highlight of subgroups in the data, with a cho- sen value of a cluster number (k) determined with a within- cluster sum of squares method. The clustering analysis has been performed with the Factoextra package (Kassambara and Mundt, 2020). For XRF data used on for the PCA, due to the low detection signal of a part of the data, a regular- ized imputation of the missing values has been applied, using the missMDA (Josse and Husson, 2016) and FactoMineR (Lê et al., 2008) packages. Missing-value imputation reduces the loss of information caused by missing values, lowering the ability to discern patterns (Dray and Josse, 2015). Regular- ized imputation entails ﬁlling in missing values with values selected from a Gaussian distribution, with mean and stan- 2.6.2 Quantitative reconstruction of climate parameters: a multi-method approach Various methods have been developed to quantify climatic parameters from the pollen signal (see the review of Cheva- lier et al., 2020), and multi-method approaches have been developed to increase the reliability of palaeoclimatic recon- structions (Brewer et al., 2008; Peyron et al., 2005, 2011, 2013, 2017; Salonen et al., 2019; Robles et al., 2022, 2023). These methods were initially developed to calibrate the re- lationship between modern pollen data (soils, mosses) and current climate parameters. Our multi-method approaches include (1) transfer functions based on linear regressions (weighted averaging partial least squares regression, WA- PLS) between pollen taxa and climate parameters, (2) as- semblage approaches based on the analogy principle between fossil and modern assemblages (modern analogue technique, The other two methods (RF and BRT) have been devel- oped more recently in palaeoclimatology and are based on machine learning (Salonen et al., 2019). These methods use regression trees to divide pollen data by successive separa- tions of samples according to their abundance in the pollen spectrum. The random forest method (Breiman, 2001; Prasad et al., 2006) is based on the estimation and combination of many regression trees; each tree is estimated from a set of pollen samples by bootstrapping (Chevalier et al., 2020). Boosted regression trees (De’Ath, 2007; Elith et al., 2008) differ from RF in the deﬁnition of the modern database. For RF, each sample has the same probability of being selected; https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 2133 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2134 Figure 3. Geochemical data from the CAN02 sequence. (a) Peat accumulation rate (PAR) calculated from the age–depth model (cmyr−1). (b) Organic matter content (OMC; %) derived from loss on ignition (LOI). (c) Zr relative counts, standardized according to Ti. (d) First dimension (PC1XRF) extracted from the principal component analysis (PCA) performed on the XRF data. (e) Second dimension (PC2XRF) extracted from the principal component analysis (PCA) performed on the XRF data. (f) Principal component analysis (PCA) of the XRF signal. The principal components are grouped into three clusters. Samples are coloured according to the age gradient (yrs cal BP). In (a) to (e), the shaded period corresponds to the period between 6600 and 4700 cal BP, when the accumulation rate increases, and detrital activity decreases (light-grey period), and between 4700 and 3000 cal BP, when detrital activity increases, and detrital input dynamics changes (dark- grey period). Figure 3. Geochemical data from the CAN02 sequence. (a) Peat accumulation rate (PAR) calculated from the age–depth model (cmyr−1). (b) Organic matter content (OMC; %) derived from loss on ignition (LOI). (c) Zr relative counts, standardized according to Ti. (d) First dimension (PC1XRF) extracted from the principal component analysis (PCA) performed on the XRF data. (e) Second dimension (PC2XRF) extracted from the principal component analysis (PCA) performed on the XRF data. (f) Principal component analysis (PCA) of the XRF signal. The principal components are grouped into three clusters. Samples are coloured according to the age gradient (yrs cal BP). In (a) to (e), the shaded period corresponds to the period between 6600 and 4700 cal BP, when the accumulation rate increases, and detrital activity decreases (light-grey period), and between 4700 and 3000 cal BP, when detrital activity increases, and detrital input dynamics changes (dark- grey period). 3.1 Radiocarbon dating and age–depth model The CAN02 sequence covers the Late Glacial from ca. 15 000 cal BP (171 cm depth) to −80 cal BP (surface) (Fig. 2 and Table 2). The average accumulation rate is 0.01 cmyr−1 between 170–113 cm and 10 times higher between 110– 85 cm (0.13 cmyr−1), with a maximum value of 0.18 cmyr−1 at 95 cm depth (Fig. 2, shaded period). The accumulation rate drops back to initial values between 85–15 cm. It increases again to 0.03 cmyr−1 from 15 to the core top. Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 3.3.3 Palaeotemperature reconstruction All calibrations show similar trends during the Holocene, i.e. (1) cold conditions between 15 000–11 500 cal BP, followed by (2) warmer conditions until 6600 cal BP (Fig. 5c). This stable period ends with (3) a drop in temperature between 6600–5000 cal BP and is followed by (4) a new warming until 300 cal BP before (5) a new last drop in temperature (Fig. 5c). Despite similar and synchronous trends, the ab- solute values of temperature are different between calibra- tions. From the Late Glacial to the present, the peat cali- bration shows the lowest MAAT values, ranging from −5.0 to 6.9 ◦C (SD = 0.5 ◦C, n = 75), whereas multiple regres- sion (mr), Index1, MBT’5Me, and Bayesian calibrations are associated with higher MAAT values ranging from 1.59 to 11.51 ◦C (SD = 0.3 ◦C, n = 75). Among all calibrations, the multiple regression, the Bayesian calibration, and the cali- bration based on Index1 show the lowest variations (respec- tively 7.2, 6.9, and 5.5 ◦C) compared to the peat calibrations and those based on methylation indexes (respectively 11.5 and 12.9 ◦C). Penta- and hexamethylated brGDGTs show similar trends: a decrease between 15 000–6600 cal BP followed by an increase between 6600–6100 cal BP and a further de- crease between 6100 and −80 cal BP (Fig. 5a). Tetram- ethylated brGDGTs show the opposite trend: an increase between 13 800–6600, followed by a sharp decrease be- tween 6600–6300 cal BP and then a gradual increase be- tween 6300 and −80 cal BP. Pentamethylated brGDGTs dominate between 15 000–5000 cal BP, whereas tetram- ethylated brGDGTs dominate from 5000 cal BP onwards. The tetramethylated-dominated period can be divided into two subperiods, both surrounded by lower values: 5000– 2300 cal BP and 2300 cal BP onward. The CBT’ index ranges from −1.58 to −0.98 (SD = 0.05, n = 75; Fig. 5b) and shows a slight continuous decrease over time. The MBT’5Me index varies between 0.32 and 0.55 (SD = 0.01, n = 75) and shows a slightly increasing trend over time (Fig. 5c), with two periods of rapid decrease between 6500–600 cal BP and between 290 and −80 cal BP, and two well-differentiated pe- riods: 15 000–6500 and 4700–290 cal BP. 3.3.1 Compound concentrations In surface samples, the average concentrations of iso- and brGDGTs are 0.6 ± 0.5 mgg−1 [sed] (0.7 × 10−6–0.4 mgg−1 [sed]) and 0.8 ± 0.6 mgg−1 [sed] (0.9 × 10−6–2.2 mgg−1 [sed]) respec- tively. The CAN02 core samples have higher aver- age concentrations of iso- and brGDGTs than the sur- face samples: 2.1 ± 2.0 mgg−1 [sed] (53.0–1.5 mgg−1 [sed]) and 2.4 ± 2.0 mgg−1 [sed] (143.6–29.7 mgg−1 [sed]) respectively. PCA of brGDGT relative abundances reveals that PC1brGDGTs and PC2brGDGTs account for more than 58 % of the variance in brGDGT compounds (PC1brGDGTs: 45.6 %; PC2brGDGTs: 13.1 %; Fig. C1b and c). The cluster analysis revealed three cluster (Fig. C1d), delimited by depth (cluster 1: 15 000–5400 cal BP; cluster 2: 5400–2300 cal BP; cluster 3: 2300–0 cal BP), and demonstrated that the lower half of the sequence (cluster 1) is positively correlated with most brGDGT compounds (except Ia, Ib, and Ic), while the up- per half (cluster 2 and 3) is negatively correlated. That is, the lower half of the sequence (15 000–5400 cal BP) has a high abundance of most brGDGT compounds, whereas the upper half (especially 5400–2300 cal BP) has a high abundance of primarily Ia. 3.3.2 Relative abundances Average analytical errors are calculated from the aver- aged standard deviations of replicate measurements for br- and isoGDGTs (SD = 2 % and 4 % respectively, n = 75). BrGDGTs are predominant over isoGDGTs in the seven sur- face samples (including CAN0) (average abundance of 92 % and 7 % respectively). IsoGDGTs of surface samples are dominated by GDGT-0 (between 47 % and 96 %; Fig. 4a). BrGDGT relative abundances in surface samples show the dominance of tetramethylated (Ia) and pentamethylated (IIa) (36 % and 37 % respectively; Fig. 4b). For CAN02 downcore samples, average relative abundances of iso- and brGDGTs show mean values of 12 % and 88 % respectively. Downcore samples are also dominated by GDGT-0 (isoGDGTs) and tetramethylated and pentamethylated (brGDGTs) (Fig. 4a and b). Double isomers are present in very low abundances and are present only between 7800 and 6420 cal BP at two occurrences for the IIIa7Me and one occurrence for the IIa7Me. 3.3 GDGT analysis values range from 0.30 to 0.55 and are less than 0.65 (Fig. 5e). The period from 15 000 to 4700 cal BP has mean CI values of 0.38, but the period from 4700 to −80 cal BP has higher mean CI values of 0.49. The pH varies from 4.6 to 5.6 and shows a little continuous decline over time (Fig. 5f). L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2135 3.2 Sedimentological analysis ing a recovery of detrital inputs. The OMC estimated from the LOI (Fig. 3b) indicates low values (under 60 %) from 15 000 to 6600 cal BP. After this period, the OMC increases up to 90 % from 6600 to 3000 cal BP, after which it decreases to lower values (around 60 %). In more detail, from 6600 to 3000 cal BP two dynamics are observed, highlighted by Zr, PC1XRF and PC2XRF axes. The ﬁrst dynamic extends from 6600 to 4700 cal BP, during which the values of Zr and both PCXRF axes are at their lowest, and indicates a low variabil- ity (Fig. 3c–e, in light grey). The second dynamic extends from 4700 to 3000 cal BP and is characterized by higher val- ues and variability in Zr and both PCXRF axes (Fig. 3c–e, in dark grey). During this second period, the PC2XRF values be- come positive, meaning that the origin of detrital inputs may have changed between the light- and dark-grey area. The peat accumulation rate (PAR; Fig. 3a) ranges from 0.01 to 0.18 cmyr−1 with a maximum value between 6500– 6340 cal BP. The ﬁrst two principal components PC1XRF and PC2XRF explain 63.5 % and 17.2 % of the total elemental variation (Fig. 3d and e). All elements are positively corre- lated with PC1XRF, except the Zr element. The PC2XRF il- lustrates two groups of elements: elements such as Si, Pb, S, Zn, and Rb on the negative side and Ca, Mn, Sr, Fe, Ba, K, and Zr on the positive side (Fig. 3d and e). The Zr el- ement (Fig. 3c) traces the detrital activity: the higher the value, the higher the lithogenic inputs (Silva-Sanchez et al., 2014). At Canroute, Zr ranges from 0 to 0.14. High values of Zr are present from 15 000 to 6600 cal BP (0.05–0.14), re- vealing important detrital inputs, followed by a progressive lowering of detrital inputs. From 6600 to 3000 cal BP, very low values of Zr are present (0–0.08), revealing the quasi- absence of detrital inputs. From 3000 cal BP onward, the val- ues slowly increase to previous values (0.04–0.13), translat- https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 3.4 Pollen analysis Contrary to the EAPDB and TEMP- SCAND, the results obtained with the MEDTEMP show a warm Late Glacial and the absence of the thermic optimum for the four methods (Fig. 7). y p y p Zone 3 (from 6600 to ca. 4500 cal BP) is marked by an increase in the Quercus pubescens type and Tilia at the ex- pense of Corylus avellana. This period is also marked by the decrease in megaphorb taxa and by the record of Poaceae, Calluna vulgaris, and Sphagnum. Finally, Zone 4 (from ca. 4500 to −80 cal BP) is charac- terized by the mesophilous Abies alba and Fagus sylvatica, and the hydrophyte Alnus glutinosa type. Corylus avellana and the Quercus pubescens type continue to decrease, while Tilia is only slightly recorded. 3.4 Pollen analysis The MAT indicates a warming of 1.9 ◦C (Fig. 7c). BRT, MAT, and WA-PLS are the three methods with the largest temperature amplitudes. The RF shows a much less con- trasting climate signal over time (3.4 ◦C variations between 15 000 cal BP and present; Fig. 7c), a climate pattern that is different than from the results obtained with the MAT, WA- PLS, and BRT methods. Contrary to the EAPDB and TEMP- SCAND, the results obtained with the MEDTEMP show a warm Late Glacial and the absence of the thermic optimum for the four methods (Fig. 7). Zone 2 (from 11 700 to 6600 cal BP) is dominated by Corylus avellana (up to 60 %) and to a lesser extent by the Quercus pubescens type (< 30 %) that progressively in- creases. During this period, the record of steppe taxa de- creases, but hydrophytes are still present. again followed by (4) a slight warming for 5000 cal BP on- ward. Cold conditions (MAAT around 2.3 to 8.7 ◦C) are ev- idenced from 15 000 to 11 700 cal BP. The ﬁrst warming oc- curs between 11 700 and 6000 cal BP. The WA-PLS (and the MAT) indicate high values for this thermal optimum (Fig. 7a and b), while the BRT method indicates a warming of 1.5 ◦C between 12 000 and 6500 cal BP (Fig. 7d). The duration of the cooling observed around 7000–6500 cal BP is method- dependent. The cooling reconstructed with the MAT and WA-PLS is progressive, with a variation in the MAAT of −2.1 and −4.5 ◦C respectively, and is shorter for the MAT than for the WA-PLS (Fig. 7a and b). The BRT indicates a slight cooling between 6400 and 4700 cal BP, less marked than for the other two methods, with a variation of −0.9 ◦C (Fig. 7d). The last period is characterized by a slight warming trend that is particularly marked for the BRT and WA-PLS methods, with an increase of +2.4 and +2.6 ◦C respectively. The MAT indicates a warming of 1.9 ◦C (Fig. 7c). BRT, MAT, and WA-PLS are the three methods with the largest temperature amplitudes. The RF shows a much less con- trasting climate signal over time (3.4 ◦C variations between 15 000 cal BP and present; Fig. 7c), a climate pattern that is different than from the results obtained with the MAT, WA- PLS, and BRT methods. 3.4 Pollen analysis Zone 1 (from 15 000 to ca. 11 700 cal BP) of the pollen dia- gram (Fig. 6) is dominated by steppe taxa (Artemisia, Ama- ranthaceae, Poaceae), peatland (Cyperaceae up to 50 %), and megaphorb (Filipendula, Ranunculus, Succisa pratensis) but contains some occurrences (mostly of long-distance origin) of Betula, Corylus avellana, the Pinus sylvestris type, and the Quercus pubescens type. At the end of this period, the arboreal pollen represents ca. 45 % of the pollen sum, indi- cating the gradual replacement of the steppe vegetation by forests. The isomer ratio (IR) values range from 0.03 to 0.13, with the lowest values obtained between 4700 and 2300 cal BP (Fig. 5d). Throughout the record, the community index (CI) Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 2136 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures Figure 4. GDGT results: fractional abundances (%) of (a) isoGDGT and (b) brGDGT compounds for the CAN02 sequence (n = 75, blue) and surface samples (n = 6, grey). L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2136 Figure 4. GDGT results: fractional abundances (%) of (a) isoGDGT and (b) brGDGT compounds for the CAN02 sequence (n = 75, blue) and surface samples (n = 6, grey). again followed by (4) a slight warming for 5000 cal BP on- ward. Cold conditions (MAAT around 2.3 to 8.7 ◦C) are ev- idenced from 15 000 to 11 700 cal BP. The ﬁrst warming oc- curs between 11 700 and 6000 cal BP. The WA-PLS (and the MAT) indicate high values for this thermal optimum (Fig. 7a and b), while the BRT method indicates a warming of 1.5 ◦C between 12 000 and 6500 cal BP (Fig. 7d). The duration of the cooling observed around 7000–6500 cal BP is method- dependent. The cooling reconstructed with the MAT and WA-PLS is progressive, with a variation in the MAAT of −2.1 and −4.5 ◦C respectively, and is shorter for the MAT than for the WA-PLS (Fig. 7a and b). The BRT indicates a slight cooling between 6400 and 4700 cal BP, less marked than for the other two methods, with a variation of −0.9 ◦C (Fig. 7d). The last period is characterized by a slight warming trend that is particularly marked for the BRT and WA-PLS methods, with an increase of +2.4 and +2.6 ◦C respectively. 3.5 Pollen-inferred mean annual temperature To provide reliable climate reconstruction, we applied a multi-method approach tested here with three different mod- ern pollen databases to the Canroute pollen assemblages. The results based on the global EAPDB and regional TEMP- SCAND databases broadly indicate the same temperature trends, but with smaller amplitudes for the TEMPSCAND calibration. The MEDTEMP regional calibration shows op- posite trends compared to the other two databases, partic- ularly during the Late Glacial (Fig. 7). The EAPDB and TEMPSCAND databases are associated with higher values of R2 than the MEDTEMP ones (Table 3). The BRT method shows higher values of R2 (Table 3; R2 = 0.92, RMSE = 1.30), and the RF method shows lower values of R2 = 0.70 (RMSE = 1.83). For the EAPDB and TEMPSCAND modern pollen datasets, three of the methods (MAT, WA-PLS, and BRT) show a similar climatic signal (Fig. 7a, b, and d) character- ized by (1) cold temperatures during the Late Glacial, fol- lowed by (2) a warming through the Early Holocene, re- sulting in a thermic optimum accentuated depending on the methods used, then (3) a sudden cooling around 6000 cal BP, https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2137 . (a) Relative abundances of tetra-, penta-, and hexamethylated brGDGTs in the CAN02 sequence. (b) Index of n (CBT’). (c) Index of the degree of methylation (MBT’5Me). (d) Isomer ratio (IR) through time. (e) Bacterial com ugh time. (f) The pH reconstruction based on global calibration of De Jonge et al., (2014b; Soil CBT’). (g) Annual me reconstructions based on global calibrations of De Jonge et al. (2014a, b) (Index1, Soil MBT’5Me, mr), Naafs et al Me), and Dearing Crampton-Flood et al. (2020) (Soil Bayesian). Calibration errors are represented by the lateral lin ded periods highlight the signiﬁcant decrease in the abundance of tetramethylated brGDGTs and a shift in accumula 700 cal BP (light-grey area) and the decrease in IR from 4700 to 2300 cal BP (dark-grey area). Symbols: modern MA Dashed black line: current calculated MAAT at Canroute. oi org/10 5194/cp 19 2127 2023 Clim Past 19 2127 5. (a) Relative abundances of tetra-, penta-, and hexamethylated brGDGTs in the CAN02 sequence. (b) Index on (CBT’). (c) Index of the degree of methylation (MBT’5Me). (d) Isomer ratio (IR) through time. (e) Bacterial ugh time. 3.5 Pollen-inferred mean annual temperature (f) The pH reconstruction based on global calibration of De Jonge et al., (2014b; Soil CBT’). (g) Annual m reconstructions based on global calibrations of De Jonge et al. (2014a, b) (Index1, Soil MBT’5Me, mr), Naafs et M ) d D i C Fl d l (2020) (S il B i ) C lib i d b h l l Figure 5. (a) Relative abundances of tetra-, penta-, and hexamethylated brGDGTs in the CAN02 sequence. (b) Index of the degree of cyclization (CBT’). (c) Index of the degree of methylation (MBT’5Me). (d) Isomer ratio (IR) through time. (e) Bacterial community index (CI) through time. (f) The pH reconstruction based on global calibration of De Jonge et al., (2014b; Soil CBT’). (g) Annual mean temperature (MAAT) reconstructions based on global calibrations of De Jonge et al. (2014a, b) (Index1, Soil MBT’5Me, mr), Naafs et al. (2017b) (Bog MBT’5Me), and Dearing Crampton-Flood et al. (2020) (Soil Bayesian). Calibration errors are represented by the lateral lines on the right side. Shaded periods highlight the signiﬁcant decrease in the abundance of tetramethylated brGDGTs and a shift in accumulation rate from 6600 to 4700 cal BP (light-grey area) and the decrease in IR from 4700 to 2300 cal BP (dark-grey area). Symbols: modern MAATs of surface samples. Dashed black line: current calculated MAAT at Canroute. https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 Figure 5. (a) Relative abundances of tetra-, penta-, and hexamethylated brGDGTs in the CAN02 sequence. (b) Index of the degree of cyclization (CBT’). (c) Index of the degree of methylation (MBT’5Me). (d) Isomer ratio (IR) through time. (e) Bacterial community index (CI) through time. (f) The pH reconstruction based on global calibration of De Jonge et al., (2014b; Soil CBT’). (g) Annual mean temperature (MAAT) reconstructions based on global calibrations of De Jonge et al. (2014a, b) (Index1, Soil MBT’5Me, mr), Naafs et al. (2017b) (Bog MBT’5Me), and Dearing Crampton-Flood et al. (2020) (Soil Bayesian). Calibration errors are represented by the lateral lines on the right side. Shaded periods highlight the signiﬁcant decrease in the abundance of tetramethylated brGDGTs and a shift in accumulation rate from 6600 to 4700 cal BP (light-grey area) and the decrease in IR from 4700 to 2300 cal BP (dark-grey area). Symbols: modern MAATs of surface samples. Dashed black line: current calculated MAAT at Canroute. Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 2138 L. https://doi.org/10.5194/cp-19-2127-2023 3.5 Pollen-inferred mean annual temperature d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures Figure 6. Simpliﬁed pollen diagram of the CAN02 sequence grouping selected tree taxa (green), herbaceous taxa (yellow), hydrophytes (blue), and AP/NAP ratio (black). Percentages were calculated on pollen sums excluding spores. The dashed black lines delineate four zones based on CONISS. The shaded period highlights the period of decrease in megaphorb taxa to the beneﬁt of Calluna vulgaris and Sphagnum (6600–4500 cal BP). L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2138 Figure 6. Simpliﬁed pollen diagram of the CAN02 sequence grouping selected tree taxa (green), herbaceous taxa (yellow), hydrophytes (blue), and AP/NAP ratio (black). Percentages were calculated on pollen sums excluding spores. The dashed black lines delineate four zones based on CONISS. The shaded period highlights the period of decrease in megaphorb taxa to the beneﬁt of Calluna vulgaris and Sphagnum (6600–4500 cal BP). Table 3. Performance results of the MAT, WA-PLS, BRT, and RF methods applied to three different modern pollen databases – the modern Eurasian (EAPDB), Temperate Europe–Scandinavian (TEMPSCAND), and Mediterranean–Temperate Europe (MEDTEMP) databases – for mean annual air temperature (MAAT; ◦C); k is the number of parameters used in the methods (e.g. number of analogues for the MAT method or the number of PLS components for the WA-PLS method). The best k corresponds to the number of parameters that infer the best R2 and calibration error (RMSE) values. The chosen k corresponds to the lowest number of parameters associated with the best R2 and RMSE values. In bold: selected database and methods associated with their respective R2 and calibration error (RMSE) values. Database Methods Best k R2 Best k RMSE Chosen k Chosen R2 Chosen RMSE EAPDB MAT 4 6 4 0.87 3.09 WA-PLS 3 3 3 0.71 4.11 BRT – – – 0.92 2.55 RF – – – 0.68 4.26 TEMPSCAND MAT 3 6 3 0.87 1.41 WA-PLS 3 3 3 0.60 2.14 BRT – – – 0.92 1.30 RF – – – 0.70 1.83 MEDTEMP MAT 3 9 3 0.86 2.14 WA-PLS 3 3 3 0.67 2.92 BRT – – – 0.91 2.02 RF – – – 0.74 2.45 Clim. Past, 19, 2127–2156, 2023 eira et al.: Reconstructing 15 000 years of southern France temperatures L. 4.2 Climate signal from pollen Different studies underline the importance of the modern pollen database used for the reconstruction of climate pa- rameters (Turner et al., 2021) and point out the advantage of regional calibration databases (Dugerdil et al., 2021a, b). The results obtained with the Eurasian pollen database (EAPDB) and regional Temperate Europe–Scandinavian pollen database (TEMPSCAND) broadly indicate simi- lar temperature trends at Canroute, while those based on the Mediterranean–Temperate Europe pollen database (MEDTEMP) shows opposite trends (Fig. 7). These dis- crepancies can be explained by the dominance in this pe- riod of taxa such as Artemisia, Amaranthaceae, and Poaceae (Fig. 6), able to constitute physiognomically and ﬂoristically similar arid steppes, both in the southern Mediterranean and Irano-Turanian warm context (e.g. Le Houérou, 2001) and in the north-eastern Eurasian cold context (e.g. Yurtsev, 1982). From 11 700 cal BP onwards, the methods performed on the three databases show similar trends but with different ampli- tudes. Signals obtained with the EAPDB and TEMPSCAND databases are particularly close, especially for the MAT and BRT methods (Fig. 7a and d), while climate results based on the MEDTEMP dataset appear as non-reliable due to the ab- sence of Mediterranean taxa in the pollen sequence (Fig. 6), as well as the poor R2 and RMSE values (Table 3). The beginning of the Holocene is marked by a strong dom- inance of Corylus avellana that constituted woodland, whose open character can be associated with a dominant mechanic erosion of the soil (Mohammad and Adams, 2010), allowing the strong detrital activity revealed by XRF until 9000 cal BP. After this date, the progressive decrease in detrital activ- ity may be attributed to the slow expansion of deciduous oaks, which replaced hazelnut open woodland across south- ern France and reduced the mechanic erosion. However, the slower establishment of dense deciduous forests compared to the southern Alps (de Beaulieu, 1977; de Beaulieu and Reille, 1983) suggests the inﬂuence of unfavourable climate conditions which slowed oak progression. The emergence of a dense mature oak forest is attested between 6600 and 4500 cal BP by both the maximal pollen record of the Quercus pubescens type and Tilia (Fig. 6) and the decrease in mineral input (Fig. 3b and c) that reveals the reduced detrital activity. 4.1 Past vegetation, peat accumulation, and detrital activity The Late Glacial steppe environment, dominated by Artemisia, Poaceae, and to a lesser extent Amaranthaceae (including the ex-Chenopodiaceae), reveals cold and dry con- ditions. However, the records of Cupressaceae (only repre- sented by Juniperus at that time) and especially Betula re- veal the ﬁrst afforestation dynamics supported by the slight warming of the Bølling–Allerød period. The end of the Late Glacial is characterized by the early expansion of temper- ate deciduous forests of Corylus avellana and the Quer- cus pubescens type, surprisingly contemporaneous with the Younger Dryas cooling event (12 900–11 700 cal BP; e.g. Broecker et al., 2010; Denton et al., 2010). Although the whole Younger Dryas is well recorded in northern regions (e.g. Duprat-Oualid et al., 2022), only the onset of the period is marked at Canroute by a peak of Artemisia and Amaran- thaceae and a punctual decrease in both Corylus avellana and the Quercus pubescens type. This pattern could be the result of a lack of accumulation, as the low PAR indicates during this period (Fig. 3a), or it could be due to a Younger Dryas event in this region that is not very marked. 4 Discussion 4 Discussion tation from groundwater. Finally, change affecting both the local hydrophytic vegetation and the surrounding landscape occurs at ca. 4500 cal BP, when the open peatland system is replaced by an alder swamp dominated by A. glutinosa (the only Alnus species present in the Massif Central), and the deciduous oak forest declines in favour of a mixed ﬁr–beech forest. While this change could be interpreted as an environ- mental shift towards “mountainous” conditions, the contem- poraneous pollen record of Plantago (not seen in Canroute) and the late Neolithic date (Miras et al., 2005) could also suggest anthropogenic impact, which likely destabilized the competitive equilibrium in favour of the mixed forest onset. 4.1 Past vegetation, peat accumulation, and detrital activity 4.1 Past vegetation, peat accumulation, and detrital activity 3.5 Pollen-inferred mean annual temperature d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2139 7. Climate signal from the three calibration sets EAPDB (blue circles and curves), MEDTEMP (red squares and curves), a D (green triangle and curve) for the MAT (a), the WA-PLS (b), the RF (c), and the BRT (d) methods. Black dashes: curren e. d i /10 5194/ 19 2127 2023 Cli P t 19 2127 21 Figure 7. Climate signal from the three calibration sets EAPDB (blue circles and curves), MEDTEMP (red squares and curves), and TEMP- CAND (green triangle and curve) for the MAT (a), the WA-PLS (b), the RF (c), and the BRT (d) methods. Black dashes: current MAAT at Figure 7. Climate signal from the three calibration sets EAPDB (blue circles and curves), MEDTEMP (red squares and curves), and TEMP- SCAND (green triangle and curve) for the MAT (a), the WA-PLS (b), the RF (c), and the BRT (d) methods. Black dashes: current MAAT at Canroute. Figure 7. Climate signal from the three calibration sets EAPDB (blue circles and curves), MEDTEMP (red squares and curves), and TEMP- SCAND (green triangle and curve) for the MAT (a), the WA-PLS (b), the RF (c), and the BRT (d) methods. Black dashes: current MAAT at Canroute. Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2140 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures site to Canroute (Fig. 1a), the calibrations based on Index1 and multi-regression appear to be the most reliable compared to their current MAAT (Fig. 8b). For the Canroute surface sample (CAN0), the calibration based on the MBT’5Me (Soil MBT’5Me) and the Bayesian calibration (Soil Bayesian) provide temperature values closer to the present tempera- tures at Canroute (Fig. 8b). Soil calibrations based on In- dex1, MBT’5Me, multi-regression, and Bayesian calibration produced the most suitable MAATs: both the Index1 and the multi-regression calibrations show a good estimate of the current MAAT, with a low scatter and a current MAAT close to the observed climatic conditions at Canroute (Fig. 8c). When applied to the whole sequence (CAN02), these two calibrations (Index 1 and mr) have the lowest standard devia- tions (respectively SD = 1.4 and 1.5 ◦C, n = 75; Fig. 8c). Re- garding the Bayesian and MBT’5Me calibrations, they show a less reliable estimate of the current MAAT and have a close standard deviation when applied to the CAN02 sequence (SD = 1.9 ◦C, n = 75; Fig. 8c). However, the Bayesian cal- ibration is associated with a lower error than the MBT’5Me- based one (RMSE = 3.8 and 4.8 ◦C respectively; Fig. 8b). MAT and BRT are two methods based on different math- ematical and ecological concepts (Chevalier et al., 2020), so their similar reconstructed trend supports the reliability of the methods and calibrations. However, the MAT shows a larger temperature range and in particular a slight thermal op- timum in the Early Holocene, between 11 700–8000 cal BP (Fig. 7a). A similar pattern, although marked by more pro- nounced positive anomalies, is shown by Martin et al. (2020) for Lake St Front, where high percentages of Corylus (< 55 %) are also recorded. Such a high proportion of Corylus does not exist in the modern pollen database assemblages of the TEMPSCAND dataset. The highest proportion (25 %– 35 %) is associated with sites located in Italy, Ireland, and Albania, explaining the bias in the signal towards higher tem- peratures for the EAPDB- and TEMPSCAND-based calibra- tions. In the CAN02 pollen sequence, the Early Holocene is also characterized by high Corylus percentages (up to 60 %; Fig. 6), which could explain the large temperature variation between the Late Glacial and Early Holocene proposed by the MAT, the only method based on the similarity of present- day and fossil assemblages. 4.3.1 Consistency of brGDGTs relative abundances with peat and soils databases The Canroute fossil samples and the surface samples from Massif Central are consistent with the global peat and soil databases that are globally dominated by tetra- and pen- tamethylated brGDGTs (Fig. 8a; Yang et al., 2014; Naafs et al., 2017a, b; Dearing Crampton-Flood et al., 2020). The use of both types of calibrations (peat and soil) therefore appears to be consistent with the brGDGT assemblages observed in the CAN02 sequence. L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures For the 10 700–6600 cal BP pe- riod, it can be suggested that the MAT method is less efﬁcient than the BRT one because Early Holocene hazelnut wood- land has no modern analogue. Nevertheless, both the MAT and BRT methods calibrated on the TEMPSCAND database can be retained in the following discussion because of the good reliability of their reconstructions. Ultimately, among the ﬁve calibrations tested, three of them (Bayesian, Index1, and mr) are retained for the interpre- tation of the climate trend at Canroute, due to the low RMSE of the Bayesian calibration and the low standard deviations of Index1 and multi-regression (mr) calibration reconstruction. 4.3 Temperature signal from brGDGTs According to various studies in peatlands, communities of brGDGT-producing Bacteria as well as brGDGT composi- tion are controlled by local hydrological conditions (Rao et al., 2022), vegetation composition (Xiong et al., 2016), and pH (De Jonge et al., 2021), whose changes are thus likely to affect temperature reconstructions (De Jonge et al., 2019). Furthermore, edaphic factors such as anoxic or oxic con- ditions have an impact on GDGT production and bacterial communities (Weber et al., 2018). Because crenarchaeol and GDGT-0 can be derived from Group I Crenarchaeota, the GDGT-0 / crenarchaeol ratio can be used to investigate the presence of methanogenic Archaea that thrive in anoxic con- ditions in sediments, whereas methanogenic Archaea syn- thesize GDGT-0, but no crenarchaeol (Blaga et al., 2009). The lower the ratio, the lower the anoxic conditions. Cre- narchaeol is also an indicator of the water-table level, which refers to the limits between the acrotelm and catotelm in peatlands and therefore is an indicator of anaerobic condi- tions (Yang et al., 2019). In the CAN02 sequence, the abun- dance GDGT-0 decreases in favour of crenarchaeol over time (Fig. C1a). This points to less anoxic conditions, indicating that the Canroute water-table level lowers over time, making the peatland surface drier. Furthermore, the brGDGT index IIIa/IIa, which investigates brGDGTs sources (Xiao et al., 2016), exhibits a signiﬁcant shift in its values throughout the sequence (0.12 to 0.46), demonstrating the effect of environ- 4.3.1 Consistency of brGDGTs relative abundances with peat and soils databases 4.2 Climate signal from pollen Such a vegetation change oc- curring on the surrounding slopes is likely to have triggered hydrological changes within the studied wetland, which ef- fectively experienced several contemporaneous changes: an abrupt acceleration of peat accumulation (Fig. 3a), an in- crease in organic matter content (from less than 40 % to more than 80 %; Fig. 3d; Joosten, 2015), and a replacement of the previous megaphorb by a bog-type peatland dominated by Calluna vulgaris and Sphagnum (Fig. 6). These changes are consistent with a loss of water runoff on the wetland sur- face, the result of either a reduced water supply resulting from a change in the river system or the natural rise in peat- land surface that progressively isolated peat-forming vege- Among the four methods, RF and WA-PLS appear as the least reliable (low R2 and high RMSE; Table 3), so the MAATs reconstructed by these two methods will not be con- sidered in the following discussion. For the MAT and BRT methods, the TEMPSCAND database calibration seems to produce a signal as reliable as the one produced with the EAPDB calibration set (close R2 and RMSE values; Ta- ble 3). The TEMPSCAND calibration produces a particu- larly close signal between the two methods, exhibits less variability, and has better R2 and RMSE values, bolstering the reliability of the reconstructions based on this calibra- tion database. Furthermore, calibrations employing regional datasets appear to be more reliable than those using global datasets (Dugerdil et al., 2021a), and because the TEMP- SCAND modern dataset is more regional than the EAPDB one, it is taken into account in the subsequent discussion. https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 2141 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 4.3.2 Selection of the most relevant brGDGT calibrations Black points with error bars next to each calibration correspond to temperature anomalies of CAN02 core samples. Figure 8. (a) Ternary plot of fractional abundances of tetra-, penta-, and hexamethylated brGDGTs for CAN02 core samples (in black) and Massif Central surface samples (in yellow and red) and for global peat (Naafs et al., 2017a; in blue) and soil databases (Yang et al., 2014; Naafs et al., 2017b; Dearing Crampton-Flood et al., 2020; in brown). (b and c) Testing of soil and peat calibrations on surface samples and CAN02 core samples. (b) Reconstructed MAAT from each calibration expressed as anomalies with respect to the mean annual temperatures measured at the sites. The standard deviation of each calibration applied to the CAN02 sequence (palaeo) is represented by the lateral lines on the right side. (c) Boxplot representing the results of the calibrations applied to the surface samples (n = 6). Black points with error bars next to each calibration correspond to temperature anomalies of CAN02 core samples. ing this time period, indicating that the change in brGDGT composition has no effect on the MBT’5Me employed for the temperature quantiﬁcation. Soil and peat pH is also related to global climate patterns via precipitation, meaning that changes in precipitation dynamics over time might cause pH variations (De Jonge et al., 2021). Pollen-based precipitation changes (MAP) at Canroute can be compared to brGDGT- based pH reconstruction to try to differentiate the effects of climate and bacterial communities on pH variation (Fig. 9). The same methods and calibration as MAAT were utilized for the MAP signal, namely the BRT and MAT methods with the TEMPSCAND calibration. The MAP and pH signals do not appear to correspond well, as the wettest periods (from 11 500–8500 cal BP and 4500 cal BP onwards) are not asso- ciated with a noticeable decrease in pH (Fig. 9). This shows mental change on brGDGT composition. This correlates well with other proxies such as geochemical and pollen data that evidence a hydroseral succession from a water-demanding megaphorb to peatland and alder-swamp plant communities. Past vegetation and detrital activity showed the presence of three different local conditions in the peat, which can re- sult in large pH ﬂuctuations because plants inﬂuence soil and peat pH (De Jonge et al., 2021). Changes in pH can alter the fractional composition of brGDGTs and the bacterial com- munity, inﬂuencing the MBT’5Me-based temperature. 4.3.2 Selection of the most relevant brGDGT calibrations The brGDGT relative abundances in the surface samples of Nassette (NAS), Lapsou (LAP), and Canroute (CAN0) are close to that of the Canroute (CAN02) sequence. For these three samples, the Bayesian calibration (Soil Bayesian; Dearing Crampton-Flood et al., 2020), the one based on MBT’5Me (Soil MBT’5Me), Index1 (Index1), and multi- regression (mr) (De Jonge et al., 2014a) show more reli- able reconstructed MAAT anomalies compared to the current MAAT of their respective location (Fig. 8b). For the sam- ples from the Caroux site (CAR-1-15 and CAR-C2), which is also a soligenous peatland and is geographically the closest https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 2142 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures Figure 8. (a) Ternary plot of fractional abundances of tetra-, penta-, and hexamethylated brGDGTs for CAN02 core samples (in black) and Massif Central surface samples (in yellow and red) and for global peat (Naafs et al., 2017a; in blue) and soil databases (Yang et al., 2014; Naafs et al., 2017b; Dearing Crampton-Flood et al., 2020; in brown). (b and c) Testing of soil and peat calibrations on surface samples and CAN02 core samples. (b) Reconstructed MAAT from each calibration expressed as anomalies with respect to the mean annual temperatures measured at the sites. The standard deviation of each calibration applied to the CAN02 sequence (palaeo) is represented by the lateral lines on the right side. (c) Boxplot representing the results of the calibrations applied to the surface samples (n = 6). Black points with error bars next to each calibration correspond to temperature anomalies of CAN02 core samples. Figure 8. (a) Ternary plot of fractional abundances of tetra-, penta-, and hexamethylated brGDGTs for CAN02 core samples (in black) and Massif Central surface samples (in yellow and red) and for global peat (Naafs et al., 2017a; in blue) and soil databases (Yang et al., 2014; Naafs et al., 2017b; Dearing Crampton-Flood et al., 2020; in brown). (b and c) Testing of soil and peat calibrations on surface samples and CAN02 core samples. (b) Reconstructed MAAT from each calibration expressed as anomalies with respect to the mean annual temperatures measured at the sites. The standard deviation of each calibration applied to the CAN02 sequence (palaeo) is represented by the lateral lines on the right side. (c) Boxplot representing the results of the calibrations applied to the surface samples (n = 6). 4.5.1 Local climate from the CAN02 record based on two independent proxies The temperatures inferred from pollen data (BRT method, TEMPSCAND modern database) and brGDGT data (Bayesian, mr, and Index1 calibrations) show very similar trends through the Holocene (Fig. 10). During the Late Glacial, cold conditions are evidenced from 15 000 to 11 700 cal BP followed by warmer conditions (plateau) with temperatures lower than those observed today from 10 500 to 6600 cal BP. Contrary to the BRT and brGDGT signal, the MAT method based on the TEMPSCAND modern database shows a slight thermic optimum from 10 500 to 8000. After the thermic optimum, the onset of a cooling trend until 6600 cal BP is evidenced (Fig. 10a). Due to a possible bias in the shaded area, the 6600–4700 cal BP period is not discussed. From 4700 to 200 cal BP, a slight warming trend is reconstructed for both proxies. Finally, from 200 cal BP onward, an abrupt cooling characterizes the brGDGT signal (Fig. 10b), while the pollen signal shows only a slight cooling trend (Fig. 10a). Consequently, the climate signal reconstructed by brGDGTs does not seem to be drastically impacted by the changing environmental context before and after the 6500– 4700 cal BP period. However, during the 6500–4700 cal BP period, the shift from a running-water-demanding vegetation to a less-demanding one (Fig. 6) seems to induce a shift in brGDGT indexes and bacterial communities. The tempera- ture values reconstructed for this period (Fig. 5g, light-grey shaded area) thus must be interpreted with caution. 4.3.2 Selection of the most relevant brGDGT calibrations CBT’ and IR show the increase in 6-methyl brGDGTs as pH in- creases. IR and CBT’ values in the CAN02 sequence are at their lowest from 5000 to 2300 cal BP (Figs. 3b and 5d, dark-grey shaded area), resulting in a modest decrease in pH values (Fig. 3e). There is no change in the MBT’5Me dur- mental change on brGDGT composition. This correlates well with other proxies such as geochemical and pollen data that evidence a hydroseral succession from a water-demanding megaphorb to peatland and alder-swamp plant communities. Past vegetation and detrital activity showed the presence of three different local conditions in the peat, which can re- sult in large pH ﬂuctuations because plants inﬂuence soil and peat pH (De Jonge et al., 2021). Changes in pH can alter the fractional composition of brGDGTs and the bacterial com- munity, inﬂuencing the MBT’5Me-based temperature. CBT’ and IR show the increase in 6-methyl brGDGTs as pH in- creases. IR and CBT’ values in the CAN02 sequence are at their lowest from 5000 to 2300 cal BP (Figs. 3b and 5d, dark-grey shaded area), resulting in a modest decrease in pH values (Fig. 3e). There is no change in the MBT’5Me dur- Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 2143 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 4700 cal BP, the result of either a reduced water supply from a change in the river system or the natural rise in peatland surface that gradually isolated peat-forming vegetation from groundwater. Two of the three proxies (detrital signal and brGDGT compounds) exhibit a second response to the hy- drological shift between 4700 and 3000 cal BP for the detrital signal and 2300 cal BP for the brGDGT compounds (Figs. 3, 5, and C1, dark-grey area). To explain the timing in the prox- ies’ responses to environmental changes, a distinct resilience, depending on the proxy, might thus be postulated. Walker et al. (2004) deﬁnes resilience as the system’s ability to absorb disturbance and reorganize while experiencing change in or- der to retain essentially the same function, structure, identity, and feedback. In this study, vegetation appears to be more resilient than the detrital signal and brGDGT compounds, returning to equilibrium faster. Both the sedimentological and brGDGT signals demonstrate a synchronous reaction to vegetation, as well as a second response after the vegeta- tion is back to equilibrium. 4.3.2 Selection of the most relevant brGDGT calibrations 4.5 Temperatures changes in the northern Mediterranean basin since the Late Glacial: a synthesis 4.5.1 Local climate from the CAN02 record based on two independent proxies 4.3.2 Selection of the most relevant brGDGT calibrations Furthermore, brGDGTs appear to have less resilience than sedimentological signals because the return to equilibrium is not recorded before 2300 cal BP, whereas the detrital signal is recorded before 3000 cal BP. However, it is unknown if this second response to environ- mental changes affects brGDGT distribution and bacterial community composition. that precipitation dynamics have little effect on pH in Can- route peatland. Precipitation, which is normally acidic, can cause a low pH in ombrotrophic peatland (water supplied primarily by precipitation) (Sennès, 2004). Canroute, on the other hand, is a soligenous peatland, and because most of its water supply originates from streams and springs (Julve, 1994), its local vegetation is less affected by precipitation dy- namics. This shows that pH variations are produced mostly by local vegetation and detrital changes, which are inﬂuenced by local hydrological conditions. Temperature reconstructions can be affected by changes in the bacterial community of brGDGT producers (De Jonge et al., 2019), which can be studied using variations in CI values (De Jonge et al., 2021). Although the CI values in the CAN02 sequence do not exceed the 0.65 thresholds established by De Jonge et al. (2021), a signiﬁcant shift in value indicates a potential change in bacterial community composition from 6600 to 4700 cal BP (Fig. 5e), implying that the temperature interpretation during this period should be done with caution. Dearing Crampton-Flood et al. (2020) used two indica- tors of the presence or absence of climate seasonality, in terms of precipitation (SoP) and temperature (TS), to inves- tigate a possible bias in the production of brGDGT com- pounds. The SoP index can aid in interpreting whether a re- gion presents a potential production bias due to variability in the timing of precipitation (Dearing Crampton-Flood et al., 2020). A low SoP value is indicative of a relatively con- stant MAP throughout the year. Canroute is associated with a low SoP value (21) and therefore does not show a hetero- geneous precipitation pattern, which can create a bias in the production of brGDGTs. A region associated with a TS value over 20 is considered to have high-temperature seasonality, which biases the proxy toward summer temperatures (Dear- ing Crampton-Flood et al., 2020). At Canroute, the TS value results in monthly mean temperature variability under 20 ◦C, which indicates a low-temperature seasonality, without bias. L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures The lack of typical Late Glacial events can be attributed to a low resolution of the record, possibly caused by bioturbation smoothing abrupt events (Bradley, 2015), a low accumula- tion rate (Fig. 3a), or a Late Glacial in the region that is not very marked. Overall, the Late Glacial climate signal trend of Canroute ﬁts well with the more regional signal when con- fronted with different sites and proxies for southern Europe. F h H l i l h i f li robust reconstruction of mean annual temperatures for Can- route. This climate pattern is consistent with both atmo- spheric climate model outputs (Mauri et al., 2014; Liu et al., 2014; Wanner, 2021; Erb et al., 2022) and pollen-based palaeoclimatic studies (Herzschuh et al., 2023), which de- picted the absence of a thermic optimum for the same lati- tudes. According to several studies, the presence of the HTM can result from a seasonality bias toward summer tempera- tures (Liu et al., 2014; Samartin et al., 2017; Wanner, 2021; Herzschuh et al., 2023). L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2144 Figure 9. CAN02 (a) pollen-inferred reconstructions of the annual precipitation (MAP; in millimetres) obtained with BRT (yellow squares and curve) and MAT (blue triangles and curve) methods based on the TEMPSCAND calibration. The solid line corresponds to locally estimated scatterplot smoothing (loess) regression curves; the shaded area corresponds to its 95 % conﬁdence interval. The dashed black line corresponds to Canroute’s current calculated MAP. (b) CBT’-based pH reconstruction (green circles and line; De Jonge et al., 2021). On the right, the lateral line represents calibration error (RMSE). The time highlighted in light grey reveals a large fall in tetramethylated brGDGT abundance and a shift in accumulation rate (6600–4700 cal BP). The era shown in dark grey highlights the decrease in CBT’ and IR values (4700–2300 cal BP). Figure 9. CAN02 (a) pollen-inferred reconstructions of the annual precipitation (MAP; in millimetres) obtained with BRT (yellow squares and curve) and MAT (blue triangles and curve) methods based on the TEMPSCAND calibration. The solid line corresponds to locally estimated scatterplot smoothing (loess) regression curves; the shaded area corresponds to its 95 % conﬁdence interval. The dashed black line corresponds to Canroute’s current calculated MAP. (b) CBT’-based pH reconstruction (green circles and line; De Jonge et al., 2021). On the right, the lateral line represents calibration error (RMSE). The time highlighted in light grey reveals a large fall in tetramethylated brGDGT abundance and a shift in accumulation rate (6600–4700 cal BP). The era shown in dark grey highlights the decrease in CBT’ and IR values (4700–2300 cal BP). Robles et al., 2023). The Late Glacial climatic changes esti- mated from the CAN02 sequence are consistent with obser- vations from southern Europe, notably with the temperature signal of the Lapsou sequence proposed by Duprat-Oualid et al. (2022), located in Cantal (central part of Massif Central). The comparison between the pollen signals from Canroute and Lapsou reveals some similarities (e.g. the chronology of the Bølling–Allerød between 14 600 and 12 900 cal BP) and some discrepancies (e.g. the dynamics of Younger Dryas are clearly less marked at Canroute). Typical millennial Late Glacial events, such as the Bølling–Allerød and Younger Dryas, cannot, however, be seen on both proxies since the brGDGT signal does not reﬂect such abrupt events (Fig. 11a). 4.4 Temporality of proxies’ resilience to environmental changes There are two distinct periods for which proxy records are impacted by environmental inﬂuences, notably a hydrolog- ical change in the peatland (Figs. 3 and 6, shaded areas). The record of vegetation (Fig. 6), detrital signal (Fig. 3), and brGDGT compounds (Fig. 5) all show a ﬁrst response to a loss of water runoff on the wetland surface between 6600– Early to Middle Holocene temperatures cooler than the present-day ones and followed by a Late Holocene warm- ing (i.e. the inverse of the HTM followed by a Late Holocene cooling), inferred by two independent proxies, appear as a https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures The presence of different patterns in the different reconstructions can be ex- plained by the type of parameter reconstructed (SST; mean temperature of the warmer month, MTWA; MAAT; etc.); by the method used (local, regional, or area-average reconstruc- tions) (Erb et al., 2022); by the proxy itself; or by local condi- tions, which can largely inﬂuence the proxy record over time. For example, the two reconstructions based on brGDGTs (Fig. 11b) do not indicate the same climate signal. Such a difference for the same proxy in the same region (Massif Central) may be due to several factors used to reconstruct the signal: local environment (peatland vs. lake) and altitude (790 vs. 1234 m). On the other hand, summer temperature proxies support the cooling trend that typically follows the HTM (Herzschuh et al., 2023; Heiri et al., 2003; Samartin et al., 2017; Jalali et al., 2016). In the case of the SSTs of the Gulf of Lion proposed by Jalali et al. (2016), the signal is based on the alkenone record, which could also be biased towards seasonal temperatures (Bader et al., 2020). Addition- ally, when Canroute’s BRT-based signal is compared to the presence of a Holocene thermal maximum (HTM) between 10 000 and 6000 cal BP. Summer and annual temperatures, reconstructed from the Swiss Alps, Gulf of Lions, Europe (40–50◦N), northern Italy, and Massif Central (Heiri et al., 2003; Jalali et al., 2016; Samartin et al., 2017; Martin et al., 2020; Herzschuh et al., 2023; Fig. 11b), suggest a HTM be- tween 10 700 and 5500 cal BP. The pollen-based annual tem- perature reconstruction by Herzchuh et al. (2023) indicates warm conditions for southern Europe (40–50◦N) but not re- ally a HTM (Fig. 11b). When compared to those sequences, only the pollen-based MAT method reconstructs a clear HTM between 10 000 and 8000 cal BP at Canroute, although it is less marked in terms of anomalies and probably biased by the high percentages of Corylus without modern analogues (Fig. 6). The BRT method and brGDGT-based reconstructions both indicate a plateau, closer to the annual climate signal pro- posed by Herzschuh et al. (2023) for Europe (40–50◦N) with the WA-PLS (Fig. 11b). From the Middle Holocene (ca. 6000 cal BP) onward, the reconstructions from Canroute in- dicate a warming trend in agreement with the larger synthe- sis by Davis et al. (2003) and Herzschuh et al. (2023). L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2145 Figure 10. CAN02 temperature reconstructions (MAAT; ◦C) obtained from (a) pollen assemblages using BRT (yellow triangles and curve) and MAT (blue squares and curve) methods based on the TEMPSCAND calibration. The solid line corresponds to locally estimated scatter- plot smoothing (loess) regression curves, and the shaded area corresponds to its 95 % conﬁdence interval. (b) The brGDGT signal for the three selected soil calibrations: Soil Bayesian (dark-blue squares and line; Dearing Crampton-Flood et al., 2020), mr (triangle symbol and pink line; De Jonge et al., 2014a), and Index1 (yellow circle and line; De Jonge et al., 2014a). On the right, lateral lines represent calibration errors (RMSE). Finally, the dashed black line corresponds to Canroute’s modern MAAT. The time highlighted in light grey reveals a large fall in tetramethylated brGDGT abundance (6600–4700 cal BP) and a shift in accumulation rate. The era shown in dark grey highlights the signiﬁcant decrease in IR (4700–2300 cal BP). Figure 10. CAN02 temperature reconstructions (MAAT; ◦C) obtained from (a) pollen assemblages using BRT (yellow triangles and curve) and MAT (blue squares and curve) methods based on the TEMPSCAND calibration. The solid line corresponds to locally estimated scatter- plot smoothing (loess) regression curves, and the shaded area corresponds to its 95 % conﬁdence interval. (b) The brGDGT signal for the three selected soil calibrations: Soil Bayesian (dark-blue squares and line; Dearing Crampton-Flood et al., 2020), mr (triangle symbol and pink line; De Jonge et al., 2014a), and Index1 (yellow circle and line; De Jonge et al., 2014a). On the right, lateral lines represent calibration errors (RMSE). Finally, the dashed black line corresponds to Canroute’s modern MAAT. The time highlighted in light grey reveals a large fall in tetramethylated brGDGT abundance (6600–4700 cal BP) and a shift in accumulation rate. The era shown in dark grey highlights the signiﬁcant decrease in IR (4700–2300 cal BP). al., 2020), the Swiss Alps (chironomids; Heiri et al., 2003), northern Italy (chironomids; Samartin et al., 2017), and the Mediterranean (alkenones; Jalali et al., 2016; Fig. 11b) in- dicate a cooling trend following the HTM. 4.5.2 Regional climate of the northern Mediterranean basin during the last 15 000 years The Canroute climate reconstruction indicates cold condi- tions for the Late Glacial and a warming for the Early Holocene (about +5 ± 1 ◦C; Fig. 11a). A similar climatic trend for this period is reconstructed from chironomid data in northern Italy (Fig. 11a), with increasing summer tem- peratures between 14 000 and 10 000 cal BP of the same or- der of magnitude (ca. +6.5 ◦C; Samartin et al., 2017). The brGDGT signal from Canroute indicates an abrupt warm- ing around 13 500 cal BP, which is also observed in south- ern Italy from the Lake Matese brGDGT record (Fig. 11a; For the Holocene, a spatio-temporal heterogeneity of cli- mate patterns is observed at the northern Mediterranean re- gion scale depending on sequences and proxies (Fig. 11). Most of the records indicate, despite a temporal disparity, the https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures In contrast, the data from Massif Central (brGDGTs; Martin et https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2146 1. Selected palaeoenvironmental records from southern Europe. (a) Late Glacial climate changes: pollen- and brG (Fig. 10), chironomid-based July temperatures from northern Italy (Samartin et al., 2017), brGDGT-based MAATs southern Italy) (Robles et al., 2023). (b) Holocene climate changes: July and January insolation for 44◦N (Wm−2) (L AATs inferred from CAN02 pollen assemblages (the dashed part corresponds to the period where Corylus strongly i btained with the MAT and over warmed the HTM) and from brGDGTs, MAAT and summer temperature of the Europ N) from pollen data (Herzschuh et al., 2023), MAATs based on brGDGTs (Martin et al., 2020), chironomid-based July t al., 2003; Samartin et al., 2017), pollen-based MAAT for south-western Europe (Davis et a., 2003), sea surface tempera the Gulf of Lions (Jalali et al., 2016). The green (Canroute) and blue curves correspond to annual reconstructed temper s correspond to seasonal (summer) reconstructed temperatures. The red box marks the Holocene thermal maximum of th ere (HTM). Temperature values are expressed as anomalies from the modern climate conditions at each site. ast 19 2127–2156 2023 https://doi org/10 5194/cp-19-2 Figure 11. Selected palaeoenvironmental records from southern Europe. (a) Late Glacial climate changes: pollen- and brGDGT-based MAATs (Fig. 10), chironomid-based July temperatures from northern Italy (Samartin et al., 2017), brGDGT-based MAATs from Lake Matese (southern Italy) (Robles et al., 2023). (b) Holocene climate changes: July and January insolation for 44◦N (Wm−2) (Laskar et al., 2004), MAATs inferred from CAN02 pollen assemblages (the dashed part corresponds to the period where Corylus strongly impacts the results obtained with the MAT and over warmed the HTM) and from brGDGTs, MAAT and summer temperature of the European region (40–50◦N) from pollen data (Herzschuh et al., 2023), MAATs based on brGDGTs (Martin et al., 2020), chironomid-based July temperature (Heiri et al., 2003; Samartin et al., 2017), pollen-based MAAT for south-western Europe (Davis et a., 2003), sea surface temperature (SST) values of the Gulf of Lions (Jalali et al., 2016). The green (Canroute) and blue curves correspond to annual reconstructed temperatures. The red curves correspond to seasonal (summer) reconstructed temperatures. The red box marks the Holocene thermal maximum of the Northern Hemisphere (HTM). Temperature values are expressed as anomalies from the modern climate conditions at each site. Clim. L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 2147 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures two signals from Herzschuh et al. (2023), i.e. the annual and July signal, only the annual signal appears consistent with Canroute’s signal, the seasonal signal indicating a different trend from 8000 cal BP onward (Fig. 11b). This supports the hypothesis of the inﬂuence of the seasonal (summer) tem- peratures in the cooling trend following the presence of the HTM in the Mediterranean region in many studies (Wanner, 2021). To conclude, in this study, the inﬂuence of seasonal tem- perature in different reconstructions, with independent prox- ies, is a solid hypothesis to explain the presence of different climatic patterns during the Holocene, particularly for the presence of the Holocene thermic maximum (HTM) in the Mediterranean region. 5 Conclusion The palaeoclimatic reconstruction of the last 15 000 years based on the CAN02 sequence has allowed us to compare the southern Massif Central climate changes to the southern Europe one. The Late Glacial and Early Holocene tempera- ture patterns at Canroute are consistent with reconstructions in Italy that show cold conditions during the Late Glacial and a warming for the Early Holocene. The brGDGT and pollen climate signal shows the presence of a Middle Holocene plateau followed by a Late Holocene warming instead of a clear Middle Holocene thermal maximum (HTM). The sim- ilar trends between the two independent proxies support the reliability of their respective reconstructions. Our study also highlights the potential causes of the differences between the reconstructions from independent proxies. The inﬂuence of local context changes, such as a decrease in water input, on the vegetation and brGDGT records has been assessed from geochemical, pollen, and brGDGT records. The multi-proxy approach points out the importance of investigating changes in the local environmental context for a better interpretation of the reconstructed climate parameters, as those changes could impact pollen and brGDGT records and thus the qual- ity of the reconstructed climate parameters. Whether for pollen or brGDGTs, the choice of (1) the method, (2) the modern dataset, and (3) the calibration is a key step to reconstruct climate parameters and has a sig- niﬁcant role in the reliability of reconstructions. Our study corroborates the role of regional calibration in the reliability of reconstructed MAATs. For brGDGTs, a selection of Eu- ropean surface samples from the global peat and soil calibra- tions could improve the reliability of MAAT reconstructions. Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2148 Appendix A Appendix A Appendix A Table A1. Location and mean current climate values of sites associated with surface samples for brGDGT analysis. The current climate parameters were extracted with the geographic information system (GIS) software QGIS (QGIS.org, 2022) from the interpolated database WorldClim2.0 (averaged over the period 1970–2000; Fick and Hijmans, 2017) for annual temperature (MAAT) and precipitation (MAP) data and the CRU TS (version 4.06) measured database (Harris et al., 2020) for monthly temperature and precipitation data (Fig. 1c). Table A1. Location and mean current climate values of sites associated with surface samples for brGDGT analysis. The current climate parameters were extracted with the geographic information system (GIS) software QGIS (QGIS.org, 2022) from the interpolated database WorldClim2.0 (averaged over the period 1970–2000; Fick and Hijmans, 2017) for annual temperature (MAAT) and precipitation (MAP) data and the CRU TS (version 4.06) measured database (Harris et al., 2020) for monthly temperature and precipitation data (Fig. 1c). Peatland Localization Sample Elevation (m) MAAT (◦C) MAP (mm) Canroute 43◦38′48′′ N, 02◦34′35′′ E CAN0 790 9.5 895 Caroux 43◦35′59′′ N, 02◦58′25′′ E CAR-1-15 1090 9.1 1044 Caroux 43◦36′06′′ N, 02◦59′01′′ E CAR-C2 1090 9.8 1078 Lapsou 45◦04′39′′ N, 03◦44′44′′ E LAP 1200 8.7 697 Lozère 44◦27′01′′ N, 03◦38′01′′ E LOZ 1700 5.5 1534 Nassette 44◦28′11′′ N, 03◦37′27′′ E NAS 1320 5.9 1445 Nassette 44◦28′11′′ N, 03◦37′27′′ E NAS-21 1320 5.9 1445 Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 2149 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures Appendix B e B1. Location of surface sites used in (a) the Eurasian Pollen Database (EAPDB) compiled by Peyron et al. (2013, 2017), (b) PSCAND (Temperate Europe–Scandinavian) database, and (c) the MEDTEMP (Mediterranean–Temperate Europe) database. Figure B1. Location of surface sites used in (a) the Eurasian Pollen Database (EAPDB) compiled by Peyron et al. (2013, 2017), (b) the TEMPSCAND (Temperate Europe–Scandinavian) database, and (c) the MEDTEMP (Mediterranean–Temperate Europe) database. https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2150 Appendix C Figure C1. (a) Crenarchaeol. (b) PC1brGDGTs of the PCA performed on the brGDGT relative abundances of the Canroute sequence. (c) PC2brGDGTs of the PCA performed on the brGDGT relative abundances of the Canroute sequence, (d) PCA results with samples coloured according to the cluster to which they belong. Figure C1. (a) Crenarchaeol. Appendix A (b) PC1brGDGTs of the PCA performed on the brGDGT relative abundances of the Canroute sequence. (c) PC2brGDGTs of the PCA performed on the brGDGT relative abundances of the Canroute sequence, (d) PCA results with samples coloured according to the cluster to which they belong. References Data availability. BrGDGT analysis and pollen-inferred climate reconstruction will be fully available on PANGAEA; the pollen counts will be shared thanks to open international databases such as the EPD and Neotoma. Ardenghi, N., Mulch, A., Koutsodendris, A., Pross, J., Kahmen, A., and Niedermeyer, E. M.: Temperature and moisture vari- ability in the eastern Mediterranean region during Marine Iso- tope Stages 11–10 based on biomarker analysis of the Tenaghi Philippon peat deposit, Quaternary Sci. Rev., 225, 105977, https://doi.org/10.1016/j.quascirev.2019.105977, 2019. Author contributions. Ld’O performed the analytical work, and LD and JA designed the R codes. Ld’O, LD, GM, AE, and OP de- signed the study. LD, GM, AE, and OP supervised the study. SDM and LB provided the study material (CAN02 sequence). SDM, CB, LB, SAA, and MS contributed to data analysis. AE pro- vided ﬁnancial support for the project. All authors contributed to the writing of the manuscript. Bader, J., Jungclaus, J., Krivova, N., Lorenz, S., Maycock, A., Raddatz, T., Schmidt, H., Toohey, M., Wu, C.-J., and Claussen, M.: Global temperature modes shed light on the Holocene temperature conundrum, Nat. Commun., 11, 4726, https://doi.org/10.1038/s41467-020-18478-6, 2020. Ball, D. F.: Loss-on-ignition as an estimate of organic matter and organic carbon in non-calcareous soils, J. Soil Sci., 15, 84–92, https://doi.org/10.1111/j.1365-2389.1964.tb00247.x, 1964. Competing interests. The contact author has declared that none of the authors has any competing interests. Birks, H. H. and Birks, H. J. B.: Multi-proxy studies in palaeolimnology, Veg. Hist. Archaeobot., 15, 235–251, https://doi.org/10.1007/s00334-006-0066-6, 2006. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims made in the text, pub- lished maps, institutional afﬁliations, or any other geographical rep- resentation in this paper. While Copernicus Publications makes ev- ery effort to include appropriate place names, the ﬁnal responsibility lies with the authors. Birks, H. J. B. and Seppä, H.: Pollen-based reconstructions of late- Quaternary climate in Europe – progress, problems, and pitfalls, Acta Palaeobotanica, 44, 317–334, 2004. Blaauw, M., Christen, J. A., Vázquez, J. E., and Goring, S.: clam: Classical Age-Depth Modelling of Cores from Deposits, RCRAN, https://doi.org/doi:10.1016/j.quageo.2010.01.002, 2022. Blaga, C. I., Reichart, G.-J., Heiri, O., and Sinninghe Damsté, J. S.: Tetraether membrane lipid distributions in water-column par- ticulate matter and sediments: a study of 47 European lakes along a north–south transect, J. Paleolimnol., 41, 523–540, https://doi.org/10.1007/s10933-008-9242-2, 2009. Acknowledgements. Appendix D Figure D1. Reconstruction of the mean annual air temperature (MAAT) based on Canroute pollen sequence signal for the four methods used with the TEMPSCAND modern pollen database (a MAT, b WA-PLS, c RF, d BRT). The plain line corresponds to locally estimated scatterplot smoothing (loess) regression curves, and the shaded area corresponds to the conﬁdence interval used for the model (95 %). The black arrows represent the direction of the climate trends for the different periods considered. Dashed black line: current MAAT at Canroute. Figure D1. Reconstruction of the mean annual air temperature (MAAT) based on Canroute pollen sequence signal for the four methods used with the TEMPSCAND modern pollen database (a MAT, b WA-PLS, c RF, d BRT). The plain line corresponds to locally estimated scatterplot smoothing (loess) regression curves, and the shaded area corresponds to the conﬁdence interval used for the model (95 %). The black arrows represent the direction of the climate trends for the different periods considered. Dashed black line: current MAAT at Canroute. Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 L. d’Oliveira et al.: Reconstructing Data availability. BrGDGT analysis reconstruction will be fully available counts will be shared thanks to open as the EPD and Neotoma. Author contributions. Ld’O perform LD and JA designed the R codes. Ld’O signed the study. LD, GM, AE, and SDM and LB provided the study m SDM, CB, LB, SAA, and MS contribu vided ﬁnancial support for the projec the writing of the manuscript. Competing interests. The contact a of the authors has any competing inter Disclaimer. Publisher’s note: Coper neutral with regard to jurisdictional cl lished maps, institutional afﬁliations, o resentation in this paper. While Copern ery effort to include appropriate place n lies with the authors. Acknowledgements. The sampling samples was performed during mentor versity of Montpellier, as part of ORP authors thank Frédéric Néri and the CE ﬁeldwork facilities; ISEM for ﬁnancia dating; Sandrine Canal for the prepara samples; and Jean-Frédéric Terral, V students for help with ﬁeldwork and w the Canroute peatland. This is ISEM c Financial support. This research is project and has been supported by th cil (ERC) under the European Union’s innovation programme (grant agreem Evin). Review statement. This paper was bourieu Nebout and reviewed by Cind mous referee. 2151 References Dugerdil, L., Ménot, G., Peyron, O., Jouffroy-Bapicot, I., Ansanay- Alex, S., Antheaume, I., Behling, H., Boldgiv, B., Develle, A.- L., Grossi, V., Magail, J., Makou, M., Robles, M., Unkelbach, J., Vannière, B., and Joannin, S.: Late Holocene Mongolian cli- mate and environment reconstructions from brGDGTs, NPPs and pollen transfer functions for Lake Ayrag: Palaeoclimate implica- tions for Arid Central Asia, Quaternary Sci. Rev., 273, 107235, https://doi.org/10.1016/j.quascirev.2021.107235, 2021b. Dearing Crampton-Flood, E., Tierney, J. E., Peterse, F., Kirkels, F. M. S. A., and Sinninghe Damsté, J. S.: BayMBT: A Bayesian cal- ibration model for branched glycerol dialkyl glycerol tetraethers in soils and peats, Geochim. Cosmochim. Ac., 268, 142–159, https://doi.org/10.1016/j.gca.2019.09.043, 2020. Duprat-Oualid, F., Bégeot, C., Peyron, O., Rius, D., Millet, L., and Magny, M.: High-frequency vegetation and climatic changes dur- ing the Lateglacial inferred from the Lapsou pollen record (Can- tal, southern Massif Central, France), Quatern. Int., 636, 69–80, https://doi.org/10.1016/j.quaint.2022.04.012, 2022. De’Ath, G.: Boosted Trees for Ecological. Modelling and Pre- diction, Ecology, 88, 243–251, https://doi.org/10.1890/0012- 9658(2007)88[243:BTFEMA]2.0.CO;2, 2007. de Beaulieu, J.-L. and Reille, M.: Paléoenvironnement tardiglaciaire et holocène des lacs de Pelléautier et Siguret (Hautes-Alpes, France). I. Histoire de la végétation d’après les analyses polliniques, Ecologia Mediterranea, 9, 19–36, 1983. Elith, J., Leathwick, J. R., and Hastie, T.: A working guide to boosted regression trees, J. Anim. Ecol., 77, 802–813, https://doi.org/10.1111/j.1365-2656.2008.01390.x, 2008. Decorsiere, J., Delamare-Deboutteville, J., and Lecerf, C.: Recon- stitution de 13 500 ans d’histoire d’une tourbière dans le Sud de la France, Orpalm Synthesis, 12, 29–39, 2019. Erb, M. P., McKay, N. P., Steiger, N., Dee, S., Hancock, C., Ivanovic, R. F., Gregoire, L. J., and Valdes, P.: Re- constructing Holocene temperatures in time and space us- ing paleoclimate data assimilation, Clim. Past, 18, 2599–2629, https://doi.org/10.5194/cp-18-2599-2022, 2022. De Jonge, C., Hopmans, E. C., Zell, C. I., Kim, J.-H., Schouten, S., and Sinninghe Damsté, J. S.: Occurrence and abun- dance of 6-methyl branched glycerol dialkyl glycerol tetraethers in soils: Implications for palaeoclimate re- construction, Geochim. Cosmochim. Ac., 141, 97–112, https://doi.org/10.1016/j.gca.2014.06.013, 2014a. Fick, S. E. and Hijmans, R. J.: WorldClim 2: new 1 km spatial reso- lution climate surfaces for global land areas, Int. J. Climatol., 37, 4302–4315, https://doi.org/10.1002/joc.5086, 2017. Grimm, E. C.: CONISS: a FORTRAN 77 program for strati- graphically constrained cluster analysis by the method of in- cremental sum of squares, Comput. Geosci.-UK, 13, 13–35, https://doi.org/10.1016/0098-3004(87)90022-7, 1987. De Jonge, C., Stadnitskaia, A., Hopmans, E. References d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2152 of soil chemistry on branched tetraether lipids in mid- and high latitude soils: Implications for brGDGT-based palaeothermometry, Geochim. Cosmochim. Ac., 310, 95–112, https://doi.org/10.1016/j.gca.2021.06.037, 2021. T., Phelps, L. N., Dawson, A., Zanon, M., Vallé, F., Nolan, C., Mauri, A., de Vernal, A., Izumi, K., Holmström, L., Marsicek, J., Goring, S., Sommer, P. S., Chaput, M., and Kupriyanov, D.: Pollen-based climate reconstruction techniques for late Quater- nary studies, Earth-Sci. Rev., 210, 33, 2020. Denton, G. H., Anderson, R. F., Toggweiler, J. R., Edwards, R. L., Schaefer, J. M., and Putnam, A. E.: The Last Glacial Termina- tion, Science, 328, 1652–1656, 2010. Davies, S. J., Lamb, H. F., and Roberts, S. J.: Micro-XRF Core Scanning in Palaeolimnology: Recent Developments, in: Micro-XRF Studies of Sediment Cores: Applications of a non- destructive tool for the environmental sciences, edited by: Croudace, I. W. and Rothwell, R. G., Springer Netherlands, Dor- drecht, 189–226, https://doi.org/10.1007/978-94-017-9849-5_7, 2015. Ding, S., Schwab, V. F., Ueberschaar, N., Roth, V.-N., Lange, M., Xu, Y., Gleixner, G., and Pohnert, G.: Identiﬁcation of novel 7-methyl and cyclopentanyl branched glycerol dialkyl glyc- erol tetraethers in lake sediments, Org. Geochem., 102, 52–58, https://doi.org/10.1016/j.orggeochem.2016.09.009, 2016. Davis, B. A. S., Brewer, S., Stevenson, A. C., and Guiot, J.: The temperature of Europe during the Holocene recon- structed from pollen data, Quaternary Sci. Rev., 22, 1701–1716, https://doi.org/10.1016/S0277-3791(03)00173-2, 2003. Dray, S. and Josse, J.: Principal component analysis with missing values: a comparative survey of methods, Plant Ecol., 216, 657– 667, https://doi.org/10.1007/s11258-014-0406-z, 2015. Dugerdil, L., Joannin, S., Peyron, O., Jouffroy-Bapicot, I., Vannière, B., Boldgiv, B., Unkelbach, J., Behling, H., and Ménot, G.: Cli- mate reconstructions based on GDGT and pollen surface datasets from Mongolia and Baikal area: calibrations and applicability to extremely cold–dry environments over the Late Holocene, Clim. Past, 17, 1199–1226, https://doi.org/10.5194/cp-17-1199-2021, 2021a. Davtian, N., Bard, E., Darfeuil, S., Ménot, G., and Rostek, F.: The Novel Hydroxylated Tetraether Index RI-OH′ as a Sea Sur- face Temperature Proxy for the 160–45 ka BP Period Off the Iberian Margin, Palaeoceanography and Palaeoclimatology, 36, e2020PA004077, https://doi.org/10.1029/2020PA004077, 2021. de Beaulieu, J.-L.: Contribution pollenanalytique à l’histoire tardiglaciaire et holocène des Alpes méridionales françaises, Doctoral thesis, Université d’Aix-Marseille III, France, 358 pp., 490842065, 1977. References The sampling of peat cores and surface samples was performed during mentored ﬁeld research of the Uni- versity of Montpellier, as part of ORPAM teaching modules. The authors thank Frédéric Néri and the CEN Languedoc-Roussillon for ﬁeldwork facilities; ISEM for ﬁnancial contribution to radiocarbon dating; Sandrine Canal for the preparation and treatment of pollen samples; and Jean-Frédéric Terral, Vincent Girard, and ORPAM students for help with ﬁeldwork and with the preliminary study of the Canroute peatland. This is ISEM contribution ISEM 2023-199. Bradley, R. S.: Paleoclimatology: reconstructing climates of the quaternary, 3rd Edn., Elsevier Academic Press, Amsterdam, 675 pp., https://doi.org/10.1016/C2009-0-18310-1, 2015. Breiman, L.: Random Forests, Mach. Learn., 45, 5–32, https://doi.org/10.1023/A:1010933404324, 2001. Brewer, S., Guiot, J., Sánchez-Goñi, M. F., and Klotz, S.: The climate in Europe during the Eemian: a multi-method ap- proach using pollen data, Quaternary Sci. Rev., 27, 2303–2315, https://doi.org/10.1016/j.quascirev.2008.08.029, 2008. Financial support. This research is a part of the DEMETER project and has been supported by the European Research Coun- cil (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 852573, PI Allowen Evin). Broecker, W. S., Denton, G. H., Edwards, R. L., Cheng, H., Alley, R. B., and Putnam, A. E.: Putting the Younger Dryas cold event into context, Quaternary Sci. Rev., 29, 1078–1081, 2010. Cartapanis, O., Jonkers, L., Moffa-Sanchez, P., Jaccard, S. L., and de Vernal, A.: Complex spatio-temporal structure of the Holocene thermal maximum, Nat. Commun., 13, 5662, https://doi.org/10.1038/s41467-022-33362-1, 2022. Review statement. This paper was edited by Nathalie Com- bourieu Nebout and reviewed by Cindy De Jonge and one anony- mous referee. Cheddadi, R., Yu, G., Guiot, J., Harrison, S. P., and Prentice, I. C.: The climate of Europe 6000 years ago, Clim. Dynam., 13, 1–9, https://doi.org/10.1007/s003820050148, 1997. Chen, Y., Zheng, F., Yang, H., Yang, W., Wu, R., Liu, X., Liang, H., Chen, H., Pei, H., Zhang, C., Pancost, R. D., and Zeng, Z.: The production of diverse brGDGTs by an Acidobacterium providing a physiological basis for paleo- climate proxies, Geochim. Cosmochim. Ac., 337, 155–165, https://doi.org/10.1016/j.gca.2022.08.033, 2022. Chevalier, M., Davis, B. A. S., Heiri, O., Seppä, H., Chase, B. M., Gajewski, K., Lacourse, T., Telford, R. J., Finsinger, W., Guiot, J., Kühl, N., Maezumi, S. Y., Tipton, J. R., Carter, V. A., Brussel, https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 L. References Lê, S., Josse, J., and Husson, F.: FactoMineR: A Package for Mul- tivariate Analysis, J. Stat. Softw., 25, 1-18, 2008. Le Houérou, H.-N.: Biogeography of the arid steppeland north of the Sahara, J. Arid Environ., 48, 103–128, 2001. Li, J., Pancost, R. D., Naafs, B. D. A., Yang, H., Zhao, C., and Xie, S.: Distribution of glycerol dialkyl glycerol tetraether (GDGT) lipids in a hypersaline lake system, Org. Geochem., 99, 113–124, https://doi.org/10.1016/j.orggeochem.2016.06.007, 2016. Hijmans, R. J., Phillips, S., Leathwick, J., and Elith, J.: dismo: Species Distribution Modeling, RCRAN, https://CRAN. R-project.org/package=dismo (last access: March 2023), 2022. Liaw, A. and Wiener, M.: Classiﬁcation and Regression by random- Forest, R News, 2, 18–22, 2002. Hopmans, E. C., Weijers, J. W. H., Schefuß, E., Herfort, L., Sin- ninghe Damsté, J. S., and Schouten, S.: A novel proxy for ter- restrial organic matter in sediments based on branched and iso- prenoid tetraether lipids, Earth Planet. Sc. Lett., 224, 107–116, https://doi.org/10.1016/j.epsl.2004.05.012, 2004. Liu, Z., Zhu, J., Rosenthal, Y., Zhang, X., Otto-Bliesner, B. L., Timmermann, A., Smith, R. S., Lohmann, G., Zheng, W., and Elison Timm, O.: The Holocene temperature co- nundrum, P. Natl. Acad. Sci. USA, 111, E3501–E3505, https://doi.org/10.1073/pnas.1407229111, 2014. Hopmans, E. C., Schouten, S., and Sinninghe Damsté, J. S.: The effect of improved chromatography on GDGT-based palaeoproxies, Org. Geochem., 93, 1–6, https://doi.org/10.1016/j.orggeochem.2015.12.006, 2016. Marcott, S. A., Shakun, J. D., Clark, P. U., and Mix, A. C.: A Reconstruction of Regional and Global Tempera- ture for the Past 11 300 Years, Science, 339, 1198–1201, https://doi.org/10.1126/science.1228026, 2013. Huguet, A., Fosse, C., Laggoun-Défarge, F., Toussaint, M.-L., and Derenne, S.: Occurrence and distribution of glycerol di- alkyl glycerol tetraethers in a French peat bog, Org. Geochem., 41, 559–572, https://doi.org/10.1016/j.orggeochem.2010.02.015, 2010. Marriner, N., Kaniewski, D., Pourkerman, M., and Devillers, B.: Anthropocene tipping point reverses long-term Holocene cool- ing of the Mediterranean Sea: A meta-analysis of the basin’s Sea Surface Temperature records, Earth-Sci. Rev., 227, 103986, https://doi.org/10.1016/j.earscirev.2022.103986, 2022. Huguet, C., Hopmans, E. C., Febo-Ayala, W., Thompson, D. H., Sinninghe Damsté, J. S., and Schouten, S.: An improved method to determine the absolute abundance of glycerol dibiphy- tanyl glycerol tetraether lipids, Org. Geochem., 37, 1036–1041, https://doi.org/10.1016/j.orggeochem.2006.05.008, 2006. Marsicek, J., Shuman, B. N., Bartlein, P. J., Shafer, S. L., and Brewer, S.: Reconciling divergent trends and millen- nial variations in Holocene temperatures, Nature, 554, 19, https://doi.org/10.1038/nature25464, 2018. References C., Cherkashov, G., Fedotov, A., and Sinninghe Damsté, J. S.: In situ pro- duced branched glycerol dialkyl glycerol tetraethers in sus- pended particulate matter from the Yenisei River, East- ern Siberia, Geochim. Cosmochim. Ac., 125, 476–491, https://doi.org/10.1016/j.gca.2013.10.031, 2014b. Guiot, J.: Methodology of the last climatic cycle reconstruction in France from pollen data, Palaeogeogr. Palaeocl., 80, 49–69, https://doi.org/10.1016/0031-0182(90)90033-4, 1990. De Jonge, C., Radujkovi´c, D., Sigurdsson, B. D., Weedon, J. T., Janssens, I., and Peterse, F.: Lipid biomarker temperature proxy responds to abrupt shift in the bacterial community composi- tion in geothermally heated soils, Org. Geochem., 137, 103897, https://doi.org/10.1016/j.orggeochem.2019.07.006, 2019. Halamka, T. A., Raberg, J. H., McFarlin, J. M., Younkin, A. D., Mulligan, C., Liu, X., and Kopf, S. H.: Production of diverse brGDGTs by Acidobacterium Solibacter usitatus in response to temperature, pH, and O2 provides a culturing perspective on br GDGT proxies and biosynthesis, Geobiology, 21, 102–118, https://doi.org/10.1111/gbi.12525, 2023. De Jonge, C., Kuramae, E. E., Radujkovi´c, D., Weedon, J. T., Janssens, I. A., and Peterse, F.: The inﬂuence Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2153 Kassambara, A. and Mundt, F.: factoextra: Extract and Visualize the Results of Multivariate Data Analyses, RCRAN, https://CRAN. R-project.org/package=factoextra (last access: July 2023), 2020. Harris, I., Osborn, T. J., Jones, P., and Lister, D.: Version 4 of the CRU TS monthly high-resolution gridded multivariate climate database, Sci. Data, 7, 109, https://doi.org/10.1038/s41597-020- 0453-3, 2020. Kaufman, D., McKay, N., Routson, C., Erb, M., Dätwyler, C., Som- mer, P. S., Heiri, O., and Davis, B.: Holocene global mean surface temperature, a multi-method reconstruction approach, Sci. Data, 7, 201, https://doi.org/10.1038/s41597-020-0530-7, 2020. Heiri, O., Lotter, A. F., Hausmann, S., and Kienast, F.: A chironomid-based Holocene summer air temperature recon- struction from the Swiss Alps, Holocene, 13, 477–484, https://doi.org/10.1191/0959683603hl640ft, 2003. Laskar, J., Robutel, P., Joutel, F., Gastineau, M., Correia, A. C. M., and Levrard, B.: A long-term numerical solution for the insola- tion quantities of the Earth, Astron. Astrophys., 428, 261–285, https://doi.org/10.1051/0004-6361:20041335, 2004. Herzschuh, U., Böhmer, T., Chevalier, M., Hébert, R., Dallmeyer, A., Li, C., Cao, X., Peyron, O., Nazarova, L., Novenko, E. Y., Park, J., Rudaya, N. A., Schlütz, F., Shumilovskikh, L. S., Tarasov, P. E., Wang, Y., Wen, R., Xu, Q., and Zheng, Z.: Re- gional pollen-based Holocene temperature and precipitation pat- terns depart from the Northern Hemisphere mean trends, Clim. Past, 19, 1481–1506, https://doi.org/10.5194/cp-19-1481-2023, 2023. References A., Gasse, F., van Kreveld, S., Holmgren, K., Lee-Thorp, J., Rosqvist, G., Rack, F., Staubwasser, M., Schneider, R. R., and Steig, E. J.: Holocene climate variability, Quaternary Res., 62, 243–255, https://doi.org/10.1016/j.yqres.2004.07.001, 2004. Pearson, E. J., Juggins, S., Talbot, H. M., Weckström, J., Rosén, P., Ryves, D. B., Roberts, S. J., and Schmidt, R.: A lacus- trine GDGT-temperature calibration from the Scandinavian Arc- tic to Antarctic: Renewed potential for the application of GDGT- palaeothermometry in lakes, Geochim. Cosmochim. Ac., 75, 6225–6238, https://doi.org/10.1016/j.gca.2011.07.042, 2011. Miras, Y., Vergne, V., Guenet, P., and Surmely, F.: Le Mas- sif central: premières traces d’anthropisation révélées par l’analyse pollinique des zones humides corrélées aux don- nées archéologiques, in: Néolithisation précoce. Premières traces d’anthropisation du couvert végétal à partir des données polliniques, edited by: Richard, H., Presses Universitaires de Franche-Comté, 89–106, https://hal.science/hal-00267413 (last access: October 2023), 2005. Peterse, F., van der Meer, J., Schouten, S., Weijers, J. W. H., Fierer, N., Jackson, R. B., Kim, J.- H., and Sinninghe Damsté, J. S.: Revised calibration of the MBT–CBT palaeotemper- ature proxy based on branched tetraether membrane lipids in surface soils, Geochim. Cosmochim. Ac., 96, 215–229, https://doi.org/10.1016/j.gca.2012.08.011, 2012. Peyron, O., Bégeot, C., Brewer, S., Heiri, O., Magny, M., Millet, L., Ruffaldi, P., Van Campo, E., and Yu, G.: Late-Glacial cli- matic changes in Eastern France (Lake Lautrey) from pollen, lake-levels, and chironomids, Quaternary Res., 64, 197–211, https://doi.org/10.1016/j.yqres.2005.01.006, 2005. Mohammad, A. G. and Adam, M. A.: The impact of vegetative cover type on runoff and soil erosion under different land uses, CATENA, 81, 97–103, https://doi.org/10.1016/j.catena.2010.01.008, 2010. Moore, P. D.: The ecology of peat-forming processes: a review, Int. J. Coal Geol., 12, 89–103, 1989. Peyron, O., Goring, S., Dormoy, I., Kotthoff, U., Pross, J., de Beaulieu, J.-L., Drescher-Schneider, R., Vannière, B., and Magny, M.: Holocene seasonality changes in the central Mediter- ranean region reconstructed from the pollen sequences of Lake Accesa (Italy) and Tenaghi Philippon (Greece), Holocene, 21, 131–146, https://doi.org/10.1177/0959683610384162, 2011. Muller, S. D., Brémond, L., Girard, V., and Terral, J.-F.: Végéta- tion de la tourbière de Canroute, dans les Monts de Lacaune (Le Margnès, Tarn), Orpalm Synthesis, 11, 9–13, 2018. Naafs, B. D. A., Gallego-Sala, A. V., Inglis, G. N., and Pancost, R. D.: Reﬁning the global branched glyc- erol dialkyl glycerol tetraether (brGDGT) soil tem- perature calibration, Org. Geochem., 106, 48–56, https://doi.org/10.1016/j.orggeochem.2017.01.009, 2017a. References Jalali, B., Sicre, M.-A., Bassetti, M.-A., and Kallel, N.: Holocene climate variability in the North-Western Mediterranean Sea (Gulf of Lions), Clim. Past, 12, 91–101, https://doi.org/10.5194/cp-12- 91-2016, 2016. Martin, C., Ménot, G., Thouveny, N., Davtian, N., Andrieu- Ponel, V., Reille, M., and Bard, E.: Impact of human activi- ties and vegetation changes on the tetraether sources in Lake St Front (Massif Central, France), Org. Geochem., 135, 38–52, https://doi.org/10.1016/j.orggeochem.2019.06.005, 2019. Joosten, H.: Current soil carbon loss and land degradation globally: where are the hotspots and why there?, CABI Books, 224–234, https://doi.org/10.1079/9781780645322.0224, 2015. Martin, C., Ménot, G., Thouveny, N., Peyron, O., Andrieu- Ponel, V., Montade, V., Davtian, N., Reille, M., and Bard, E.: Early Holocene thermal maximum recorded by branched tetraethers and pollen in Western Europe (Mas- sif Central, France), Quaternary Sci. Rev., 228, 106109, https://doi.org/10.1016/j.quascirev.2019.106109, 2020. Josse, J. and Husson, F.: missMDA: A Package for Handling Miss- ing Values in Multivariate Data Analysis, J. Stat. Softw., 70, 1– 31, https://doi.org/10.18637/jss.v070.i01, 2016. Juggins, S.: rioja: Analysis of Quaternary Science Data, RCRAN, https://cran.r-project.org/package=rioja (last access: March 2023), 2023. Martínez-Sosa, P., Tierney, J. E., Stefanescu, I. C., Dear- ing Crampton-Flood, E., Shuman, B. N., and Routson, C.: A global Bayesian temperature calibration for lacus- trine brGDGTs, Geochim. Cosmochim. Ac., 305, 87–105, https://doi.org/10.1016/j.gca.2021.04.038, 2021. Julve, P.: Les tourbières de France : répartition, caractères biogéo- graphiques, fonctionnement écologique et dynamique, valeur patrimoniale (French Peat-bogs : localisation, biogeographic characters, ecological functionning, patrimonial value), Bul- letin de l’Association de géographes français, 71, 287–293, https://doi.org/10.3406/bagf.1994.1748, 1994. p g j g Mauri, A., Davis, B. A. S., Collins, P. M., and Kaplan, J. O.: The in- ﬂuence of atmospheric circulation on the mid-Holocene climate of Europe: a data–model comparison, Clim. Past, 10, 1925–1938, https://doi.org/10.5194/cp-10-1925-2014, 2014. Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2154 https://doi.org/10.5067/MEaSUREs/SRTM/SRTMGL1.003, 2013. Mauri, A., Davis, B. A. S., Collins, P. M., and Kaplan, J. O.: The climate of Europe during the Holocene: a gridded pollen-based reconstruction and its multi- proxy evaluation, Quaternary Sci. Rev., 112, 109–127, https://doi.org/10.1016/j.quascirev.2015.01.013, 2015. Pearson, A. and Ingalls, A. E.: Assessing the Use of Archaeal Lipids as Marine Environmental Proxies, Annu. Rev. Earth Pl. Sc., 41, 359–384, https://doi.org/10.1146/annurev-earth-050212- 123947, 2013. Mayewski, P. A., Rohling, E. E., Curt Stager, J., Karlén, W., Maasch, K. A., Meeker, L. D., Meyerson, E. References Peyron, O., Magny, M., Goring, S., Joannin, S., de Beaulieu, J.- L., Brugiapaglia, E., Sadori, L., Garﬁ, G., Kouli, K., Ioakim, C., and Combourieu-Nebout, N.: Contrasting patterns of cli- matic changes during the Holocene across the Italian Penin- sula reconstructed from pollen data, Clim. Past, 9, 1233–1252, https://doi.org/10.5194/cp-9-1233-2013, 2013. Naafs, B. D. A., Inglis, G. N., Zheng, Y., Amesbury, M. J., Biester, H., Bindler, R., Blewett, J., Burrows, M. A., Del Castillo Torres, D., Chambers, F. M., Cohen, A. D., Ever- shed, R. P., Feakins, S. J., Gałka, M., Gallego-Sala, A., Gan- dois, L., Gray, D. M., Hatcher, P. G., Honorio Coronado, E. N., Hughes, P. D. M., Huguet, A., Könönen, M., Laggoun- Défarge, F., Lähteenoja, O., Lamentowicz, M., Marchant, R., McClymont, E., Pontevedra-Pombal, X., Ponton, C., Pour- mand, A., Rizzuti, A. M., Rochefort, L., Schellekens, J., De Vleeschouwer, F., and Pancost, R. D.: Introducing global peat- speciﬁc temperature and pH calibrations based on brGDGT bacterial lipids, Geochim. Cosmochim. Ac., 208, 285–301, https://doi.org/10.1016/j.gca.2017.01.038, 2017b. Peyron, O., Combourieu-Nebout, N., Brayshaw, D., Goring, S., Andrieu-Ponel, V., Desprat, S., Fletcher, W., Gambin, B., Ioakim, C., Joannin, S., Kotthoff, U., Kouli, K., Montade, V., Pross, J., Sadori, L., and Magny, M.: Precipitation changes in the Mediter- ranean basin during the Holocene from terrestrial and marine pollen records: a model–data comparison, Clim. Past, 13, 249– 265, https://doi.org/10.5194/cp-13-249-2017, 2017. Ponel, P., Guiter, F., Gandouin, E., Peyron, O., and Beaulieu, J.-L. D.: Late-Glacial palaeotemperatures and palaeopre- cipitations in the Aubrac Mountains (French Massif Cen- tral) reconstructed from multiproxy analyses (Coleoptera, chironomids and pollen), Quatern. Int., 636, 39–51, https://doi.org/10.1016/j.quaint.2022.02.005, 2022. Naafs, B. D. A., Inglis, G. N., Blewett, J., McClymont, E. L., Lau- retano, V., Xie, S., Evershed, R. P., and Pancost, R. D.: The po- tential of biomarker proxies to trace climate, vegetation, and bio- geochemical processes in peat: A review, Global Planet. Change, 179, 57–79, https://doi.org/10.1016/j.gloplacha.2019.05.006, 2019. Posit team: RStudio: Integrated Development Environment for R, Posit Software, PBC, Boston, MA, http://www.posit.co/ (last ac- cess: July 2023), 2023. Prasad, A. M., Iverson, L. R., and Liaw, A.: Newer Classiﬁ- cation and Regression Tree Techniques: Bagging and Ran- dom Forests for Ecological Prediction, Ecosystems, 9, 181–199, https://doi.org/10.1007/s10021-005-0054-1, 2006. Naafs, B. D. A., Oliveira, A. S. F., and Mulholland, A. J.: Molecular dynamics simulations support the hypothesis that the brGDGT paleothermometer is based on homeovis- cous adaptation, Geochim. Cosmochim. Ac., 312, 44–56, https://doi.org/10.1016/j.gca.2021.07.034, 2021. References QGIS.org: QGIS Geographic Information System, QGIS Associa- tion, https://www.qgis.org/ (last access: February 2023), 2022. Raberg, J. H., Harning, D. J., Crump, S. E., de Wet, G., Blumm, A., Kopf, S., Geirsdóttir, Á., Miller, G. H., and Sepúlveda, J.: NASA JPL: NASA Shuttle Radar Topography Mission Global 1 arc second, NASA EOSDIS Land Processes DAAC [data set], Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2155 southern Iberian Peninsula), Quaternary Sci. Rev., 281, 107434, https://doi.org/10.1016/j.quascirev.2022.107434, 2022. Revised fractional abundances and warm-season temperatures substantially improve brGDGT calibrations in lake sediments, Biogeosciences, 18, 3579–3603, https://doi.org/10.5194/bg-18- 3579-2021, 2021. Revised fractional abundances and warm-season temperatures substantially improve brGDGT calibrations in lake sediments, Biogeosciences, 18, 3579–3603, https://doi.org/10.5194/bg-18- 3579-2021, 2021. southern Iberian Peninsula), Quaternary Sci. Rev., 281, 107434, https://doi.org/10.1016/j.quascirev.2022.107434, 2022. Russell, J. M., Hopmans, E. C., Loomis, S. E., Liang, J., and Sin- ninghe Damsté, J. S.: Distributions of 5- and 6-methyl branched glycerol dialkyl glycerol tetraethers (brGDGTs) in East African lake sediment: Effects of temperature, pH, and new lacustrine palaeotemperature calibrations, Org. Geochem., 117, 56–69, https://doi.org/10.1016/j.orggeochem.2017.12.003, 2018. Raberg, J. H., Miller, G. H., Geirsdóttir, Á., and Sepúlveda, J.: Near-universal trends in brGDGT lipid distributions in nature, Sci. Adv., 8, eabm7625, https://doi.org/10.1126/sciadv.abm7625, 2022. Ramos-Román, M. J., De Jonge, C., Magyari, E., Veres, D., Il- vonen, L., Develle, A.-L., and Seppä, H.: Lipid biomarker (brGDGT)- and pollen-based reconstruction of temperature change during the Middle to Late Holocene transition in the Carpathians, Global Planet. Change, 215, 103859, https://doi.org/10.1016/j.gloplacha.2022.103859, 2022. Sahonero-Canavesi, D. X., Siliakus, M. F., Abdala Asbun, A., Koe- nen, M., Von Meijenfeldt, F. A. B., Boeren, S., Bale, N. J., En- gelman, J. C., Fiege, K., Strack Van Schijndel, L., Sinninghe Damsté, J. S., and Villanueva, L.: Disentangling the lipid divide: Identiﬁcation of key enzymes for the biosynthesis of membrane- spanning and ether lipids in Bacteria, Sci. Adv., 8, eabq8652, https://doi.org/10.1126/sciadv.abq8652, 2022. Rao, Z., Guo, H., Wei, S., Cao, J., and Jia, G.: Inﬂuence of water conditions on peat brGDGTs: A modern investigation and its palaeoclimatic implications, Chem. Geol., 606, 120993, https://doi.org/10.1016/j.chemgeo.2022.120993, 2022. Salonen, J. S., Korpela, M., Williams, J. W., and Luoto, M.: Machine-learning based reconstructions of primary and secondary climate variables from North American and European fossil pollen data, Sci. Rep.-UK, 9, 15805, https://doi.org/10.1038/s41598-019-52293-4, 2019. References R Core Team: R: A language and environment for statistical com- puting, R Foundation for Statistical Computing, Vienna, Austria, https://www.R-project.org (last access: August 2023), 2022. Samartin, S., Heiri, O., Joos, F., Renssen, H., Franke, J., Brönni- mann, S., and Tinner, W.: Warm Mediterranean mid-Holocene summers inferred from fossil midge assemblages, Nat. Geosci., 10, 207–212, https://doi.org/10.1038/ngeo2891, 2017. Reimer, P. J., Austin, W. E. N., Bard, E., Bayliss, A., Blackwell, P. G., Bronk Ramsey, C., Butzin, M., Cheng, H., Edwards, R. L., Friedrich, M., Grootes, P. M., Guilderson, T. P., Hajdas, I., Heaton, T. J., Hogg, A. G., Hughen, K. A., Kromer, B., Manning, S. W., Muscheler, R., Palmer, J. G., Pearson, C., van der Plicht, J., Reimer, R. W., Richards, D. A., Scott, E. M., Southon, J. R., Turney, C. S. M., Wacker, L., Adolphi, F., Büntgen, U., Capano, M., Fahrni, S. M., Fogtmann-Schulz, A., Friedrich, R., Köhler, P., Kudsk, S., Miyake, F., Olsen, J., Reinig, F., Sakamoto, M., Sookdeo, A., and Talamo, S.: The IntCal20 Northern Hemisphere Radiocarbon Age Calibration Curve (0–55 cal kBP), Radiocar- bon, 62, 725–757, https://doi.org/10.1017/RDC.2020.41, 2020. Schouten, S., Rijpstra, W. I. C., Durisch-Kaiser, E., Schu- bert, C. J., and Sinninghe Damsté, J. S.: Distribution of glycerol dialkyl glycerol tetraether lipids in the water column of Lake Tanganyika, Org. Geochem., 53, 34–37, https://doi.org/10.1016/j.orggeochem.2012.01.009, 2012. J., Reimer, R. W., Richards, D. A., Scott, E. M., Southon, J. R., rney, C. S. M., Wacker, L., Adolphi, F., Büntgen, U., Capano, , Fahrni, S. M., Fogtmann-Schulz, A., Friedrich, R., Köhler, Schouten, S., Hopmans, E. C., and Sinninghe Damsté, J. S.: The organic geochemistry of glycerol dialkyl glyc- erol tetraether lipids: A review, Org. Geochem., 54, 19–61, https://doi.org/10.1016/j.orggeochem.2012.09.006, 2013. Renssen, H., Seppä, H., Crosta, X., Goosse, H., and Roche, D. M.: Global characterization of the Holocene thermal maximum, Quaternary Sci. Rev., 48, 7–19, https://doi.org/10.1016/j.quascirev.2012.05.022, 2012. Sennès, V.: Les tourbières de l’Europe de l’ouest, Travaux du Laboratoire de Géographie Physique Appliquée, 22, 11–25, https://doi.org/10.3406/tlgpa.2003.1008, 2004. Seppä, H. and Bennett, K. D.: Quaternary pollen analysis: recent progress in palaeoecology and palaeoclimatology, Prog. Phys. Geog., 27, 548–579, 2003. References ter Braak, C. J. F. and Juggins, S.: Weighted averaging partial least squares regression (WA-PLS): an improved method for recon- structing environmental variables from species assemblages \| SpringerLink, Hydrobiologia, 269/270, 485–502, 1993. Xiao, W., Wang, Y., Zhou, S., Hu, L., Yang, H., and Xu, Y.: Ubiquitous production of branched glycerol dialkyl glycerol tetraethers (brGDGTs) in global marine environments: a new source indicator for brGDGTs, Biogeosciences, 13, 5883–5894, https://doi.org/10.5194/bg-13-5883-2016, 2016. Turner, M. G., Wei, D., Prentice, I. C., and Harrison, S. P.: The impact of methodological decisions on climate re- constructions using WA-PLS, Quaternary Res., 99, 341–356, https://doi.org/10.1017/qua.2020.44, 2021. Xiong, Q., Pan, K., Zhang, L., Wang, Y., Li, W., He, X., and Luo, H.: Warming and nitrogen deposition are inter- active in shaping surface soil microbial communities near the alpine timberline zone on the eastern Qinghai–Tibet Plateau, southwestern China, Appl. Soil Ecol., 101, 72–83, https://doi.org/10.1016/j.apsoil.2016.01.011, 2016. van Andel, T. H., Zangger, E., and Demitrack, A.: Land Use and Soil Erosion in Prehistoric and Historical Greece, J. Field Ar- chaeol., 17, 379–396, 1990. Walker, B., Holling, C. S., Carpenter, S. R., and Kinzig, A. P.: Re- silience, Adaptability and Transformability in Social-ecological Systems, Ecol. Soc., 9, art5, https://doi.org/10.5751/ES-00650- 090205, 2004. Yang, H., Pancost, R. D., Dang, X., Zhou, X., Evershed, R. P., Xiao, G., Tang, C., Gao, L., Guo, Z., and Xie, S.: Correlations between microbial tetraether lipids and environmental variables in Chinese soils: Optimizing the palaeo-reconstructions in semi- arid and arid regions, Geochim. Cosmochim. Ac., 126, 49–69, https://doi.org/10.1016/j.gca.2013.10.041, 2014. Wanner, H.: Late-Holocene: Cooler or warmer?, Holocene, 31, 1501–1506, https://doi.org/10.1177/09596836211019106, 2021. Watson, B. I., Williams, J. W., Russell, J. M., Jackson, S. T., Shane, L., and Lowell, T. V.: Temperature variations in the southern Great Lakes during the last deglaciation: Comparison between pollen and GDGT proxies, Quaternary Sci. Rev., 182, 78–92, https://doi.org/10.1016/j.quascirev.2017.12.011, 2018. Yang, H., Xiao, W., Słowakiewicz, M., Ding, W., Ayari, A., Dang, X., and Pei, H.: Depth-dependent variation of archaeal ether lipids along soil and peat proﬁles from southern China: Implications for the use of isoprenoidal GDGTs as environmental tracers, Org. Geochem., 128, 42–56, https://doi.org/10.1016/j.orggeochem.2018.12.009, 2019. Weber, Y., Sinninghe Damsté, J. S., Zopﬁ, J., De Jonge, C., Gilli, A., Schubert, C. J., Lepori, F., Lehmann, M. F., and Niemann, H.: Redox-dependent niche differentiation provides evidence for multiple bacterial sources of glycerol tetraether lipids in lakes, P. Natl. Acad. Sci. USA, 115, 10926–10931, https://doi.org/10.1073/pnas.1805186115, 2018. Yurtsev, B. References Robles, M., Peyron, O., Brugiapaglia, E., Ménot, G., Dugerdil, L., Ollivier, V., Ansanay-Alex, S., Develle, A.-L., Toza- lakyan, P., Meliksetian, K., Sahakyan, K., Sahakyan, L., Perello, B., Badalyan, R., Colombié, C., and Joannin, S.: Impact of climate changes on vegetation and hu- man societies during the Holocene in the South Caucasus (Vanevan, Armenia): A multiproxy approach including pollen, NPPs and brGDGTs, Quaternary Sci. Rev., 277, 107297, https://doi.org/10.1016/j.quascirev.2021.107297, 2022. Silva-Sánchez, N., Martínez Cortizas, A., and López-Merino, L.: Linking forest cover, soil erosion and mire hydrology to late- Holocene human activity and climate in NW Spain, Holocene, 24, 714–725, https://doi.org/10.1177/0959683614526934, 2014. Sinninghe Damsté, J. S.: Spatial heterogeneity of sources of branched tetraethers in shelf systems: The geochem- istry of tetraethers in the Berau River delta (Kaliman- tan, Indonesia), Geochim. Cosmochim. Ac., 186, 13–31, https://doi.org/10.1016/j.gca.2016.04.033, 2016. Robles, M., Peyron, O., Ménot, G., Brugiapaglia, E., Wulf, S., Appelt, O., Blache, M., Vannière, B., Dugerdil, L., Paura, B., Ansanay-Alex, S., Cromartie, A., Charlet, L., Guédron, S., de Beaulieu, J.-L., and Joannin, S.: Climate changes during the Late Glacial in southern Europe: new insights based on pollen and brGDGTs of Lake Matese in Italy, Clim. Past, 19, 493–515, https://doi.org/10.5194/cp-19-493-2023, 2023. Smith, A. C., Wynn, P. M., Barker, P. A., Leng, M. J., Noble, S. R., and Tych, W.: North Atlantic forcing of moisture delivery to Europe throughout the Holocene, Sci. Rep.-UK, 6, 24745, https://doi.org/10.1038/srep24745, 2016. Sugita, S., Parshall, T., and Calcote, R.: Detecting differences in vegetation among paired sites using pollen records, Holocene, 16, 1123–1135, 2006. Rodrigo-Gámiz, M., García-Alix, A., Jiménez-Moreno, G., Ramos- Román, M. J., Camuera, J., Toney, J. L., Sachse, D., Anderson, R. S., and Sinninghe Damsté, J. S.: Palaeoclimate reconstruc- tion of the last 36 kyr based on branched glycerol dialkyl glyc- erol tetraethers in the Padul palaeolake record (Sierra Nevada, Sun, Q., Chu, G., Liu, M., Xie, M., Li, S., Ling, Y., Wang, X., Shi, L., Jia, G., and Lü, H.: Distributions and temperature dependence of branched glycerol dialkyl glycerol tetraethers in recent lacus- https://doi.org/10.5194/cp-19-2127-2023 Clim. Past, 19, 2127–2156, 2023 L. d’Oliveira et al.: Reconstructing 15 000 years of southern France temperatures 2156 trine sediments from China and Nepal, J. Geophys. Res.-Biogeo., 116, G01008, https://doi.org/10.1029/2010JG001365, 2011. trine sediments from China and Nepal, J. Geophys. Res.-Biogeo., 116, G01008, https://doi.org/10.1029/2010JG001365, 2011. Wickham, H.: Data Analysis, in: ggplot2: Elegant Graphics for Data Analysis, edited by: Wickham, H., Springer International Publishing, Cham, 189–201, https://doi.org/10.1007/978-3-319- 24277-4_9, 2016. References A.: 9 – Relics of the xerophyte vegetation in Beringia in northeastern Asia, in: Paleoecology of Beringia, edited by: Hopkins, D. M., Matthews, J. V., Schweger, C. E., and Young, S. B., Academic Press, 157–177, https://doi.org/10.1016/B978- 0-12-355860-2.50018-1, 1982. Weijers, J. W. H., Schouten, S., Linden, M., Geel, B., and Sinninghe Damsté, J. S.: Water table related variations in the abundance of intact archaeal membrane lipids in a Swedish peat bog, FEMS Microbiol. Lett., 239, 51–56, https://doi.org/10.1016/j.femsle.2004.08.012, 2004. Zeng, Z., Chen, H., Yang, H., Chen, Y., Yang, W., Feng, X., Pei, H., and Welander, P. V.: Identiﬁcation of a protein responsible for the synthesis of archaeal membrane-spanning GDGT lipids, Nat. Commun., 13, 1545, https://doi.org/10.1038/s41467-022-29264- x, 2022. Weijers, J. W. H., Schouten, S., Spaargaren, O. C., and Sinninghe Damsté, J. S.: Occurrence and distribution of tetraether mem- brane lipids in soils: Implications for the use of the TEX86 proxy and the BIT index, Org. Geochem., 37, 1680–1693, https://doi.org/10.1016/j.orggeochem.2006.07.018, 2006. Zhao, B., Russell, J. M., Tsai, V. C., Blaus, A., Parish, M. C., Liang, J., Wilk, A., Du, X., and Bush, M. B.: Evaluating global temperature calibrations for lacustrine branched GDGTs: Seasonal variability, paleoclimate implica- tions, and future directions, Quaternary Sci. Rev., 310, 108124, https://doi.org/10.1016/j.quascirev.2023.108124, 2023. Weijers, J. W. H., Schouten, S., van den Donker, J. C., Hop- mans, E. C., and Sinninghe Damsté, J. S.: Environmen- tal controls on bacterial tetraether membrane lipid distri- bution in soils, Geochim. Cosmochim. Ac., 71, 703–713, https://doi.org/10.1016/j.gca.2006.10.003, 2007. Zheng, Y., Li, Q., Wang, Z., Naafs, B. D. A., Yu, X., and Pancost, R. D.: Peatland GDGT re- cords of Holocene climatic and biogeo- chemical responses to the Asian Monsoon, Org. Geochem., 87, 86–95, https://doi.org/10.1016/j.orggeochem.2015.07.012, 2015. Weijers, J. W. H., Panoto, E., van Bleijswijk, J., Schouten, S., Ri- jpstra, W. I. C., Balk, M., Stams, A. J. M., and Damsté, J. S. S.: Constraints on the Biological. Source(s) of the Orphan Branched Tetraether Membrane Lipids, Geomicrobiol. J., 26, 402–414, https://doi.org/10.1080/01490450902937293, 2009. Zheng, Y., Pancost, R. D., Naafs, B. D. A., Li, Q., Liu, Z., and Yang, H.: Transition from a warm and dry to a cold and wet climate in NE China across the Holocene, Earth Planet. Sc. Lett., 493, 36– 46, https://doi.org/10.1016/j.epsl.2018.04.019, 2018. Clim. Past, 19, 2127–2156, 2023 https://doi.org/10.5194/cp-19-2127-2023
W2015363851.txt	https://zenodo.org/records/2367879/files/article.pdf	de		Die Hemmung verschiedener Giftwirkungen auf das befruchtete Seeigelei durch Hemmung der Oxydationen in demselben	Development, genes and evolution	1,911	public-domain	1,211	808 Referate. toxisch wirkt. Der zweiten Serie wurde zu dieser LSsung (auf 50 ccm) 0,7 ccm 3/s m CaC12, tier dritten 1,1 ccm m/2 KC1, tier vierten 1,1 ccm KC1 -Jr- 0,7 ccm 3/3 m CaC12 hinzugesetzt. Dis Versuche wurden in diesen vier LSsungen bei der n e u t r a l e n , a l k a l i s c h en and s an r en Reaktion durchgefiihrt. Die Expositionszeit ist stets in den r Protokollen angegeben. Aus diesen Versuchen geht horror: 1) In dieser n e u t r a l e n L~sung ist die entgiftende Wirkung des K -+- Ca hauptsiichlich auf das Kalium zuriickzufiihren und das Calcium spielt dabei Bur eine untergeordnete Rolle. 2) Die entgiftende Wirkung von Ca in einer a l k a l i s c h e n Chlornatrium15sung ist grSl3er als die yon K, and die entgiftendc Wirkung von K -}- Ca ist grSl3er als die Summe der Einzelwirkung yon K und Ca. 3) In den s a u r e n LSsungen verh~lt sich die eBtgiftende Wirkung ungef~hr so wie in alkalischen. Endlich hat LOEB in dieser Arbeit festgestellt, dab der Zusatz yon etwas Alkali zu einer neutraleu Mischung you l~aC1 -p KC1 die LSsung giftiger, w~ihrend derselbe Zusatz zu einer neutralen Misehung yon l~aC1 -{- CaC12 die L~isung fiir die Entwicklung giinstiger macht. E. Godlewski inn. (Krakau). J. LOEB, Die H e m m u n g v e r s c h i e d e n e r G i f f w i r k u n g e n a u f das b e f r u c h tote S e e i g e l e i durch H e m m u n g der O x y d a t i o n c n in demselben. Biochem. Zeitschr. Bd. 29. 1910. Der Verf. schlie~t seine Untersuchungen an die Resultate an, die er und teilweise WA~BVRG friiher bekommen haben, dal3 ni~mlich die giftige Wirkung gewisser Substanzen d u r c h U n t e r d r i i c k u n g d e r 0 x y d a t i o n s v o r g i i n g e gehemmt werden kann. In den friiheren Arbeiten hat der Verf. in dieser Beziehung die giftige Wirkung der hypertonischen, hyperalkalischen LSsungen und der neutralen LSsung yon 5TaC1, LiC1, KC1 u. a. untersucht. In d~r v6rliegenden Arbeit hat er seine Studien noch bedeutend erweitert. Die Versuche wurden an Arbacic~-Eiern durchgefiihrt. Die Unterdriickung tier 0xydationsprozesse wurde entweder dutch dic Leitung eines Stromes yon Wasserstoff durch die Kulturflaschen odor durch Zusatz yon 6 Tropfen einer 1/I0 NaCbI- odor KCN-Li~sung zustande gebracht. Die Temperatur variierte moist zwischen 20--24 ~ C. Zuerst hat LOEB durch Unterdriicknng der 0xydationen die Giftwirkung der Salze bTaC1,KC1, MgC12, and der Misehung der Salze, wie NaC1 ~ •aC12 u. a., oft Bach mehrerc Stunden dauernder Exposition gepriift. Alle diese Salze, die bekanntlich im Seewasser enthalten sind, wenn sie einzeln wirken and die Eier etwas l~tnger dieser Wirkung ausgesetzt sind, beeintr}ichtigen die Entwicklungsfiihigkeit bzw. heben dieselbe vollkommen auf. Durch die Unterdriickung der Oxydation wurde diese giftige Wirkung bedeutend gehemmt. Die Untersuchungen des Verf. wurden sodann auf diejenigen Salze erweitert, welche im Seewasser nicht enthalten sind, wie CaCl.~ und tIgC12. Das Resultat war hier auch, wenigstens teilweise, positiv. Die N i c h t l e i t e r , wie Traubenzuckerliisung, wirken ebenfalls in bestimmten hSheren Konzentrationen giftig. Abet auch diese giftige Wirkung kann durch die Hemmung der 0xydationsvorg~nge neutralisiert werden. Die weiteren Experimente yon LOEB zeigen, dal3 auch die giftige Wirkung Referate. 809 der h y p e r t o n i s eh en L~isungen (u des Seewassers mit destilliertem Wasser oder Alkohol) dutch Sauerstoffmangel vermindert werden kann. Ist n~mlich die Konzentration des Seewassers auf die H~ilfte reduziert and die Oxydation dabei unterdriickt, so leben die befruchteten Eier etwa siebenmal so lange, als wenn die Oxydationen nicht gehemmt sind. Sehr interessant sind auch die Versuche des Verf. mit den n a r k o t i s c h e n M i t t e l n , insbesondere mit Chloralhydrat und Phenylurethan. Die cytolisierende Wirkung dieser Substanzen kann durch Vertreibung des Sauerstoffes bzw. dutch Unterdrtickung der 0xydation aufgehoben werden. Daraus sehliel~t der Verf:, dal3 die s c h i ~ d i g e n d e W i r k u n g der N a r k o t i k a n i c h t a u f e i n e r V e r m i n d e r u n g der 0 x y d a t i o n e n b e r u h e n k a n n , da j a die S a u e r s t o f f entziehung gerade diese schEdigenden Wirkungen aufhebt. Die Hemmang der Giftwirkungen verschiedener Substanzen durch die Unterdriickung der Oxydation hat LOEB auch bei der Regeneration bei Hydroidpolypen festgestellt. Diese neuen, ftir die allgemeine Biologie bedeutsamen Entdeckungen yon J. LO~B verdienen besondere Beachtung. E. Godlewski jun. (Krakau). WILH., B a u u n d E n t s t e h u n g der W i r b e l t i e r g e l e n k e , eine morphologische u n d histogenetische U n t e r s u c h u n g . Mit 230 Abb. im Text u. 10 lithogr. Tafeln. J e n a , Gust. Fischcr~ 1910. 349 S. Lcuosc~, Preis M. 2 7 . - - . In dem grol~angelegten, tiefgriindigen Werke LUBOSO~s iiber die Morphologie und Histogenese der Gelenke tiberwiegt der Ausdehnung nach entspreehend dem Inhaltsplane des Buches natiirlich die deskriptive vergleichend-anatomische histologische Behandlung der Gelenke, doch ist in allen Teilen des Werkes zu erkennen, dab der Verf. im Grunde die c a u s a l e Erforsehung der Gelenkform sich zum Ziele gesetzt hat. ~Sit vollem Recht glaubt er aber, das eausale Versti~ndnis nur auf eine griindliche~deskriptive Kenntnis der Gelenkeinrichtungen stiitzen zu dtirfen, und diesem Bestreben verdanken wir die auBerordentlich wertvolle Darstellung des Verf. tiber die Gelenkeinrichtungen der Fische, Amphibien und Sauropsiden, die neben griindlicher Beriicksichtigung der Literatur auch eine Fiille neuer eigner Untersuchungen des Verf. bringt. Der II. Abschnitt des Werkes behandelt ,die histogenetischen Vorg~nge bei der Entstehung der Wirbeldergelenke,,, wobei der Verf. versucht, die Umwandlung der hyalinen und basophilen Intercetlularsubstanz des Knorpels in aeidophile und weiterhin fibrilli~re Grundsubstanz, die zur Zerkliiftung und Spattbildung fiihrt, direkt dutch die Wirkung yon Biegung und Drillung zu erkliiren. Die verscbiedenen Formen der Gelenkbildung bei den verscbiedenen Tieren glaubt Verfi abet nicht auf Unterschiede im M e c h a n i m u s der einwirkenden Biegungs- und DrillungskrEfte, sondern auf Unterschiede im C h e m i s m u s der betr. Knorpet zurtickftihren zu sollen. Groi3e Bedeutung legt Verf. der yon ihm nachgewiesenen Tatsache bei, dab bei der phylo- und ontogenetischen Gelenkbildung stets der Gelenkkopf zuerst (und aus festerem Gewebe) gebildet wird und weist darauf hin, dal~ das auch bei den kiinstlichen Gelenkschliffen R. FICKS der Fall gewesen ist. TrotzArchiv f. En~wickhngsmechaniL XXXI. 59
https://openalex.org/W2773229067	https://www.scielo.br/j/pab/a/VdwMp34hzNwSb66ZRQmqCnw/?lang=en&format=pdf	English	null	Solidifying agents and activated charcoal for in vitro culture of Solanum sessiliflorum	Pesquisa Agropecuária Brasileira	2,017	cc-by	2,268	Pesq. agropec. bras., Brasília, v.52, n.11, p.1123-1126, nov. 2017 DOI: 10.1590/S0100-204X2017001100019 Notas Científicas Solidifying agents and activated charcoal for in vitro culture of Solanum sessiliflorum Filipe Almendagna Rodrigues(1), Renata Alves Lara Silva Rezende(1), Moacir Pasqual(1) and Maria Teresa Gomes Lopes(2) (1)Universidade Federal de Lavras, Departamento de Agricultura, Laboratório de Cultura de Tecidos Vegetais, Campus Universitário, Caixa Postal 3037, CEP 37200-000 Lavras, MG, Brazil. E-mail: filipealmendagna@yahoo.com.br, renata_vga@yahoo.com.br, mpasqual@dag.ufla.br (2)Universidade Federal do Amazonas, Faculdade de Ciências Agrárias, Departamento de Produção Animal e Vegetal, Avenida General Rodrigo Otávio Jordão Ramos, no 1.200, Coroado I, CEP 69067-005 Manaus, AM, Brazil. E-mail: mtglopes@ufam.edu.br Abstract ‒ The objective of this work was to evaluate the effects of the solidifying agents agar and phytagel and of activated charcoal on the in vitro cultivation of two maná cubiu (Solanum sessiliflorum) varieties: Thaís and Santa Luzia. The phytotechnical characteristics analyzed included number of leaves, number of roots, shoot and root length, and fresh matter of shoot and root. Regardless of the variety, phytagel was superior to agar as a culture medium. A greater number of leaves and longer shoots were observed in the Santa Luzia variety, in the absence of charcoal. The Thaís variety showed longer shoots and roots in the presence of charcoal. Index terms: maná cubiu, micropropagation, plant tissue culture, phytagel, Solanaceae. Agentes solidificantes e carvão ativado no cultivo in vitro de Solanum sessiliflorum Resumo ‒ O objetivo deste trabalho foi avaliar os efeitos dos agentes solidificantes ágar e phytagel e do carvão ativado no cultivo in vitro de duas variedades de maná cubiu (Solanum sessiliflorum): Thaís e Santa Luzia. As características fitotécnicas analisadas incluíram número de folhas, número de raízes, comprimento de parte aérea e raiz, e massa de matéria fresca de parte aérea e raiz. Independentemente da variedade, o phytagel foi superior ao ágar como meio de cultura. Observou-se maior número de folhas e comprimento da parte aérea na variedade Santa Luzia, na ausência de carvão. A variedade Thaís apresentou maior comprimento da parte aérea e da raiz na presença de carvão. Termos para indexação: maná cubiu, micropropagação, cultura de tecidos vegetais, phytagel, Solanaceae. Solidifying agents are used in micropropagation because they provide ideal support conditions for plantlets, although the overall expenditure of in vitro production increases as these are expensive products. Agar and phytagel are the most commonly used solidifying agents in tissue culture. Agar is extracted from red algae, mainly from the species Gelidium amansii Lamouroux, and consists of a complex mixture of polysaccharides, especially agarose and agaropectins. Phytagel is a polysaccharide produced by the bacterium Sphingomonas elodea (ex. Pseudomonas elodea) and is composed of molecules of keto- glucuronate, rhamnose, and cellobiose (George, 1993). According to Babbar et al. (2005), the substitution of Maná cubiu (Solanum sessiliflorum Dunal) is a fruit species belonging to the Solanaceae family and originating in Western Amazon. The fruit has nutritional and medicinal characteristics due to active principle niacin (vitamin B3), which plays a role in cell defense, besides being rich in fiber, phosphorus, iron, potassium, vitamin C, and pectin (Silva Filho et al., 2005). The technique of micropropagation is one of the most practical applications of tissue culture and has a great impact, since large numbers of seedlings can be propagated using this method, and the crop can be obtained in a very short period of time (Dutra et al., 2009). Pesq. agropec. bras., Brasília, v.52, n.11, p.1123-1126, nov. 2017 DOI: 10.1590/S0100-204X2017001100019 1124 F.A. Rodrigues et al. To evaluate the effect of activated charcoal, the MS culture medium was solidified using 1.8 g L-1 phytagel (using the same concentration as in the experiment on solidifying agents) and the pH was adjusted to 5.8 before autoclaving (121°C and 1.0 atm for 20 min). Agentes solidificantes e carvão ativado no cultivo in vitro de Solanum sessiliflorum The concentrations of activated charcoal used were 0 g L-1 (control) and 2.0 g L-1. The segments were inoculated in flasks containing 50 mL of the culture medium. After inoculation, the flasks were kept in a growth room with artificial light provided by special daylight fluorescent tubes, maintained at a mean irradiance of 42 W m-2, photoperiod of 16 hours, and temperature of 25±2°C. agar with other polysaccharides, such as phytagel, improves the production of shoots and reduces the cost of micropropagation by up to 90%. Therefore, the use of alternate solidifying agents becomes important in order to increase the production of plants in vitro.i Activated charcoal is coal powder that has been finely milled in order to increase the adsorption area of the particles. Its addition to the culture medium promotes darkening of the medium. Moreover, it participates in the adsorption of substances released by the medium, such as the impurities in agar, growth regulators, organic compounds, or phenols released by the damaged tissues in vitro (George & Sherrington, 1984). Thus, activated charcoal is beneficial to the development of plants in vitro. The experiment was performed in a completely randomized design, using a 2×2 factorial arrangement, activated charcoal (absence: 0 g L-1 and presence: 2.0 g L-1), and maná cubiu varieties (Thaís and Santa Luzia), in six replicates. Each replicate consisted of a single flask containing two explants, which totaled to 12 explants per treatment. After 30 days from the beginning of the experiment, the following phytotechnical characteristics were evaluated: leaf number, number of roots, shoot length, root length, and fresh matter shoot and root. The objective of this work was to evaluate the effects of the solidifying agents agar and phytagel and of activated charcoal on the in vitro cultivation of two maná cubiu (S. sessiliflorum) varieties: Thaís and Santa Luzia. The experiments were conducted at the plant tissue culture laboratory of the Department of Agriculture of Universidade Federal of Lavras (Ufla), in the municipality of Lavras, in the state of Minas Gerais, Brazil. Stem segments (bearing a 1-cm long bud) extracted from maná cubiu plants already established in vitro were used as explants. The data obtained were subjected to the statistical software Sisvar (Ferreira, 2011), and the means were compared using Scott-Knott’s test, at 5% probability. Pesq. agropec. bras., Brasília, v.52, n.11, p.1123-1126, nov. 2017 DOI: 10.1590/S0100-204X2017001100019 Agentes solidificantes e carvão ativado no cultivo in vitro de Solanum sessiliflorum For the evaluation of the effects of the solidifying agents, the basic culture medium used was MS (Murashige & Skoog, 1962) and the pH was adjusted to 5.8 before autoclaving (121°C and 1.0 atm for 20 min). For solidification of the culture medium, agar (HiMedia Laboratories, Mumbai, India) or phytagel (Sigma-Aldrich, Inc., St. Louis, MO, USA) was used. The concentrations used were: 5.5 g L-1 agar and 1.8 g L-1 phytagel; agar was used as a control treatment. The experiment was conducted in four replicates of three tubes each. The stem segments were inoculated in test tubes containing 15 mL of the culture medium and then maintained in a growth room with mean irradiance of 42 W m-2, photoperiod of 16 hours, and temperature of 25±2°C. Phytagel was superior to agar for all studied variables, independent by of the variety (Table 1). According to Chevreau et al. (1997), phytagel is highly purified and contains no foreign substances, whereas agar may contain substances that inhibit the development of plants in vitro. Chapla et al. (2009) also evaluated the effect of agar and phytagel on the in vitro growth of Miltonia flavescens Lindl. As in the present experiment, the authors obtained superior results when the plants were grown in a medium containing phytagel as the solidifying agent. Therefore, the use of purer solidifying agents promotes better results in in vitro culture. In general, roots of the Thaís variety were longer than those of the Santa Luzia variety (Table 1). However, when the isolated effect of the solidifying agents was evaluated, it was possible to observe larger increments in root length in media containing phytagel. Chapla et al. (2009) also found longer root length in M. flavescens cultured in media containing phytagel, compared with agar. The experimental design was completely randomized, in a 2×2 factorial arrangement, with two varieties of maná cubiu (Thaís and Santa Luzia) and two solidifying agents (agar and phytagel). After 60 days from the beginning of the experiment, the number of leaves, number of roots, shoot length, root length, mass of fresh shoot matter, and mass of fresh root matter of the plants were evaluated. In the absence of activated charcoal, shoots of the Santa Luzia variety were longer (Table 2). This result Solidifying agents and activated charcoal for in vitro culture of Solanum sessiliflorum 1125 Table 1. References corroborates with that of Unemoto et al. (2006), who evaluated the effect of the addition of activated charcoal on the culture medium and found that the absence of this product favored an increase in the shoot length of rainha-do-abismo [Sinningia leucotricha (Hoehne) Moore] plantlets. However, contrary results were found by Chapla et al. (2009), who observed longer shoots in media containing activated charcoal. BABBAR, S.B.; JAIN, R.; WALIA, N. Guar gum as a gelling agent for plant tissue culture media. In Vitro Cellular and Developmental Biology – Plant, v.41, p.258-261, 2005. DOI: 10.1079/IVP2005628. CHAPLA, P.I.; BESSON, J.C.F.; OLIVEIRA, L.K.; SILVA, J.M. da; ROCHA, A.C. de S.; STEFANELLO, S. pH, carvão ativado e agentes geleificantes do meio de cultura no crescimento in vitro de Miltonia flavescens Lindl. Plant Cell Culture and Micropropagation, v.5, p.87-93, 2009. There was no significant difference in root length between the Thaís and Santa Luzia varieties in the absence of activated charcoal. However, the presence of activated charcoal promoted an increase in root length in the Thaís variety when compared with Santa Luzia (Table 2). Villa et al. (2007) also reported longer roots in 'Ébano' blackberry when activated charcoal was present in the culture medium. CHEVREAU, E.; MOURGUES, F.; NEVEU, M.; CHEVALIER, M. Effect of gelling agents and antibiotics on adventitious bud regeneration from in vitro leaves of pear. In vitro Cell and Developmental Biology – Plants, v.33, p.173-179, 1997. DUTRA, L.F.; WENDLING, I.; BRONDANI, G.E. A micropropagação de eucalipto. Pesquisa Florestal Brasileira, v.58, p.49-59, 2009. DOI: 10.4336/2009.pfb.58.49. FERREIRA, D.F. Sisvar: a computer statistical analysis system. Ciência e Agrotecnologia, v.35, p.1039-1042, 2011. DOI: 10.1590/ S1413-70542011000600001. GEORGE, E.F. (Ed.). Plant propagation by tissue culture: part 1: the technology. 2nd ed. Edington: Exegetics, 1993. 574p. Regardless of the variety studied, greater root length was observed in the absence of activated charcoal in the culture medium (Table 2). Chapla et al. (2009) also verified an increased root length in M. flavescens under the same conditions. Therefore, Maná cubiu showed better growth in vitro when the MS medium was solidified using phytagel, in the absence of activated charcoal. GEORGE, E.F.; SHERRINGTON, P.D. Plant propagation by tissue culture. Basingstone: Exegetics, 1984. 709p. MURASHIGE, T.; SKOOG, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, v.15, p.473-479, 1962. DOI: 10.1111/j.1399- 3054.1962.tb08052.x. SILVA FILHO, D.F. da; YUYAMA, L.K.O.; AGUIAR, J.P.L.; OLIVEIRA, M.C.; MARTINS L.H.P. Caracterização e avaliação Pesq. Agentes solidificantes e carvão ativado no cultivo in vitro de Solanum sessiliflorum Phytotechnical characteristics of the maná cubiu (Solanum sessiliflorum) varieties Santa Luzia and Thais in medium containing the solidifying agents agar and phytagel(1). Variety Number of leaves Shoot length (cm) Fresh matter of shoots (g) Fresh matter of roots (g) Root length (cm) Medium Variety ME Agar Phytagel Agar Phytagel Agar Phytagel Agar Phytagel main effect (ME) Agar Phytagel Santa Luzia 6.2bB 10.7aA 1.7aB 11.9bA 0.133aB 0.775aA 0.022aB 0.131aA 10.9b 11.45B 15.42A Thaís 7.7aA 8.4bA 3.0aB 6.0aA 0.227aB 0.473bA 0.021aB 0.092bA 16.0a CV (%) 15.96 31.19 29.49 49.84 32.62 (1)Means followed by equal letters, lowercase in the columns and uppercase in the rows, do not differ significantly by Scott-Knott’s test, at 5% probability. characteristics of the maná cubiu (Solanum sessiliflorum) varieties Santa Luzia and Thais in medium ng agents agar and phytagel(1). (1)Means followed by equal letters, lowercase in the columns and uppercase in the rows, do not differ significantly by Scott-Knott’s test, at 5% probability. Table 2. Phytotechnical characteristics of two varieties of maná cubiu (Solanum sessiliflorum) in the presence and absence of activated charcoal(1). Activated charcoal Number of leaves Shoot length (cm) Number of roots Root length (cm) Fresh matter of shoots (g) Fresh matter of roots (g) Santa Luzia Thaís Santa Luzia Thaís Santa Luzia Thaís Santa Luzia Thaís Santa Luzia Thaís Santa Luzia Thaís With 4.47bA 4.20aA 1.37bA 1.58aA 2.60bA 1.63bB 2.45bB 7.57aA 0.107bA 0.149aA 0.008bA 0.028bA Without 5.10aA 4.03aB 3.13aA 1.80aB 4.03aA 2.37aB 9.91aA 7.90aA 0.341aA 0.184aB 0.161aA 0.102aB CV (%) 16.15 45.66 42.5 60.22 81.02 90.93 (1)Means followed by equal letters, lowercase in the columns and uppercase in the rows, do not differ significantly by Scott-Knott’s test, at 5% probability. Table 2. Phytotechnical characteristics of two varieties of maná cubiu (Solanum sessiliflorum) in of activated charcoal(1). Pesq. agropec. bras., Brasília, v.52, n.11, p.1123-1126, nov. 2017 DOI: 10.1590/S0100-204X2017001100019 References agropec. bras., Brasília, v.52, n.11, p.1123-1126, nov. 2017 DOI: 10.1590/S0100-204X2017001100019 1126 F.A. Rodrigues et al. do potencial agronômico e nutricional de etnovariedades de cubiu (Solanum sessiliflorum Dunal) da Amazônia. Acta Amazônica, v.35, p.399-406, 2005. DOI: 10.1590/S0044-59672005000400003. Moore-(Gesneriaceae). Acta Scientiarum. Agronomy, v.28, p.503-506, 2006. VILLA, F.; PASQUAL, M.; PIO, L.A.S.; ASSIS, F.A.; TEODORO, G.S. Influência do carvão ativado e BAP na multiplicação in vitro de duas frutíferas de clima temperado. Revista Ceres, v.54, p.118- 124, 2007. UNEMOTO, L.K.; FARIA, R.T. de; MENEGUCE, B.; ASSIS, A.M. de. Estabelecimento de um protocolo para a propagação in vitro de rainha-do-abismo, Sinningia leucotricha (Hoehne) UNEMOTO, L.K.; FARIA, R.T. de; MENEGUCE, B.; ASSIS, A.M. de. Estabelecimento de um protocolo para a propagação in vitro de rainha-do-abismo, Sinningia leucotricha (Hoehne) Received on January 15, 2017 and accepted on April 10, 2017
https://openalex.org/W4362625764	https://www.frontiersin.org/articles/10.3389/fmed.2023.1121558/pdf	English	null	The prevalence of symptoms and its correlation with sex in polish COVID-19 adult patients: Cross-sectional online open survey	Frontiers in medicine	2,023	cc-by	12,031	OPEN ACCESS OPEN ACCESS EDITED BY Amirali Karimi, Tehran University of Medical Sciences, Iran REVIEWED BY Vinh-Long Tran-Chi, Chulalongkorn University, Thailand Mohammad Amin Salehi, Tehran University of Medical Sciences, Iran CORRESPONDENCE Pawel Lewek pawel.lewek@umed.lodz.pl SPECIALTY SECTION This article was submitted to Family Medicine and Primary Care, a section of the journal Frontiers in Medicine RECEIVED 11 December 2022 ACCEPTED 23 February 2023 PUBLISHED 05 April 2023 CITATION Lewek P, Banaś I, Witkowski K, Lewek J and Kardas P (2023) The prevalence of symptom and its correlation with sex in polish COVID- adult patients: Cross-sectional online open survey. Front. Med. 10:1121558. doi: 10.3389/fmed.2023.1121558 OPEN ACCESS EDITED BY Amirali Karimi, Tehran University of Medical Sciences, Iran REVIEWED BY Vinh-Long Tran-Chi, Chulalongkorn University, Thailand Mohammad Amin Salehi, Tehran University of Medical Sciences, Iran Pawel Lewek 1, Izabela Banaś 1, Konrad Witkowski 1, Joanna Lewek 2,3 and Przemyslaw Kardas 1 1 Department of Family Medicine, Medical University of Lodz, Łódź, Poland, 2 Department of Preventive Cardiology and Lipidology, Chair of Nephrology and Hypertension, Medical University of Lodz, Łódź, Poland, 3 Department of Cardiology and Adult Congenital Heart Diseases, Polish Mother's Memorial Hospital Research Institute (PMMHRI), Łódź, Poland Background: The understanding and treatment of COVID-19 has improved rapidly since December 2019 when SARS-CoV-2 was sequenced. However most papers on its symptomatology focus on hospitalized patients and address only a limited number of major presentations. Although differences depending on sex of COVID-19 patients have been previously confirmed (higher ICU admission and higher death rate for men), no publication has focused on sex-related differences in COVID-19 symptomatology. Lewek P, Banaś I, Witkowski K, Lewek J and Kardas P (2023) The prevalence of symptoms and its correlation with sex in polish COVID-19 adult patients: Cross-sectional online open survey. Objective: The aim of the study was to present a reliable list of COVID-19 symptoms and identify any differences in symptom prevalence depending on sex. Methods: A sample of Polish patients suffering from COVID-19 were surveyed using a cross-sectional anonymous online survey in Polish available on a web- based surveying platform (Survey Monkey). The survey included 20 questions asking about COVID-19 symptoms, days of occurrence (from day 1 until day 14 and “15 days or more”) and patient characteristics including sex, age, height, weight, place of residence and type of therapy received during COVID-19. The survey was made available during the third COVID-19 wave in Poland. TYPE Original Research PUBLISHED 05 April 2023 DOI 10.3389/fmed.2023.1121558 TYPE Original Research PUBLISHED 05 April 2023 DOI 10.3389/fmed.2023.1121558 Abbreviations: Avg, average; COVID-19, coronavirus disease 2019; ECDC, European Centre for Disease Prevention and Control; LQ, lower quartile; Min, minimum; Max, maximum; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SD, standard deviation; UQ, upper quartile. 1. Introduction From this initial discovery, new cases soon began to be reported all over the world. The virus showed significant infectivity, reaching over 100 mln cases globally in one year (4). The first case in Poland was confirmed on March 4, 2021, and this was to rise to over 5.5 mln cases and over 100,000 deaths by the end of February 2022 (5). In addition to symptoms and clinical presentation, the influence of sex on COVID-19 manifestation is of great interest. It was found soon after the outbreak of epidemic that the course of the disease was depended on the sex of the patient. For example, 73% of the 116 deceased COVID-19 patients from Wuhan Tongji Hospital in China in January–February 2020 were men (20). Data from South Korea also showed a 2:1 female to male ratio of COVID-19 cases, but with twice the mortality among men (21). A report from the Italian National Institute of Health from April 2020 stated that approximately 70% of COVID-19 deaths were men, while the National Centre for Health Statistics in the US reported that 59% out of 37,308 deaths were men (22). Another study found the case fatality ratio for men to women to be 1.7 in Italy, Spain and Sweden and 1.4 in Germany (23). Finally, a meta-analysis of over 3.1 million COVID-19 cases confirmed that despite there being no sex-related difference in the chance of being infected with COVID-19, men were three times more likely to require treatment in the intensive care unit and to have a higher chance death (24). Among the reported symptoms of COVID-19, the most common were fever (83–99%), headache (10–28%), cough (20–82%), fatigue (44–70%) and sore throat (14–19%) in adults (6, 7) and fever (46–64%) and cough (32–56%) in children (8). Although many papers have described COVID-19 manifestations, most have focused on selected symptoms (7), and to our knowledge, none have summarized all the possible signs of COVID-19 in a single population. Moreover most of the available studies only assess hospitalized patients (9) or fail to mention their origin (10). For example, one meta-analysis of COVID-19 symptoms (11) included research from 33 articles conducted on 15,244 hospitalized patients and only 9,011 non-hospitalized patients; in addition, these present only limited number of symptoms, ranging from 13 (12) to 23 (13). OPEN ACCESS The link to the survey was distributed across social networks. Participation was open to anyone willing, without any incentives. The data was analyzed statistically. © 2023 Lewek, Banaś, Witkowski, Lewek and Kardas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: Survey responses were collected from 2,408 participants (56.9% women) aged 18–90 (42 ± 12), 84.7% living in cities, who took part in the study between December 2020 and February 2021. Out of 54 predefined symptoms, the three most prevalent were fatigue (reported by 87.61% respondents), anosmia (73.74%) and headache (69.89%). Women were found to be more symptomatic than men, 31 symptoms occurred more often in women (including anosmia, headache and myalgias, p < 0.05). Subfebrility, fever and hemoptysis were more prevalent in men. Twelve symptoms (incl. hypothermia, sneezing and nausea) lasted longer in women than men (p < 0.05). Fatigue, cough, nasal dryness, xerostomia and polydipsia were the longest lasting symptoms of COVID-19 (lasted over 14 days). Conclusion: Our study presents a wide range of symptoms, which may enable better recognition of COVID-19, especially in an outpatient setting. Understanding these differences in the symptomatology of community and hospitalized patients may help diagnose and treat patients faster and more accurately. Our findings also confirmed differences in symptomatology of COVID-19 between men and Frontiers in Medicine 01 frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 10.3389/fmed.2023.1121558 women, which may lay the foundation for a better understanding of the different courses of this disease in the sexes. Further studies are necessary to understand whether a different presentation correlates with a different outcome. COVID-19, symptoms, survey, questionnaire, sex correlation 2. Aim Moreover publications based on hospital rather than community settings may upwardly bias the estimates of symptom prevalence, and not reflect the true numbers in the general population (14). Hospitalized patients also differ significantly from the general population in terms of disease severity, aggravation of symptoms and type of care received. In addition, while most COVID-19 patients are consulted in primary care, which uses different decision making processes from hospitals, most current publications on hospitalized patients focus on major symptoms and lab tests. Therefore it is crucial The primary aim of the study was to build a reliable list of possible COVID-19 symptoms based on a survey of COVID-19 positive patients about the symptoms experienced during the first 14 days of infection. The secondary aim was to identify sex-based differences in symptom prevalence. 1. Introduction Another metanalysis included 24,410 hospitalized patients from four continents; however, no data was included from Central or Eastern Europe (14). While the paper presents a high number of symptoms (n = 30), the authors concede that clear differences exist between countries, and this prevents generalization of findings (14). As such, studies are needed on single populations. While it is important to present individual symptoms, such studies run the risk of ignoring the full picture of the disease, and becoming restricted to the most common cases. Despite this, to date, no studies appear to have examined differences in COVID-19 symptoms according to sex. Moreover, no individual publication has examined the vast collection of known COVID-19 symptoms in a single population, including non-hospitalized patients. Therefore, the aim of this article is to provide an overview of the symptomatology of COVID-19, and to support physicians, especially those of outpatients, in the diagnostic process. 1. Introduction to have a full overview of all possible symptoms of COVID-19 to allow precise diagnosis; this is particularly true for primary care physicians. Other publications on COVID-19 symptoms have examined population type and ethnicity (15), machine learning (16), mobile applications (17) and social media (18, 19). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent of COVID-19, which was discovered for the first time in December 2019 in Wuhan, China (1, 2), although COVID-19 is believed to have spread unnoticed throughout the Asian region much earlier (3). From this initial discovery, new cases soon began to be reported all over the world. The virus showed significant infectivity, reaching over 100 mln cases globally in one year (4). The first case in Poland was confirmed on March 4, 2021, and this was to rise to over 5.5 mln cases and over 100,000 deaths by the end of February 2022 (5). Among the reported symptoms of COVID-19, the most common were fever (83–99%), headache (10–28%), cough (20–82%), fatigue (44–70%) and sore throat (14–19%) in adults (6, 7) and fever (46–64%) and cough (32–56%) in children (8). Although many papers have described COVID-19 manifestations, most have focused on selected symptoms (7), and to our knowledge, none have summarized all the possible signs of COVID-19 in a single population. Moreover most of the available studies only assess hospitalized patients (9) or fail to mention their origin (10). For example, one meta-analysis of COVID-19 symptoms (11) included research from 33 articles conducted on 15,244 hospitalized patients and only 9,011 non-hospitalized patients; in addition, these present only limited number of symptoms, ranging from 13 (12) to 23 (13). Another metanalysis included 24,410 hospitalized patients from four continents; however, no data was included from Central or Eastern Europe (14). While the paper presents a high number of symptoms (n = 30), the authors concede that clear differences exist between countries, and this prevents generalization of findings (14). As such, studies are needed on single populations. While it is important to present individual symptoms, such studies run the risk of ignoring the full picture of the disease, and becoming restricted to the most Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent of COVID-19, which was discovered for the first time in December 2019 in Wuhan, China (1, 2), although COVID-19 is believed to have spread unnoticed throughout the Asian region much earlier (3). frontiersin.org 3.2. Survey design A cross-sectional online open survey was performed on a convenience sample of Polish-speaking individuals with internet access who were willing to take part in the study. The first question asked about previous participation in the survey: if answered “yes,” the participant was not allowed to continue. It also asked whether the respondent was completing the survey on behalf of other person (e.g., elderly, child), which was allowed. Authenticity of the answer was not verified otherwise, it was based on patient’s declaration. The initial screen included a welcome message, introduction about the authors of the survey, explanation of study purpose, information about anonymization and possibility to withdraw at any stage, a request to give sincere and honest answers, as well as ethical board approval and lack of sponsorship of any kind. Participants were also informed about the time required to fill in the survey and given basic information on how to navigate the survey. 3.3. Completion and completeness rate Completion rate (ratio of users who finished the survey/users who agreed to participate) and completeness rate (measure for how completely questionnaires were filled, without questionnaires left blank) were counted after results were obtained. Questionnaires completed partially (i.e., not to the end) were included in the final analysis; however, the numbers of participants differed depending on the question. While it was obligatory to answer all questions, the participant was able to quit at any point. The answers were registered to the last question that had been answered. Responses to the question were recorded when respondent clicked the navigation buttons in the survey (e.g., ‘next’ button), therefore only fully completed questions were included in the final analysis. 3.4. Statistical analysis The Shapiro–Wilk test was conducted to check the normality of the data distribution. Some variables were converted into categorical variables: BMI, city of residence, stated needs for diverse types of therapy during COVID-19 infection. For continuous variables, the obtained results were reported as mean ± standard deviation for Gaussian distribution or median with 25–75% percentiles if not. Discrete variables were presented as proportions. Comparisons between groups were conducted with the Student’s t-test for independent variables, the Mann–Whitney U test or χ2 test with Yates correction and Kruskal-Wallis test, as appropriate. For all calculations, p-values <0.05 were considered statistically significant. The statistical analysis was performed using STATISTICA v.13 software (TIBCO Software Inc., Palo Alto, CA, USA). The second screen included two questions about exclusion criteria. To be included in the final analysis, the participants had to meet the following criteria: no previous participation in the survey, self-reported positive RT-PCR test confirming SARS-CoV-2 infection, answer all questions about the experienced COVID-19 symptoms. The survey included 20 questions (only selected questions were used for the following analysis) and was presented on five pages with two to nine questions on each page. All questions were available for every participant (no adaptive questioning was applied). Answering every question was mandatory and the participant was alerted if a question was missed. On the third screen, the participants were presented with a list of 54 pre-defined symptoms, and were asked to indicate which ones they had experiences, and on which days of the illness (day 1 to 14 or “day 15 and beyond”). A “no symptoms” option was also available. One of the questions included a list of COVID-19 symptoms based on available publications (25–28), authors’ clinical experience, and symptoms additionally proposed by participants during the pilot stage of the study. The symptoms were not defined or explained (except temperature and skin lesions, see below), but were self-explanatory, and written in lay language (e.g., ‘lack of smell’ instead of ‘anosmia’). The body temperature was divided into ‘lowered temperature’ (hypothermia, defined in a survey as temperature 36,1 deg.C or lower), subfebrility (37.0–37.9 Centigrades) and fever (≥38.0 deg.C). 3.5. Pilot The preliminary version of the questionnaire was piloted on a group of seven family physicians and 10 patients in order to check for errors, ensure that the survey was clear and to generally polish it. 3.1. Recruitment A cross-sectional study was performed using a bespoke questionnaire based on the previous experience of the authors and a Frontiers in Medicine 02 frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 10.3389/fmed.2023.1121558 literature review of COVID-19 cases and reported symptoms. The survey was made available in Polish through a web based surveying platform (Survey Monkey) during the 3rd COVID-19 wave in Poland, between December 23, 2020 and February 28, 2021. The invitation to the survey was distributed across social networks including related Facebook groups and the commentary fields of news web sites devoted to the topic of COVID-19 (Supplementary material S1). Participation in the study was voluntary. The link to the survey was not password protected and hence was open to anyone who was willing to complete it. (The survey announcement is presented in Supplementary material S2). The survey was completely anonymous, IP numbers were not collected, cookies were not used to assign any unique user identifier to each client computer. No incentives (monetary or non-monetary) were offered for participation in the survey. Although this division is not common in English literature, it is widely used and understood by Polish patients and as such was proposed in the survey. This followed the understanding of normal axillary temperature range of 36.2–36.9 Centigrade and fever definitions given in the literature (29, 30). Skin lesions were defined in the survey by adding an example ‘e.g. rash, pimples’. Our findings were reported according to the CHERRIES checklist for on-line survey reporting (31). Although this division is not common in English literature, it is widely used and understood by Polish patients and as such was proposed in the survey. This followed the understanding of normal axillary temperature range of 36.2–36.9 Centigrade and fever definitions given in the literature (29, 30). Skin lesions were defined in the survey by adding an example ‘e.g. rash, pimples’. Our findings were reported according to the CHERRIES checklist for on-line survey reporting (31). Until the final submission, participants were able to go back and change any answer to any question. The contact details to the principal investigator were provided at the final screen of the survey. Frontiers in Medicine 3.5.1. Ethics approvalh The study design was reviewed by the ethical commission as research involving human subjects. According to decision no. RNN/319/20/KE, December 15, 2020 of the Bioethical Commission of the Medical University of Lodz, the study protocol and survey 03 frontiersin.org frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 FIGURE 1 SARS-CoV-2 variant distribution in Poland reported during the time of survey response collection. Based on the data available from: (32). −16.49% and 1,370 women—56.89%, 641 no data given—26.62%) with an age range from 18 to 90 years.h design was approved as an observational study. The participant was informed that by joining and starting the survey, he or she was giving their informed consent to complete the survey anonymously, and that he or she accepted that the data entered may be used on the basis of this informed consent for collective data analysis without additional consent. All data was collected anonymously, thus privacy and confidentiality of participants was adequately protected. Participants were informed that filling in the questionnaire is completely voluntary, and no compensation financial or otherwise was provided. The characteristics of the participants are provided in Table 1. Most of the participating men were overweight while most women were of normal weight (Table 2). Most participants were living cities with populations of 10,001 to 100,000 inhabitants (Table 2) and did not require hospitalization, oxygen therapy, respiratory therapy or a stay in the isolation unit (Table 2). Among all the respondents, the three most prevalent symptoms were fatigue (87.61%), anosmia (73.74%) and headache (69.89%) (Figure 2, Table 3). These symptoms were present in most patients on day 5 (67.32% of surveyed patients), day 7 (56.64%) and day 2 (46.14%) respectively (Figure 5). Most patients reported fatigue (58.07%), headache (44.12%) and myalgias (37.39%) on the first day (Figure 5). Frontiers in Medicine 4. Results Statistically significant differences in the prevalence of COVID-19 symptoms were found between man and women (Figure 3). Women were generally more symptomatic than men, with 31 symptoms occurred significantly more often in women than in men; their frequencies in descending order were as follows (as given in Table 4 and Figure 4): anosmia (reported by 76% of women), headache (74%), myalgias (62%), lack of appetite (52%), back pain (52%), chills (48%), arthralgias (45%), hyperosmia (39%), chest tightness (36%), rhinorrhea (34%), sore throat (34%), dizziness (33%), nasal dryness (33%), sinusitis (33%), insomnia (31%), palpitations (31%), chest pain (29%), amnesia (28%), ophthalmalgia (28%), limbs pain (27%), xerostomia (24%), nausea (23%), diaphoresis (22%), abdominal pain/ache (21%), sneezing (21%), hoarseness (20%), polydipsia (20%), alopecia (19%), ear pain/ache (11%) and vomiting (8%). Only three symptoms were more prevalent in men: subfebrility (reported by 70% of men), fever (55%) and hemoptysis (4%). The survey responses were collected between December 23, 2020 (week 52 of 2020) and February 28, 2021 (week 08 of 2021) during the dominance of SARS-CoV-2 alpha variant (Figure 1) (32). A total number of 7,034 participants started the survey. All confirmed that they were taking part in the survey for the first time or writing the survey on behalf of other person (child, elderly). The completion rate was 1783/7034100% = 25.25%. Completeness was 1740/7034100% = 24.74%. In total, 1740 participants answered all questions. As partially-completed questionnaires were included in the final analysis, the number of participants is presented with each result. In general, the later questions in the survey elicited fewer responses. Some questionnaires were excluded for the following reasons: participants claimed that they had answered the survey previously (80 surveys excluded), participants reported that they have not been tested for SARS-CoV-2 (n = 2,579), participants were younger than 18 years (n = 15), no answer was given to questions about symptoms (or question about ‘lack of symptoms’) (n = 1951) or duplicates of the same answer were found (n = 1). Therefore, 2,408 surveys were included in the final analysis, representing 2,408 patients (397 men Seven symptoms occurred in over 50% of participating men: fatigue (86%), subfebrility (70%), anosmia (65%), myalgias (56%), headache (55%), fever (55%) and cough (53%). Nine symptoms were 04 frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 FIGURE 2 Thirty most common COVID-19 symptoms reported by survey participants. Man figure source: freepik.com. Frontiers in Medicine 4. Results FIGURE 2 Thirty most common COVID-19 symptoms reported by survey participants. Man figure source: freepik.com. TABLE 1 Characteristics of studied population (N = 397 men, N = 1,370 women). TABLE 1 Characteristics of studied population (N = 397 men, N = 1,370 women). declared by over 50% of women: fatigue (88%), anosmia (76%), headache (74%), subfebrility (64%), myalgias (62%), ageusia (59%), cough (53%), lack of appetite (52%) and back pain (52%). Regarding the duration of COVID-19 symptoms 12 lasted significantly longer in women than in men: hypothermia, fatigue, sneezing, nausea, vomiting, lack of appetite, hyperhidrosis, TABLE 1 Characteristics of studied population (N = 397 men, N = 1,370 women). Median (LQ- UQ) (men) (Men) Mean ± SD Median (LQ- UQ) (women) (Women) Mean ± SD p Median (LQ- UQ) total Total Mean ± SD Age (years) 41 (34–52) 42 ± 12 42 (34–50) 42 ± 12 0.592 42 (34–50) 42 ± 12 Height (cm) 180 (175–185) 180 ± 7 167 (162–170) 166 ± 8 <0.001 168 (164–175) 169 ± 9 Weight (kg) 88 (78–97) 89 ± 17 68 (60–80) 71 ± 15 <0.001 72 (62–85) 75 ± 17 Body mass index (kg/m2) 26.87 (24.34–29.73) 27.59 ± 4.94 24.75 (21.71–28.58) 25.64 ± 5.36 <0.001 25.31 (22.31–29.05) 26.08 ± 5.33 TABLE 1 Characteristics of studied population (N = 397 men, N = 1,370 women). Median (LQ- UQ) (men) (Men) Mean ± SD Median (LQ- UQ) (women) (Women) Mean ± SD p Median (LQ- UQ) total Total Mean ± SD Age (years) 41 (34–52) 42 ± 12 42 (34–50) 42 ± 12 0.592 42 (34–50) 42 ± 12 Height (cm) 180 (175–185) 180 ± 7 167 (162–170) 166 ± 8 <0.001 168 (164–175) 169 ± 9 Weight (kg) 88 (78–97) 89 ± 17 68 (60–80) 71 ± 15 <0.001 72 (62–85) 75 ± 17 Body mass index (kg/m2) 26.87 (24.34–29.73) 27.59 ± 4.94 24.75 (21.71–28.58) 25.64 ± 5.36 <0.001 25.31 (22.31–29.05) 26.08 ± 5.33 Regarding the duration of COVID-19 symptoms 12 lasted significantly longer in women than in men: hypothermia, fatigue, sneezing, nausea, vomiting, lack of appetite, hyperhidrosis, declared by over 50% of women: fatigue (88%), anosmia (76%), headache (74%), subfebrility (64%), myalgias (62%), ageusia (59%), cough (53%), lack of appetite (52%) and back pain (52%). 4. Results declared by over 50% of women: fatigue (88%), anosmia (76%), headache (74%), subfebrility (64%), myalgias (62%), ageusia (59%), cough (53%), lack of appetite (52%) and back pain (52%). 05 Frontiers in Medicine frontiersin.org frontiersin.org 10.3389/fmed.2023.1121558 Lewek et al. TABLE 2 Body mass, place of residence, and needs of therapy among studied population. TABLE 2 Body mass, place of residence, and needs of therapy among studied population. Body mass characteristics of studied population (p < 0.001) Body mass index category (range in kg/m2)* n total n men n women % % men % women Normal (18.5 to <25.0) 799 126 673 45.22 31.74 49.12 Underweight (<18.5) 43 2 41 2.43 0.50 2.99 Overweight (25.0 to <30.0) 575 178 397 32.54 44.17 28.81 Obese I (30.0 to <35.0) 232 63 169 13.13 15.63 12.26 Obese II (35.0 to <40.0) 86 19 67 4.87 4.71 4.86 Obese III (≥40.0) 32 9 23 1.81 2.23 1.67 Place of residence of studied population (p = 0.55) City with more than 1 mln inhabitants 342 76 266 19.35% 19.14% 19.42% City from 500.001 to 1 mln inhabitants 296 74 222 16.53% 18.64% 16.20% City from 100.001 to 500.000 inhabitants 352 83 269 19.92% 20.91% 19.64% City from 10.001 to 100.000 inhabitants 423 82 341 23.94% 20.65% 24.89% City up to 100.000 inhabitants 84 21 63 4.75% 5.29% 4.60% Rural area 270 61 209 15.28% 15.37% 15.26% Stated needs for diverse types of therapy during COVID-19 infection Stay at the hospital 84 35 49 3.49% 41.67% 58.33% Oxygen therapy 73 29 44 3.03% 39.73% 60.27% Respirator application 6 3 3 0.25% 50.00% 50.00% Stay in isolation unit 22 12 10 0.91% 54.55% 45.45% None of the above 1,653 356 1,297 68.65% 21.54% 78.46% BMI range based on (31). Body mass characteristics of studied population (p < 0.001) Body mass characteristics of studied population (p < 0.001) mass characteristics of studied population (p < 0.001 patient, and therefore easier self-isolation and better control of the spread of this disease, thus being highly clinically and epidemiologically important. diaphoresis, palpitations, insomnia, hypersomnia and xerostomia. The minimum duration of each symptom was 1 day, maximum was ‘15 days or more’. Only 21 participants (1.19%) chose ‘no symptoms’ in the survey: 2.02% of responding women and 0.95% of responding man. The difference was not statistically significant (p = 0.084). 4. Results In the studied population, the most prevalent symptoms of COVID-19 infection were fatigue, anosmia, headache, subfebrility and myalgia; these were reported by 61–88% participants. These findings are different from those obtained in other studies of COVID-19 symptoms. For example, one of the first publications summarizing the findings associated with hospitalized COVID-19 patients found cough to be the most common symptom (presented by 67.8% patients) followed by fatigue (38.1% patients) (9). Amongst the population in our study, cough was not that prevalent, being the seventh most common symptom (53%). This difference could be due to the fact that Guan’s study was based mostly on hospitalized patients (93.6%) while only 4.7% were admitted to the hospital in the present study (9). Frontiers in Medicine frontiersin.org 5. Discussion Despite the growing knowledge of the SARS-CoV-2 virus acquired since the end of 2019, its symptoms are not well recognized and a full list is still being researched. As the symptoms of COVID-19 are mostly very similar to those of other infectious diseases, it is problematic to differentiate the condition from other respiratory tract diseases (34). It may be hard to tell the difference between novel coronavirus infection and common cold or influenza, especially in primary care where diagnostic procedures are limited and number of cases is significant, especially in infectious seasons. Hence, there is a need for a better understanding of the symptoms, their occurrence in time and their relationship with patient sex. Knowing the prevalence of COVID-19 symptoms in a single population will help diagnose COVID-19 on many levels. Knowing the specific combination of symptoms that are most likely to occur in COVID-19 will make it easier to isolate COVID-19 patients from a group of similar infectious diseases at the clinical stage, especially in primary care. It will also help in screening patients who need to be diagnosed further, e.g., using antigen tests. Better knowledge of the symptoms characteristic of a given disease is associated with easier recognition at the stage of the In general the prevalence of COVID-19 symptoms differ depending on the studied population. For example, in an Iranian study of 23,749 patients from 74 hospitals in Teheran, the most prevalent symptoms were cough (50.51%), respiratory distress (43.55%), muscular pain or fatigue (38.94%), gastrointestinal problems (10.4%) and headache (4.72%) (35). A Chinese meta- analysis of 38 studies with 3,062 patients found these to be fever (80.4%), cough (63.1%) and fatigue (46.0%) but not anosmia or ageusia (36). Similar findings were described in meta-analysis of 54 mainly Chinese publications, where fever (81.2%), cough (58.5%) and fatigue (38.5%) were listed as the most prevalent symptoms (10). Other Chinese meta-analyses listed fever, fatigue and cough as the most common symptoms (37, 38). In our population, fever was 06 frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 TABLE 3 Prevalence of symptoms and their duration in studied population (N = 2,408). TABLE 3 Prevalence of symptoms and their duration in studied population (N = 2,408). TABLE 3 Prevalence of symptoms and their duration in studied population (N = 2,408). No. Frontiers in Medicine Frontiers in Medicine 07 frontiersin.org 5. Discussion Symptom n Percentage (%) Duration of the symptom in days (median (LQ-UQ)) Minimum duration in days Maximum duration in days Duration of the symptom in days average (± SD) Mode in days 1 Fatigue 1,548 87.61 11 (6–15) 1 15 9.88 (±3.19) 15 2 Anosmia 1,303 73.74 9 (6–12) 1 15 8.5 1(±3.35) 10 3 Headache 1,235 69.89 5 (3–9) 1 15 6.03 (±4.18) 3 4 Subfebrility 1,154 65.31 3 (2–5) 1 15 4.21 (±4.72) 2 5 Myalgias 1,077 60.95 5 (3–8) 1 15 5.61 (±3.93) 3 6 Ageusia 1,002 56.71 8 (5–11) 1 15 8.11 (±4.09) 7 7 Cough 942 53.31 8 (4–13) 1 15 8.28 (±4.26) 15 8 Lack of appetite 885 50.08 8 (5–11) 1 15 7.92 (±4.09) 5 9 Back pain 873 49.41 5 (3–8) 1 15 5.75 (±3.97) 3 10 Chills 820 46.41 4 (2–7) 1 15 5.01 (±4.00) 2 11 Shivers 796 45.05 3 (2–6) 1 15 4.28 (±4.47) 2 12 Fever 768 43.46 3 (2–5) 1 15 3.95 (±3.64) 1 13 Arthralgia 764 43.24 5 (2–8) 1 15 5.59 (±3.85) 1 14 Hypersomnia 653 36.96 7 (4–11) 1 15 7.46 (±3.17) 3.7 15 Chest tightness 617 34.92 5 (2–8) 1 15 5.55 (±3.17) 1 16 Rhinorrhea 574 32.48 5 (3–8) 1 15 5.96 (±3.78) 3 17 Sore throat 573 32.43 3 (2–5) 1 15 4.25 (±4.00) 3 18 Sinusitis 556 31.47 5 (3–8) 1 15 6.06 (±3.96) 3 19 Dizziness 545 30.84 5 (3–8) 1 15 6.11 (±4.65) 1 20 Nasal dryness 536 30.33 7 (4–111) 1 15 7.79 (±4.10) 15 21 Depression/depressed mood 529 29.94 7 (4–11) 1 15 7.36 (±4.41) 1 22 Hyperhidrosis 522 29.54 7 (4–10) 1 15 7.14 (±3.15) 5 23 Diarrhea 521 29.49 3 (1–5) 1 15 3.67 (±4.42) 1 24 Insomnia 516 29.2 6 (3–10) 1 15 6.84 (±3.75) 1 25 Palpitations 498 28.18 5 (3–9) 1 15 6.20 (±3.78) 1 26 Hypothermia 496 28.07 4 (2–8) 1 15 5.6 (±2.42) 1 27 Chest pain 484 27.39 5 (3–9) 1 15 5.84 (±3.09) 1 28 Limbs pain 460 26.03 5 (3–8) 1 15 6.06 (±3.95) 3 29 Amnesia 455 25.75 4 (1–8) 1 15 5.35 (±4.02) 1 30 Ophthalmalgia 450 25.47 5 (3–7) 1 14 5.44 (±3.90) 3 31 Dyspnoea 411 23.26 5 (3–8) 1 15 5.96 (±4.12) 1 32 Xerostomia 404 22.86 7 (4–12) 1 15 7.96 (±3.92) 15 33 Diaphoresis 372 21.05 5 (3–8) 1 15 5.69 (±2.90) 4 34 Nausea 365 20.66 4 (2–7) 1 15 5.26 (±4.12) 1 35 Sneezing 351 19.86 4 (2–7) 1 15 5.13 (±4.13) 3 36 Abdominal pain/ache 347 19.64 3 (2–6) 1 15 4.61 (±4.32) 1 37 Hoarseness 339 19.19 5 (2–9) 1 15 5.98 (±4.16) 1 38 Shoulder pain 336 19.02 5 (3–8) 1 15 6.00 (±4.51) 3 39 Skin hyperaesthesia 323 18.28 5 (3–7) 1 15 5.57 (±4.28) 3 40 Polydipsia 308 17.43 8 (5–12) 1 15 8.05 (±4.17) 15 41 Alopecia 274 15.51 4 (1–8) 1 13 5.31 (±3.99) 1 (Continued) 07 Frontiers in Medicine frontiersin.org 10.3389/fmed.2023.1121558 Lewek et al. TABLE 3 (Continued) No. 5. Discussion Symptom n Percentage (%) Duration of the symptom in days (median (LQ-UQ)) Minimum duration in days Maximum duration in days Duration of the symptom in days average (± SD) Mode in days 42 Metallic taste 272 15.39 5 (3–8) 1 14 5.97 (±4.58) 1 43 Limb numbness 225 12.73 4 (1–8) 1 15 5.37 (±3.50) 1 44 Skin lesions 187 10.58 4 (2–7) 1 15 4.82 (±3.88) 1 45 Conjunctivitis 187 10.58 4 (2–7) 1 15 5.33 (±4.00) 1 46 Ear ache/pain 171 9.68 3 (1–5) 1 15 3.65 (±4.28) 1 47 Blood pressure elevation 168 9.51 5 (3–8) 1 15 5.97 (±4.14) 3 48 Odontalgia 139 7.87 4 (2–6) 1 15 4.51 (±4.58) 1 49 Vomiting 125 7.07 2 (1–4) 1 15 2.82 (±4.31) 1 50 Blood pressure reduction 99 5.6 6 (2–10) 1 15 6.33 (±4.33) 1 51 Urinary incontinence 61 3.45 5 (2–10) 1 13 5.72 (±3.49) 1 52 Aphonia 47 2.66 2 (1–5) 1 15 3.53 (±3.57) 1 53 Toes discoloration 38 2.15 2 (1–8) 1 15 4.47 (±4.38) 1 54 Hemoptysis 37 2.09 2 (1–5) 1 15 3.24 (±3.96) 1 Total 2,408 100.00 the wide range of angiotensin converting enzyme 2 (ACE2) expression in human tissues, with the small intestine, testis, kidneys and heart being the highest (43). ACE2 acts to degrade angiotensin II into angiotensin (1–7) which ultimately leads to a decrease in blood pressure (44). Symptoms believed to be specific for COVID-19, i.e., anosmia and ageusia, were not that prevalent the first day (15.27 and 11.93% respectively), but were reported by more than 40% of patients on day five and day six, respectively, (Figure 4). The prevalence of olfactory dysfunctions has been found to range from 5.6 to 98% of COVID-19 patients, with differences between China/Asia and Europe/USA (45), probably due to different expression of the ACE2 receptor in global populations (46). Although they may precede, follow or co-occur with other general symptoms (47), these symptoms were present in more than 50% of participants in the present study, starting from day 5. This suggests that in order to differentiate COVID-19 in the first days of the disease, the patient should be asked about fatigue, rather than anosmia, ageusia or nasal discharge at the first visit, especially in primary care. These symptoms are more common than anosmia or ageusia in the first days of COVID-19. 5. Discussion Olfactory and gustatory problems become more prevalent starting from day 5. ranked 12 among the most common presentations; however, a subfebrility, i.e., a high temperature, was reported by nearly twice as many survey participants as fever (1,154 vs. 768). Similarly, fever (73.5%) and cough (61.0%) were found to be the most common symptoms in an Egyptian review of 1773 COVID-19 patients (39). A meta-analysis by Ghayda et al. found the most common symptoms to be fever (prevalence - 77%), cough (60%) and fatigue/myalgia (31%); the authors also reported gastrointestinal symptoms including diarrhea (6%) and nausea/vomiting (5%) (40), which were much more common in the present study: 29% for diarrhea, 21% for nausea and 7% for vomiting.i In some cases, our present findings were quite divergent from those of previous studies. In one meta-analysis, the most common symptoms were found to be fever (97%), cough (70%) and myalgia/fatigue (39%), while our respective findings were fever/cough 43%/53% and myalgia/ fatigue 61%/88%, also diarrhea was less prevalent than in our study (8% vs. 29%) (41). Another meta-analysis found the pooled prevalence of loss of appetite, nausea/vomiting, diarrhea and abdominal pain/ discomfort to be 27, 10, 12 and 9%, respectively (42), compared to 50, 28, 29 and 20% in our study. These differences may be due to variation in populations, regions or SARS-CoV-2 variants among others. In addition, our study mostly includes outpatient subjects, which may also affect the results, since other studies rely on inpatients data mostly. We may only suspect that there may be a correlation between the patient presentation and his further path in the health care system. For example, COVID-19 patients with muscle pain and digestive symptoms are less likely to be hospitalized than those with fever and cough, however this needs further investigation. Our study confirmed previous findings that anosmia and ageusia are more frequent COVID-19 dysfunctions in women than men (47– 50). This may be due to the fact that activation of the Toll-like receptor (TLR), a protein responsible for pathogen recognition in the human innate immune system (51), is related to X chromosomes; this may lead to various inflammatory conditions and clinical courses in both sexes (52). No statistically significant differences in the duration of anosmia or ageusia were observed between men and women; in both cases, the mean duration of symptoms was eight days, which is consistent with the findings of other authors (45). Frontiers in Medicine frontiersin.org Frontiers in Medicine 5. Discussion Because smell and Nonetheless, SARS-CoV-2 infection presents with variety of symptoms from different body systems. This is most likely the effect of 08 frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 FIGURE 3 Comparison of 20 most common symptoms reported by men and women. Differences between all presented symptoms were statistically significant (p < 0.05). Source of figures: freepik.com. FIGURE 3 Comparison of 20 most common symptoms reported by men and women. Differences between all presented symptoms were statistically significant (p < 0.05). Source of figures: freepik.com. taste disorders are more prominent in COVID-19 than other seasonal viral diseases (e.g., influenza (53)) it is of high importance for physicians to recognize it as one of the most common and major symptoms of COVID-19. persisting symptoms lasted over 14 days, may be assumed to be prolonged and be a part of condition known as long COVID (54). Although prolonged illness is well documented in hospitalized patients (55), it also affects individuals with mild infection who do not need hospitalization (56). Neither nasal dryness, xerostomia or polydipsia were not included in a list of 48 clinical symptoms given by a previous meta-analysis (57). Thanks to our study, these symptoms The longest-lasting symptoms were fatigue, cough, nasal dryness, xerostomia and polydipsia (15 days or more). 5. Discussion Although our study focuses on the first two weeks of COVID-19 infection, these six 09 frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 Symptom Presence of symptom in men (%) Presence of symptom in women (%) p Duration of symptom in men1 Min Max Avg3 Mode Duration of symptom in women2 Min Max Avg3 Mode p Fatigue 85.64 88.18 0.177 10 (5–14) 1 15 9.34 (±4.65) 15 11 (6–15) 1 15 10.02 (±4.73) 15 0.007 Subfebrility 69.77 64.01 0.034 3 (2–5) 1 15 3.97 (±3.17) 1 3 (2–5) 1 15 4.29 (±3.40) 2 0.183 Anosmia 65.24 76.20 0.000 8 (6–12) 1 15 8.41 (±3.98) 6 9 (6–12) 1 15 8.54 (±4.01) 10 0.600 Myalgias 55.67 62.48 0.014 5 (3–8) 1 15 5.71 (±3.71) 1 4 (3–8) 1 15 5.58 (±3.98) 3 0.252 Headache 55.42 74.09 0.000 5 (3–8) 1 15 5.68 (±4.06) 3 5 (3–9) 1 15 6.11 (±4.30) 3 0.222 Fever 54.66 40.22 0.000 3 (2–6) 1 15 4.26 (±3.37) 1 3 (2–5) 1 15 3.83 (±3.11) 2 0.138 Cough 53.15 53.36 0.941 8 (4–12) 1 15 8.15 (±4.62) 15 8 (4–13) 1 15 8.32 (±4.66) 15 0.663 Ageusia 48.11 59.20 0.000 7 (5–11) 1 15 7.91 (±3.95) 7 8 (5–11) 1 15 8.15 (±3.96) 10 0.418 Shivers 42.57 45.77 0.260 3 (2–6) 1 14 3.98 (±3.04) 2 3 (2–6) 1 15 4.36 (±3.20) 2,3 0.074 Lack of appetite 41.56 52.55 0.000 6 (4–10) 1 15 7.20 (±4.22) 5 8 (5–11) 1 15 8.08 (±3.96) 7 0.007 Back pain 40.30 52.04 0.000 4 (2–7) 1 15 5.40 (±3.96) 1 5 (3–8) 1 15 5.82 (±4.01) 3 0.154 Chills 39.80 48.32 0.003 4 (2–6) 1 15 4.68 (±3.55) 1 4 (2–7) 1 15 5.08 (±3.84) 2 0.242 Arthralgia 34.76 45.69 0.000 5 (3–8) 1 15 5.88 (±4.14) 1 4 (2–8) 1 15 5.53 (±4.08) 1 0.295 Hyperhidrosis 31.74 28.91 0.276 6 (3–9) 1 15 6.31 (±4.10) 1 7 (4–10) 1 15 7.41 (±4.10) 5 0.008 Hypersomnia 29.22 39.20 0.000 6 (4–10) 1 15 7.22 (±4.36) 3,4,7 7 (4–11) 1 15 7.52 (±4.27) 3,7 0.414 Chest tightness 28.97 36.64 0.005 5 (2–8) 1 15 5.23 (±3.65) 1 5 (2–8) 1 15 5.62 (±4.04) 1 0.542 Rhinorrhea 27.96 33.80 0.029 5 (3–9) 1 15 6.20 (±4.26) 3 5 (3–8) 1 15 5.91 (±4.07) 3 0.567 Depression/depressed mood 27.46 30.66 0.220 7 (4–11) 1 15 7.37 (±4.56) 1 7 (4–11) 1 15 7.35 (±4.59) 1 0.978 Diarrhea 27.2 30.15 0.258 3 (1–4.5) 1 15 3.61 (±3.03) 1 3 (1–5) 1 15 3.68 (±3.11) 1 0.966 Sore throat 26.45 34.16 0.004 3 (2–5) 1 15 4.50 (±3.75) 3 3 (2–5) 1 15 4.20 (±3.40) 3 0.529 Hypothermia 25.95 28.69 0.284 3 (2–6) 1 15 4.54 (±3.80) 1 5 (3–8) 1 15 5.88 (±4.24) 3 0.001 Sinusitis 25.44 33.21 0.003 5 (3–7) 1 15 5.56 (±3.57) 3 5 (3–8) 1 15 6.18 (±3.90) 3 0.157 Dyspnoea 22.67 23.43 0.752 5 (3–9) 1 15 6.26 (±4.30) 1 5 (3–8) 1 15 5.87 (±3.79) 3 0.667 Nasal dryness 21.91 32.77 0.000 7 (3–11) 1 15 7.22 (±4.50) 1 7 (5–11) 1 15 7.90 (±4.40) 15 0.186 Insomnia 21.91 31.31 0.000 5 (2–8) 1 15 6.00 (±4.31) 1 6 (3–10) 1 15 7.01 (±4.54) 15 0.053 Limbs pain 21.66 27.30 0.024 5 (3–9) 1 15 5.91 (±3.89) 3,5 5 (3–8) 1 15 6.10 (±4.03) 3 0.755 Dizziness 21.41 33.58 0.000 5 (2–8) 1 15 5.96 (±4.23) 1 5 (3–8) 1 15 6.14 (±4.13) 1 0.625 Chest pain 20.91 29.27 0.001 5 (2–8) 1 15 5.77 (±4.24) 1 5 (3–9) 1 15 5.86 (±4.06) 1 0.708 10 Frontiers in Medicine 10 frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 Symptom Presence of symptom in men (%) Presence of symptom in women (%) p Duration of symptom in men1 Min Max Avg3 Mode Duration of symptom in women2 Min Max Avg3 Mode p Amnesia 18.89 27.74 0.000 4 (1–7) 1 15 5.00 (±4.34) 1 5 (1–8) 1 15 5.42 (±4.13) 1 0.298 Xerostomia 18.39 24.16 0.016 6 (3–10) 1 15 6.92 (±4.42) 1 8 (4–12) 1 15 8.19 (±4.59) 15 0.032 Palpitations 17.38 31.31 0.000 4 (2–7) 1 15 5.03 (±4.02) 1,2 6 (3–9) 1 15 6.39 (±4.15) 1,5 0.004 Ophthalmalgia 17.38 27.81 0.000 5 (3–7) 1 15 5.57 (±3.41) 5 5 (3–7) 1 15 5.42 (±3.60) 3 0.574 Diaphoresis 16.62 22.34 0.014 4 (2–5) 1 15 4.55 (±3.33) 4 5 (3–9) 1 15 5.93 (±4.00) 3 0.011 Shoulder pain 16.12 19.85 0.095 6 (3–8) 1 15 5.80 (±3.57) 3 5 (3–8) 1 15 6.05 (±3.94) 3 0.861 Hoarseness 15.87 20.15 0.057 4 (2–8) 1 15 5.67 (±4.69) 1 5 (2–9) 1 15 6.05 (±4.36) 1 0.294 Sneezing 15.37 21.17 0.011 3 (2–5) 1 15 4.46 (±3.74) 1 4 (2–7) 1 15 5.27 (±3.74) 3 0.050 Skin hyperaesthesia 15.11 19.2 0.064 5 (3–7.5) 1 13 5.25 (±3.48) 1,3 5 (3–7) 1 15 5.64 (±3.50) 3,4 0.370 Abdominal pain/ache 13.6 21.39 0.001 3 (2–5) 1 15 4.06 (±3.30) 2,3 4 (2–6) 1 15 4.71 (±3.69) 1 0.219 Metallic taste 12.85 16.13 0.110 5 (3–9) 1 15 6.24 (±4.27) 1,3 5 (3–8) 1 15 5.91 (±3.89) 1 0.718 Nausea 11.34 23.36 0.000 3 (1–6) 1 14 3.93 (±3.47) 1 5 (2–8) 1 15 5.45 (±3.79) 1,2,3 0.003 Limb numbness 10.08 13.5 0.071 3 (1–8) 1 15 5.13 (±4.74) 1 4 (2–8) 1 15 5.42 (±4.19) 1 0.338 Polydipsia 9.57 19.71 0.000 7 (4–10) 1 15 6.92 (±3.81) 4,6,8,10 8 (5–12) 1 15 8.21 (±4.38) 15 0.095 Skin lesions 9.07 11.02 0.265 4 (1–6.5) 1 15 4.56 (±3.97) 1 4 (2–7) 1 15 4.88 (±3.95) 1 0.510 Conjunctivitis 8.82 11.02 0.194 4 (2–7) 1 14 4.60 (±3.44) 1 5 (2.5–7.5) 1 15 5.49 (±4.10) 1 0.279 Blood pressure elevation 8.06 9.93 0.264 7 (3–9) 1 15 6.84 (±4.11) 3 4 (3–8) 1 15 5.76 (±4.13) 3 0.144 Odontalgia 6.80 8.18 0.371 4 (1–6) 1 15 4.37 (±3.97) 1 4 (2–6) 1 15 4.54 (±3.38) 1 0.496 Ear ache/pain 6.30 10.66 0.010 3 (1–4) 1 12 3.36 (±3.08) 1 3 (1–5) 1 15 3.70 (±3.20) 1 0.467 Blood pressure reduction 4.79 5.84 0.422 6 (1–8) 1 11 5.37 (±3.65) 1 6 (3–10) 1 15 6.56 (±4.46) 1 0.394 Alopecia 4.79 18.61 0.000 3 (1–5) 1 15 3.84 (±3.73) 1 5 (1–8) 1 15 5.42 (±4.41) 1 0.104 Vomiting 4.28 7.88 0.014 1 (1–3) 1 4 1.82 (±1.24) 1 2 (1–4) 1 13 2.97 (±2.53) 1 0.038 Hemoptysis 4.28 1.46 0.001 2 (2–5) 1 9 3.00 (±2.12) 2 2 (1–5) 1 13 3.45 (±3.47) 1 0.582 Aphonia 2.77 2.63 0.876 2 (1–5) 1 11 3.36 (±3.17) 1 3 (1–5) 1 14 3.58 (±3.19) 1 0.825 Toes discoloration 2.77 1.97 0.333 3 (1–10) 1 14 5.36 (±5.01) 1 2 (1–7) 1 14 4.11 (±3.75) 1 0.702 Urinary incontinence 2.77 3.65 0.398 2 (1–5) 1 15 4.18 (±4.45) 1,2 6 (2–10) 1 14 6.06 (±4.25) 1 0.179 1Duration of the symptom in days in men [median (LQ-UQ)]. frontiersin.org Frontiers in Medicine 5. Discussion 11 Frontiers in Medicine frontiersin.org frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 FIGURE 4 Prevalence of COVID-19 symptoms among men and women in studied population. p<0.05, **p<0.005. ranking psychiatric symptom was depression, which was ranked 21 with a similar prevalence (30%). It is possible that, in our population, psychiatric problems were not the most common within the first two weeks of COVID-19, but could become more apparent over the course of longer-term infection as other symptoms subside. Similarly, may be added to the rich symptomatology of novel coronavirus infection. Lopez-Leon et al. found study fatigue and headache to be the most prevalent symptoms of long-COVID (80 and 58% respectively), similar to our study; however, attention disorder was also listed in third place (27%) (57). In the present study, the highest Frontiers in Medicine 12 frontiersin.org 10.3389/fmed.2023.1121558 Lewek et al. FIGURE 5 The course of the 10 most commonly reported symptoms of COVID-19 in the first 14 days of the disease, as reported by survey participants [n = 2,408]. alopecia was also not that prevalent in our study (15.5%) compared to Lopez-Leon (25%) (57).if Our findings confirm previous observations that sex affects the COVID-19 presentation. Sex-based differences were also found during the MERS outbreak in 2013–2014, with the fatality rate being 52% in men, while in women it was 23% (72). Other factors may also be involved in these observed differences: for example, the presence of comorbidities such as hypertension, cardiovascular disease, chronic obstructive pulmonary disease, which are more prevalent in men than women (73). These may also be caused by higher smoking and alcohol consumption observed in men (74, 75). In Poland, although 54.3% of COVID-19 cases were confirmed in women, 56.5% of COVID-19 deaths were confirmed in men, which also supports the thesis of gender differences in COVID-19 presentation (76). Nonetheless, the evidence for influence of patient’s sex on COVID-19 course is increasing and our study also supports this thesis with its findings. Our findings suggest that women suffering from COVID-19 are more symptomatic within the first 14 days of the disease and that many of their symptoms persist longer than for men. Many sex-dependent factors affecting COVID-19 have been confirmed previously. A large cohort study of 17 million patients in England found a hazard ratio of 1.59 for higher risk of death due to COVID-19 in men (58). 5. Discussion Higher ratio of male-to-female death was also confirmed in France, Spain, Italy, Switzerland, Germany and China (59). Although the cause of these differences is currently unknown, it is thought that differences in sex hormones may contribute to different immunologic responses between the sexes (60). Takahashi et al. report that cytokines IL-8, IL-18, CCL5 were significantly elevated in male COVID-19 patients, and that CD38 and HLA-DR-positive activated T cells in female COVID-19 patients (61). Meng et al. found significant differences between male and female COVID-19 patients in eleven laboratory parameters including glucose, CRP and creatinine concentration (62). Frontiers in Medicine Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data availability statement The major limitation of the present study is that it was not possible to verify clinically the symptoms provided by survey participants. The survey was conducted online and subjects reported the symptoms based on their subjective recognition: the study was anonymous to encourage the participants to present honest answers. What is more IP addresses where not collected due to ethical reasons (identification of participants). Indeed, the instructions on the first screen emphasized the need to provide ‘sincere and true answers’ together with an explanation. The table of symptoms was also large (55 options of symptoms in rows and 15 day options in the columns, making 825 options), although this may discourage anyone whose aim was other than to share real data about his illness. In addition, the survey included 20 questions on nine screens, and no incentive was offered for completion; as such, a high level of motivation and persistence was needed to finish, and the respondent was less likely to provide intentionally false answers. Out of 7,034 who started the survey 66% were not qualified to the final analysis due to exclusion criteria, which shows our strict approach to include only valuable data. Moreover this was a cross-sectional survey conducted on specific sample of Polish population. Although important correlations were found, generalization of our findings is not entirely possible and needs additional research. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Author contributions PL conceived and planned the study and collected the data. PL and PK designed the questionnaire and contributed to the writing of the manuscript. IB designed the figures and contributed to the interpretation of the results and to the discussion. KW worked on the manuscript and contributed to the interpretation of the results. JL performed statistical analysis and contributed to the interpretation of the obtained results. PK contributed to planning the study and supervised the project. All authors discussed the results and contributed to the final manuscript. Although COVID-19 may be asymptomatic (45) only 2% of men and 1% of women in our study declared that they had no symptoms. It is possible that it was because of the sample bias - only those who suffered from COVID-19 symptoms were willing to take part. Although the alpha SARS-CoV-2 variant was on the rise during data collection (Figure 1) we would rather not connect all the listed symptoms with this particular variant. According to ECDC data, the percentage of sequenced cases in Poland ranged from 0.2% in week 52–2020 to 1% in week 08–2021 (32). Hence, we are reluctant to confirm any link between reported symptoms and the alpha variant; however, we may assume that the symptoms presented in this publication refer to alpha variant. Even so, more studies sequencing the COVID-19 variants and collecting presented symptoms are needed to identify characteristic disease presentations and any symptomatic differences between variants. Acknowledgments We acknowledge Edward Lowczowski for English language assistance. 5.1. Strengths and limitations The strength of our study not mentioned earlier is the nature of its population: it included over 2000 patients with diagnosed COVID-19. Out of 7,034 participants who started the survey, 36.7% were disqualified for analysis due to lack of testing for SARS-CoV-2. It is possible that many of the respondents were COVID-19 convalescents, as the survey announcements were targeted at this group; if so, the high disqualification rate suggests that vast number of patients were convinced that they had been suffering from COVID-19 without any medical evidence. As antigen tests have only been available in Poland since May 2020 (77), it was assumed that the patients claiming a positive test for SARS-CoV-2 would have received an RT-PCR test. Therefore, only participants with a positive PCR test were included: those who had undergone antigen tests, with positive COVID-19 antibodies in blood serum, or those who had suffered from non-COVID related infection, were not included in the final analysis. Restricting inclusion to only PCR-positive patients improves the reliability of the symptoms given by patients and limits the results of the study to true positive COVID-19 patients. ACE2 is more strongly expressed in men than women, particularly under pathological conditions (63), its tissue expression is also higher in men than women (64); moreover, angiotensin II receptor type 1 (AT1R; which SARS-CoV-2 binds to) is downregulated by estrogens (65). In addition to AT1R, the expression of TMPRSS2, a protein that primes SARS-CoV-2 entry into cells, is upregulated by androgens (66). Low levels of baseline serum testosterone in men may also contribute to increased cardiovascular risk in COVID-19 (67). This hormone also affects cytokine production (e.g., suppresses IL-6, IL-1beta and TNF-alpha, enhances production of IL-10) (68), suppresses T-helper cells, enhances regulatory T-cell differentiation (69), and reduces B-cell proliferation and humoral response (70). Moreover men with lower testosterone levels are predisposed to pulmonary and systematic inflammation and develop worse general parameters (67). The response to pathogens also differs between sexes: adult women demonstrate twice the protective antibody response after vaccination against various viral diseases compared with men (71). 13 Frontiers in Medicine frontiersin.org Lewek et al. Lewek et al. 10.3389/fmed.2023.1121558 Publisher’s note COVID-19 research on its symptomatology is evolving. Our study have found that out of 54 identified symptoms occurring during the first 14 days of infection, the most common were fatigue, anosmia and headache. Symptom presentation was also found to differ between the sexes, shedding new light on the nature of COVID-19 presentation in men and women. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Although studies have summarized the symptoms of long COVID-19, there is still lack of good quality data concerning the symptoms of acute SARS-CoV-2 infection. In response, our findings describe not only a broad symptomatology of acute COVID-19 but also its dependence on sex. The full clinical profile of COVID-19 cannot be limited to a few major symptoms. Future studies are therefore needed to examine its presentation against the background of other factors such as COVID variant, geographical location or population type. Ethics statement The studies involving human participants were reviewed and approved by the Bioethical Commission of the Medical University of Lodz, decision number RNN/319/20/KE, December 15, 2020. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Frontiers in Medicine References Sierpiński R, Pinkas J, Jankowski M, Zgliczyński WS, Wierzba W, Gujski M, et al. Sex differences in the frequency of gastrointestinal symptoms and olfactory or taste disorders in 1942 nonhospitalized patients with coronavirus disease 2019 (COVID-19). Pol Arch Intern Med. (2020) 130:501–5. doi: 10.20452/pamw.15414 29. Ring EF, McEvoy H, Jung A, Zuber J, Machin G. New standards for devices used for the measurement of human body temperature. J Med Eng Technol. (2010) 34:249–53. doi: 10.3109/03091901003663836 8. Viner RM, Ward JL, Hudson LD, Ashe M, Patel SV, Hargreaves D, et al. Systematic review of reviews of symptoms and signs of COVID-19 in children and adolescents. Arch Dis Child. (2020) 30. Crawford DC, Hicks B, Thompson MJ. Which thermometer? Factors influencing best choice for intermittent clinical temperature assessment. J Med Eng Technol. (2006) 30:199–211. doi: 10.1080/03091900600711464 9. Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, et al. Covid-19, clinical characteristics of coronavirus disease 2019 in China. N Engl J Med. (2020) 382:1708–20. doi: 10.1056/NEJMoa2002032 31. Eysenbach G. Improving the quality of web surveys: the checklist for reporting results of internet E-surveys (CHERRIES). J Med Internet Res. (2004) 6:e34. doi: 10.2196/ jmir.6.3.e34 32. E.C.f.D.P.a. Control, data on SARS-CoV-2 variants in the EU/EEA. European centre for disease prevention and control. (2021). 10. Alimohamadi Y, Sepandi M, Taghdir M, Hosamirudsari H. Determine the most common clinical symptoms in COVID-19 patients: a systematic review and meta- analysis. J Prev Med Hyg. (2020) 61:E304–12. doi: 10.15167/2421-4248/ jpmh2020.61.3.1530 33. Centers, and f.D.C.a. Prevention., Overweight & Obesity. European centre for disease prevention and control. (2022). 11. Fernández-de-Las-Peñas C, Palacios-Ceña D, Gómez-Mayordomo V, Florencio LL, Cuadrado ML, Plaza-Manzano G, et al. Prevalence of post-COVID-19 symptoms in hospitalized and non-hospitalized COVID-19 survivors: a systematic review and meta-analysis. Eur J Intern Med. (2021) 92:55–70. doi: 10.1016/j. ejim.2021.06.009 34. D.o.V.D. National Center for Immunization and Respiratory Diseases (NCIRD), symptoms of COVID-19. Centers for Disease Control and Prevention. (2021). 35. Jamshidi E, Asgary A, Tavakoli N, Zali A, Dastan F, Daaee A, et al. Symptom prediction and mortality risk calculation for COVID-19 using machine learning. Front. Artif Intell. (2021) 4:673527 12. Galván-Tejada CE, Herrera-García CF, Godina-González S, Villagrana-Bañuelos KE, Amaro JDL, Herrera-García K, et al. Persistence of COVID-19 symptoms after recovery in Mexican population. Int J Environ Res Public Health. (2020) 17. doi: 10.3390/ ijerph17249367 36. Zhu J, Ji P, Pang J, Zhong Z, Li H, He C, et al. References Clinical characteristics of 3062 COVID-19 patients: a meta-analysis. J Med Virol. (2020) 92:1902–14. doi: 10.1002/ jmv.25884 37. Sun P, Qie S, Liu Z, Ren J, Li K, Xi J. Clinical characteristics of hospitalized patients with SARS-CoV-2 infection: a single arm meta-analysis. J Med Virol. (2020) 92:612–7. doi: 10.1002/jmv.25735 13. Stavem K, Ghanima W, Olsen MK, Gilboe HM, Einvik G. Persistent symptoms 1.5-6 months after COVID-19 in non-hospitalised subjects: a population-based cohort study. Thorax. (2021) 76:405–7. doi: 10.1136/thoraxjnl-2020-216377 38. Li LQ, Huang T, Wang YQ, Wang ZP, Liang Y, Huang TB, et al. COVID-19 patients’ clinical characteristics, discharge rate, and fatality rate of meta-analysis. J Med Virol. (2020) 92:577–83. doi: 10.1002/jmv.25757 14. Grant MC, Geoghegan L, Arbyn M, Mohammed Z, McGuinness L, Clarke EL, et al. The prevalence of symptoms in 24,410 adults infected by the novel coronavirus (SARS-CoV-2; COVID-19): a systematic review and meta-analysis of 148 studies from 9 countries. PLoS One. (2020) 15:e0234765. doi: 10.1371/journal. pone.0234765 39. El-Anwar MW, Elzayat S, Fouad YA. ENT manifestation in COVID-19 patients. Auris Nasus Larynx. (2020) 47:559–64. doi: 10.1016/j.anl.2020.06.003 15. Jones J, Sullivan PS, Sanchez TH, Guest JL, Hall EW, Luisi N, et al. Similarities and differences in COVID-19 awareness, concern, and symptoms by race and ethnicity in the United States: cross-sectional survey. J Med Internet Res. (2020) 22:e20001. doi: 10.2196/20001 40. Ghayda RA, Lee J, Lee JY, Kim DK, Lee KH, Hong SH, et al. Correlations of clinical and laboratory characteristics of COVID-19: a systematic review and meta-analysis. Int J Environ Res Public Health. (2020) 17. doi: 10.3390/ijerph17145026 41. Rodriguez-Morales A.J., Cardona-Ospina J.A., Gutiérrez-Ocampo E., Villamizar-Peña R., Holguin-Rivera Y., Escalera-Antezana J.P., Alvarado-Arnez L.E., Bonilla-Aldana D.K., Franco-Paredes C., Henao-Martinez A.F., Paniz-Mondolfi A., Lagos-Grisales G.J., Ramírez- Vallejo E., Suárez J.A., Zambrano L.I., Villamil-Gómez W.E., Balbin-Ramon G.J., Rabaan A.A., Harapan H., Dhama K., Nishiura H., Kataoka H., Ahmad T., Sah R.L.A.N.o.C.D.-C.-R.L.-E.a. https://www.lancovid.org, Clinical, laboratory and imaging features of COVID-19: a systematic review and meta-analysis. Travel Med Infect Dis 34 (2020) 101623. 16. Izquierdo JL, Ancochea JSavana COVID-19 Research Group, Soriano JB. Clinical characteristics and prognostic factors for intensive care unit admission of patients with COVID-19: retrospective study using machine learning and natural language processing. J Med Internet Res. (2020) 22:e21801. doi: 10.2196/21801 17. Schmeelk S, Davis A, Li Q, Shippey C, Utah M, Myers A, et al. Monitoring symptoms of COVID-19: review of Mobile apps. JMIR Mhealth Uhealth. (2022) 10:e36065. doi: 10.2196/36065 42. Supplementary material The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed.2023.1121558/ full#supplementary-material 14 frontiersin.org frontiersin.org 10.3389/fmed.2023.1121558 10.3389/fmed.2023.1121558 Lewek et al. References 1. Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 395:565–74. doi: 10.1016/S0140-6736(20)30251-8 23. Mangia C, Russo A, Civitelli S, Gianicolo EAL. Sex/gender differences in COVID-19 lethality: what the data say, and do not say. Epidemiol Prev. (2020) 44:400–6. doi: 10.19191/EP20.5-6.S2.145 2. CfDCa Prevention. Coronavirus disease 2019 (COVID-19). Centers for Disease Control and Prevention. (2022). Available from: https://www.cdc.gov/coronavirus/2019- ncov/your-health/about-covid-19/basics-covid-19.html 24. Peckham H, de Gruijter NM, Raine C, Radziszewska A, Ciurtin C, Wedderburn LR, et al. Male sex identified by global COVID-19 meta-analysis as a risk factor for death and ITU admission. Nat Commun. (2020) 11:6317. doi: 10.1038/s41467-020-19741-6 3. He X, Lau EHY, Wu P, Deng X, Wang J, Hao X, et al. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat Med. (2020) 26:672–5. doi: 10.1038/ s41591-020-0869-5 25. Smith DS, Richey EA, Brunetto WL. A symptom-based rule for diagnosis of COVID-19. SN Compr Clin Med. (2020) 2:1947–54. doi: 10.1007/s42399-020-00603-7 26. Struyf T., Deeks J.J., Dinnes J., Takwoingi Y., Davenport C., Leeflang M.M.G., Spijker R., Hooft L., Emperador D., Dittrich S., Domen J., Horn S.R.A., Van den Bruel A., Cochrane C.-D.T.A.G., Signs and symptoms to determine if a patient presenting in primary care or hospital outpatient settings has COVID-19 disease. Cochrane Database Syst Rev 2020 (2020), doi: 10.1002/14651858.CD013665. 4. University JH. COVID-19 cases tracking. Johns Hopkins Coronavirus Resource Center. (2022). Available from: https://coronavirus.jhu.edu/map.html 4. University JH. COVID-19 cases tracking. Johns Hopkins Coronavirus Resource Center. (2022). Available from: https://coronavirus.jhu.edu/map.html 5. E.C.f.D.P.a. Control, COVID-19 situation update for the EU/EEA. European centre for disease prevention and control. (2020). 5. E.C.f.D.P.a. Control, COVID-19 situation update for the EU/EEA. European centre for disease prevention and control. (2020). 27. Rossman H, Keshet A, Shilo S, Gavrieli A, Bauman T, Cohen O, et al. A framework for identifying regional outbreak and spread of COVID-19 from one-minute population- wide surveys. Nat Med. (2020) 26:634–8. doi: 10.1038/s41591-020-0857-9 6. Wang C, Chudzicka-Czupała A, Tee ML, Núñez MIL, Tripp C, Fardin MA, et al. A chain mediation model on COVID-19 symptoms and mental health outcomes in Americans. Asians and Europeans Sci Rep. (2021) 11:6481. doi: 10.1038/ s41598-021-85943-7 28. A. Carfì, R. Bernabei, F. Landi, and G.A.C.-P.-A.C.S. Group. Persistent symptoms in patients after acute COVID-19. JAMA. (2020) 324:603–5. 7. References Cheung KS, Hung IFN, Chan PPY, Lung KC, Tso E, Liu R, et al. Gastrointestinal manifestations of SARS-CoV-2 infection and virus load in fecal samples from a Hong Kong cohort: systematic review and meta-analysis. Gastroenterology. (2020) 159:81–95. doi: 10.1053/j.gastro.2020.03.065 18. Jeon J, Baruah G, Sarabadani S, Palanica A. Identification of risk factors and symptoms of COVID-19: analysis of biomedical literature and social media data. J Med Internet Res. (2020) 22:e20509. doi: 10.2196/20509 43. Li MY, Li L, Zhang Y, Wang XS. Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues. Infect Dis Poverty. (2020) 9:45. doi: 10.1186/ s40249-020-00662-x 19. Ding Q, Massey D, Huang C, Grady CB, Lu Y, Cohen A, et al. Tracking Self- reported symptoms and medical conditions on social media during the COVID-19 pandemic: Infodemiological study. JMIR Public Health Surveill. (2021) 7:e29413. doi: 10.2196/29413 44. Hu Y, Liu L, Lu X. Regulation of angiotensin-converting enzyme 2: a potential target to prevent COVID-19? Front Endocrinol (Lausanne). (2021) 12:725967. doi: 10.3389/fendo.2021.725967 20. Chen T, Wu D, Chen H, Yan W, Yang D, Chen G, et al. Clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study. BMJ. (2020) 368:m1091. doi: 10.1136/bmj.m1091 20. Chen T, Wu D, Chen H, Yan W, Yang D, Chen G, et al. Clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study. BMJ. (2020) 368:m1091. doi: 10.1136/bmj.m1091 45. Kang YJ, Cho JH, Lee MH, Kim YJ, Park CS. The diagnostic value of detecting sudden smell loss among asymptomatic COVID-19 patients in early stage: the possible early sign of COVID-19. Auris Nasus Larynx. (2020) 47:565–73. doi: 10.1016/j. anl.2020.05.020 21. Dudley JP, Lee NT. Disparities in age-specific morbidity and mortality from SARS- CoV-2 in China and the Republic of Korea. Clin Infect Dis. (2020) 71:863–5. doi: 10.1093/cid/ciaa354 21. Dudley JP, Lee NT. Disparities in age-specific morbidity and mortality from SARS- CoV-2 in China and the Republic of Korea. Clin Infect Dis. (2020) 71:863–5. doi: 10.1093/cid/ciaa354 22. Spagnolo PA, Manson JE, Joffe H. Sex and gender differences in health: what the COVID-19 pandemic can teach us. Ann Intern Med. (2020) 173:385–6. doi: 10.7326/ M20-1941 46. Cao Y, Li L, Feng Z, Wan S, Huang P, Sun X, et al. Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations. Cell Discov. (2020) 6:11. doi: 10.1038/s41421-020-0147-1 46. Frontiers in Medicine References (2020) 58:302–3. doi: 10.4193/Rhin20.140 50. Heidari F, Karimi E, Firouzifar M, Khamushian P, Ansari R, Mohammadi Ardehali M. Anosmia as a prominent symptom of COVID-19 infection. Rhinology. (2020) 58:302–3. doi: 10.4193/Rhin20.140 65. Regitz-Zagrosek V, Oertelt-Prigione S, Seeland U, Hetzer R. Sex and gender differences in myocardial hypertrophy and heart failure. Circ J. (2010) 74:1265–73. doi: 10.1253/circj.CJ-10-0196 51. Fitzgerald KA, Kagan JC. Toll-like receptors and the control of immunity. Cells. (2020) 180:1044–66. doi: 10.1016/j.cell.2020.02.041 66. Clinckemalie L, Spans L, Dubois V, Laurent M, Helsen C, Joniau S, et al. Androgen regulation of the TMPRSS2 gene and the effect of a SNP in an androgen response element. Mol Endocrinol. (2013) 27:2028–40. doi: 10.1210/me.2013-1098 52. Lefèvre N, Corazza F, Valsamis J, Delbaere A, De Maertelaer V, Duchateau J, et al. The number of X chromosomes influences inflammatory cytokine production following toll-like receptor stimulation. Front Immunol. (2019) 10:1052. doi: 10.3389/ fimmu.2019.01052 52. Lefèvre N, Corazza F, Valsamis J, Delbaere A, De Maertelaer V, Duchateau J, et al. The number of X chromosomes influences inflammatory cytokine production following toll-like receptor stimulation. Front Immunol. (2019) 10:1052. doi: 10.3389/ fimmu.2019.01052 67. Giagulli VA, Guastamacchia E, Magrone T, Jirillo E, Lisco G, De Pergola G, et al. Worse progression of COVID-19 in men: is testosterone a key factor? Andrology. (2021) 9:53–64. doi: 10.1111/andr.12836 53. Beltrán-Corbellini Á, Chico-García JL, Martínez-Poles J, Rodríguez-Jorge F, Natera-Villalba E, Gómez-Corral J, et al. Acute-onset smell and taste disorders in the context of COVID-19: a pilot multicentre polymerase chain reaction based case-control study. Eur J Neurol. (2020) 27:1738–41. doi: 10.1111/ene.14273 53. Beltrán-Corbellini Á, Chico-García JL, Martínez-Poles J, Rodríguez-Jorge F, Natera-Villalba E, Gómez-Corral J, et al. Acute-onset smell and taste disorders in the context of COVID-19: a pilot multicentre polymerase chain reaction based case-control study. Eur J Neurol. (2020) 27:1738–41. doi: 10.1111/ene.14273 68. Norata GD, Tibolla G, Seccomandi PM, Poletti A, Catapano AL. Dihydrotestosterone decreases tumor necrosis factor-alpha and lipopolysaccharide- induced inflammatory response in human endothelial cells. J Clin Endocrinol Metab. (2006) 91:546–54. doi: 10.1210/jc.2005-1664 54. Tenforde MW, Kim SS, Lindsell CJ, Billig Rose E, Shapiro NI, Files DC, et al. Symptom duration and risk factors for delayed return to usual health among outpatients with COVID-19 in a multistate health care systems network - United States, march-June 2020. MMWR Morb Mortal Wkly Rep. (2020) 69:993–8. doi: 10.15585/mmwr.mm6930e1 69. Roved J, Westerdahl H, Hasselquist D. References Sex differences in immune responses: hormonal effects, antagonistic selection, and evolutionary consequences. Horm Behav. (2017) 88:95–105. doi: 10.1016/j.yhbeh.2016.11.017 70. Girón-González JA, Moral FJ, Elvira J, García-Gil D, Guerrero F, Gavilán I, et al. Consistent production of a higher TH1:TH2 cytokine ratio by stimulated T cells in men compared with women. Eur J Endocrinol. (2000) 143:31–6. 55. Grasselli G, Zangrillo A, Zanella A, Antonelli M, Cabrini L, Castelli A, et al. Baseline characteristics and outcomes of 1591 patients infected with SARS-CoV-2 admitted to ICUs of the Lombardy region. Italy Jama. (2020) 323:1574–81. doi: 10.1001/ jama.2020.5394 71. Klein SL, Jedlicka A, Pekosz A. The Xs and Y of immune responses to viral vaccines. Lancet Infect Dis. (2010) 10:338–49. doi: 10.1016/S1473-3099(10)70049-9 56. Townsend L, Dowds J, O’Brien K, Martin-Loeches I, Nadarajan P, Bannan C. Reply: the impact of acute illness severity on post-COVID-19 sequelae remains an unsettled question. Ann Am Thorac Soc. (2021) 18:1754. doi: 10.1513/ AnnalsATS.202103-303LE 72. Alghamdi IG, Hussain II, Almalki SS, Alghamdi MS, Alghamdi MM, El-Sheemy MA. The pattern of Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive epidemiological analysis of data from the Saudi Ministry of Health. Int J Gen Med. (2014) 7:417–23. doi: 10.2147/IJGM.S67061 57. Lopez-Leon S, Wegman-Ostrosky T, Perelman C, Sepulveda R, Rebolledo PA, Cuapio A, et al. More than 50 long-term effects of COVID-19: a systematic review and meta-analysis. Sci Rep. (2021) 11:16144. doi: 10.1038/s41598-021-95565-8 73. G.D.a.I.I.a.P. Collaborators, Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study 2017. Lancet 392 (2018) 1789–1858. 58. Williamson EJ, Walker AJ, Bhaskaran K, Bacon S, Bates C, Morton CE, et al. Factors associated with COVID-19-related death using OpenSAFELY. Nature. (2020) 584:430–6. doi: 10.1038/s41586-020-2521-4 74. Abuse NIOD. Are there gender differences in tobacco smoking? National Institute on Drug Abuse website. (2021). 59. Gebhard C, Regitz-Zagrosek V, Neuhauser HK, Morgan R, Klein SL. Impact of sex and gender on COVID-19 outcomes in Europe. Biol Sex Differ. (2020) 11:29. doi: 10.1186/s13293-020-00304-9 75. Erol A, Karpyak VM. Sex and gender-related differences in alcohol use and its consequences: contemporary knowledge and future research considerations. Drug Alcohol Depend. (2015) 156:1–13. doi: 10.1016/j.drugalcdep.2015.08.023 60. Bartz D, Chitnis T, Kaiser UB, Rich-Edwards JW, Rexrode KM, Pennell PB, et al. References Cao Y, Li L, Feng Z, Wan S, Huang P, Sun X, et al. Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations. Cell Discov. (2020) 6:11. doi: 10.1038/s41421-020-0147-1 Frontiers in Medicine 15 frontiersin.org Lewek et al. 10.3389/fmed.2023.1121558 Lewek et al. 47. Lechien JR, Chiesa-Estomba CM, De Siati DR, Horoi M, Le Bon SD, Rodriguez A, et al. Olfactory and gustatory dysfunctions as a clinical presentation of mild-to- moderate forms of the coronavirus disease (COVID-19): a multicenter European study. Eur Arch Otorhinolaryngol. (2020) 277:2251–61. doi: 10.1007/s00405-020-05965-1 47. Lechien JR, Chiesa-Estomba CM, De Siati DR, Horoi M, Le Bon SD, Rodriguez A, et al. Olfactory and gustatory dysfunctions as a clinical presentation of mild-to- moderate forms of the coronavirus disease (COVID-19): a multicenter European study. Eur Arch Otorhinolaryngol. (2020) 277:2251–61. doi: 10.1007/s00405-020-05965-1 62. Meng Y, Wu P, Lu W, Liu K, Ma K, Huang L, et al. Sex-specific clinical characteristics and prognosis of coronavirus disease-19 infection in Wuhan, China: a retrospective study of 168 severe patients. PLoS Pathog. (2020) 16:e1008520. doi: 10.1371/journal.ppat.1008520 48. Giacomelli A, Pezzati L, Conti F, Bernacchia D, Siano M, Oreni L, et al. Self- reported olfactory and taste disorders in patients with severe acute respiratory coronavirus 2 infection: a cross-sectional study. Clin Infect Dis. (2020) 71:889–90. doi: 10.1093/cid/ciaa330 48. Giacomelli A, Pezzati L, Conti F, Bernacchia D, Siano M, Oreni L, et al. Self- reported olfactory and taste disorders in patients with severe acute respiratory coronavirus 2 infection: a cross-sectional study. Clin Infect Dis. (2020) 71:889–90. doi: 10.1093/cid/ciaa330 63. Chappell MC, Marshall AC, Alzayadneh EM, Shaltout HA, Diz DI. Update on the angiotensin converting enzyme 2-angiotensin (1-7)-MAS receptor axis: fetal programing, sex differences, and intracellular pathways. Front Endocrinol (Lausanne). (2014) 4:201. 49. Klopfenstein T, Kadiane-Oussou NJ, Toko L, Royer PY, Lepiller Q, Gendrin V, et al. Features of anosmia in COVID-19. Med Mal Infect. (2020) 50:436–9. doi: 10.1016/j. medmal.2020.04.006 49. Klopfenstein T, Kadiane-Oussou NJ, Toko L, Royer PY, Lepiller Q, Gendrin V, et al. Features of anosmia in COVID-19. Med Mal Infect. (2020) 50:436–9. doi: 10.1016/j. medmal.2020.04.006 64. Zhao Y, Zhao Z, Wang Y, Zhou Y, Ma Y, Zuo W. Single-cell RNA expression profiling of ACE2, the receptor of SARS-CoV-2. Am J Respir Crit Care Med. (2020) 202:756–9. doi: 10.1164/rccm.202001-0179LE 50. Heidari F, Karimi E, Firouzifar M, Khamushian P, Ansari R, Mohammadi Ardehali M. Anosmia as a prominent symptom of COVID-19 infection. Rhinology. References Clinical advances in sex- and gender-informed medicine to improve the health of all: a review. JAMA Intern Med. (2020) 180:574–83. doi: 10.1001/jamainternmed.2019.7194 76. The COVID-19 Sex-Disaggregated Data Tracker. The sex, gender and COVID-19 project. (2021). 77. U.S.F.a.D. Administration, coronavirus (COVID-19) update: FDA authorizes first antigen test to help in the rapid detection of the virus that causes COVID-19 in patients. US Food and Drug Administration. (2021). 61. Takahashi T, Ellingson MK, Wong P, Israelow B, Lucas C, Klein J, et al. Sex differences in immune responses that underlie COVID-19 disease outcomes. Nature. (2020) 588:315–20. 16 16 Frontiers in Medicine frontiersin.org
https://openalex.org/W3092239664	https://link.springer.com/content/pdf/10.1007/JHEP04(2021)226.pdf	English	null	Two point functions in defect CFTs	The Journal of high energy physics/The journal of high energy physics	2,021	cc-by	19,912	Received: January 20, 2021 Accepted: March 14, 2021 Published: April 22, 2021 Received: January 20, 2021 Accepted: March 14, 2021 Published: April 22, 2021 Open Access, c⃝The Authors. Article funded by SCOAP3. Two point functions in defect CFTs JHEP04(2021)226 Christopher P. Herzog and Abhay Shrestha Department of Mathematics, King’s College London, The Strand, London WC2R 2LS, U.K. E-mail: christopher.herzog@kcl.ac.uk, abhay.shrestha@kcl.ac.uk Christopher P. Herzog and Abhay Shrestha Department of Mathematics, King’s College London, The Strand, London WC2R 2LS, U.K. Christopher P. Herzog and Abhay Shrestha Department of Mathematics, King’s College London, The Strand, London WC2R 2LS, U.K. E-mail: christopher.herzog@kcl.ac.uk, abhay.shrestha@kcl.ac.uk Abstract: This paper is designed to be a practical tool for constructing and investigating two-point correlation functions in defect conformal ﬁeld theory, directly in physical space, between any two bulk primaries or between a bulk primary and a defect primary, with ar- bitrary spin. Although geometrically elegant and ultimately a more powerful approach, the embedding space formalism gets rather cumbersome when dealing with mixed symmetry tensors, especially in the projection to physical space. The results in this paper provide an alternative method for studying two-point correlation functions for a generic d-dimensional conformal ﬁeld theory with a ﬂat p-dimensional defect and d −p = q co-dimensions. We tabulate some examples of correlation functions involving a conserved current, an energy momentum tensor and a Maxwell ﬁeld strength, while analysing the constraints arising from conservation and the equations of motion. A method for obtaining bulk-to-defect correlators is also explained. Some explicit examples are considered: free scalar theory on Rp × (Rq/Z2) and a free four dimensional Maxwell theory on a wedge. Keywords: Boundary Quantum Field Theory, Conformal Field Theory ArXiv ePrint: 2010.04995 ArXiv ePrint: 2010.04995 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. 1 Introduction Our interest is to improve fundamental understanding of defect conformal ﬁeld theory (dCFT). Such an improvement has a host of possible applications. Conformal ﬁeld theories with defects and boundaries describe physical systems at a phase transition, for example water inside a container at the end-point of the liquid-gas critical line, or an antiferromag- netic spin system with disorder at the Néel-dimer transition. Topological defects may exist astrophysically, as relics from phase transitions in the early universe. Thus an improved understanding of dCFT has experimental applications both in condensed matter and cos- mology. There are more fundamental theoretical reasons to pursue such a study as well, associated with D-branes in string theory and twist defects for computing entanglement entropy in a quantum information context. In this work, we study symmetry constraints on two-point correlation functions in dCFT. JHEP04(2021)226 Given substantial existing work [1–5] using conformal symmetry to constrain corre- lation functions in dCFT, one may legitimately ask why do more? Our answer is largely personal, that in attempting to investigate some speciﬁc examples of dCFT correlation func- tions involving tensors with mixed symmetries, for example a Maxwell ﬁeld strength and a stress tensor, we found the embedding space formalism employed by refs. [1–5] challenging to work with. The embedding space formalism requires an initial step of constructing a correlation function as a polynomial in embedding space, and a second step, which is not always necessary, of projecting the polynomial to a tensorial object in physical space. The polynomials in embedding space are simpler objects than their tensorial physical space counter-parts. In many situations, it may be enough to work with these polynomials, in which case the embedding space formalism remains a superior approach to ours. An old idea, it was originally developed in the context of CFT without boundaries or defects [6–9]. The conformal group on Rd with d > 2 is the orthogonal group O(d + 1, 1) and its action is not linear. However, we know that SO(d + 1, 1) acts naturally (and linearly) on Rd+1,1. This fact can be exploited by embedding the spacetime into Rd+1,1 and looking at the linearised action of the conformal group on this embedding space. The uplift simpliﬁes the constraints on the n point correlation functions arising from conformal symmetry. Sometimes one would like access to the physical space correlation functions as well, however. Two point functions in defect CFTs https://doi.org/10.1007/JHEP04(2021)226 Contents 1 Introduction 1 2 Conformal ﬁeld theory with a ﬂat defect 2 2.1 O(d) vectors, bi-vectors and rank-2 tensors 3 2.2 Comment about one and higher point functions 6 3 Bulk-bulk two point functions 7 3.1 ⟨JO⟩ 7 3.2 ⟨JJ⟩ 9 3.3 ⟨TO⟩ 10 3.4 ⟨TJ⟩ 11 3.5 ⟨TT⟩ 12 3.6 Constraints from Maxwell’s equation in d = 4 on ⟨FO⟩and ⟨FF⟩ 13 Contents 1 Introduction 1 2 Conformal ﬁeld theory with a ﬂat defect 2 2.1 O(d) vectors, bi-vectors and rank-2 tensors 3 2.2 Comment about one and higher point functions 6 3 Bulk-bulk two point functions 7 3.1 ⟨JO⟩ 7 3.2 ⟨JJ⟩ 9 3.3 ⟨TO⟩ 10 3.4 ⟨TJ⟩ 11 3.5 ⟨TT⟩ 12 3.6 Constraints from Maxwell’s equation in d = 4 on ⟨FO⟩and ⟨FF⟩ 13 4 Bulk-defect two point functions 15 4.1 Tensor structures for the defect limit 15 4.2 ⟨V ˆO⟩ 17 4.3 ⟨V ˆV ⟩ 17 4.4 ⟨S ˆO⟩ 18 4.5 ⟨F ˆV ⟩ 19 4.6 ⟨F ˆF⟩ 19 5 Free ﬁeld defect CFT 20 5.1 Free scalar theory on Rp × (Rq/Z2) 20 5.2 Free Maxwell theory on a wedge 23 5.3 Maxwell theory on a R × (R3/Z2) 24 6 Conclusion and further discussion 25 A Useful identities for ⟨JO⟩, ⟨JJ⟩, ⟨T O⟩, and ⟨F O⟩ 27 B Tensor structures for ⟨T T ⟩ 28 C Taking the defect limit of ⟨V O⟩, ⟨V V ′⟩, ⟨SO⟩, ⟨F V ′⟩and ⟨F F ′⟩ 28 D ⟨T T ⟩in the free Maxwell theory for d = 4, q = 2 30 E Embedding space to physical space 31 JHEP04(2021)226 4 Bulk-defect two point functions 4.1 Tensor structures for the defect limit 4.2 ⟨V ˆO⟩ 4.3 ⟨V ˆV ⟩ 4.4 ⟨S ˆO⟩ 4.5 ⟨F ˆV ⟩ 4.6 ⟨F ˆF⟩ 4 Bulk-defect two point functions 4.1 Tensor structures for the defect limit 4.2 ⟨V ˆO⟩ 4.3 ⟨V ˆV ⟩ 4.4 ⟨S ˆO⟩ 4.5 ⟨F ˆV ⟩ 4.6 ⟨F ˆF⟩ 5 Free ﬁeld defect CFT 20 5.1 Free scalar theory on Rp × (Rq/Z2) 20 5.2 Free Maxwell theory on a wedge 23 5.3 Maxwell theory on a R × (R3/Z2) 24 6 Conclusion and further discussion 25 A Useful identities for ⟨JO⟩, ⟨JJ⟩, ⟨T O⟩, and ⟨F O⟩ 27 B Tensor structures for ⟨T T ⟩ 28 C Taking the defect limit of ⟨V O⟩, ⟨V V ′⟩, ⟨SO⟩, ⟨F V ′⟩and ⟨F F ′⟩ 28 D ⟨T T ⟩in the free Maxwell theory for d = 4, q = 2 30 E Embedding space to physical space 31 – i – 1 Introduction In our case, we wanted to use Feynman diagrams to investigate the eﬀect of small interactions on the structure of correlation functions of free dCFT’s. As perturbation theory in CFT is typically performed in physical space, it is often more convenient to have a presentation of the conformal symmetry constraints on the physical space correlation functions. (It is possible to lift the perturbative results to embedding space, contracting the tensor structures with polarization vectors.) To refer to “the” embedding space formalism is already inaccurate, as there are at least two distinct variants of the formalism for mixed symmetry tensors, refs. [4] and [5]. The ingredients that make up the polynomial are diﬀerent in the two cases. Ref. [4] reproduces the antisymmetry of the correlation function through the use of Grassman variables. This approach has the advantage of producing simpler polynomials, but the disadvantage of making the projection to physical space more diﬃcult to implement. Ref. [5], in contrast, – 1 – encodes the antisymmetry of the correlators directly into bosonic building blocks for the polynomials. While the projection to physical space is then as straightforward as in the fully symmetric case, the building blocks themselves are more involved. A further issue for us in both cases was that the projection procedure depends on whether the indices in the resultant tensors are tangent or perpendicular to the defect. The aim of this paper is to introduce a formalism where bulk correlation functions can be written directly in physical space in a uniform way where parallel and perpendicular in- dices are treated on the same footing. As such, we follow in the footsteps of McAvity and Os- born [10] who developed a similar but simpler formalism for dealing with correlation func- tions of boundary CFT. We hope that the formalism we present may be helpful to others. JHEP04(2021)226 The work is organised as follows. Section 2 begins with a brief review of conformal maps to establish notation and conventions. The second half of section 2 presents our method for writing down dCFT correlation functions directly in physical space, extending the boundary CFT formalism of ref. [10]. Section 3 uses the formalism to present several speciﬁc examples of two-point correlation functions involving a scalar operator O, a conserved current Jµ, a stress tensor Tµν, and a Maxwell ﬁeld (in four dimensions) Fµν. 1 Introduction These tensorial objects are speciﬁed by symmetry constraints typically up to several functions of two invariant cross ratios. In section 4, we investigate the limit where one of the operators in these bulk two-point functions approaches the defect and hence derive the independent tensor structures required for writing any bulk-to-defect two point correlation function. These are speciﬁed up to a handful of constants and we also present several examples. Sections 5 looks at two speciﬁc examples of dCFT, a free scalar on Rp × (Rq/Z2), a free Maxwell ﬁeld on R2 × (R2/ZN) for N = 2 and 4 and also a free Maxwell ﬁeld on R × (R3/Z2). As supplemental material, we have provided a Mathematica notebook [11] that deﬁnes some of the tensor structures we introduce and computes the ⟨Fµν(x)Fλρ(x′)⟩correlation function. 2 Conformal ﬁeld theory with a ﬂat defect + y2q. (2.2) (2.2) (The deﬁning property of a cross-ratio is ξa(x, x′) = ξa(φ(x), φ(x′)).) Certain formulae are more simply expressed using particular rational combinations of ξ1 and ξ2. In particular, we will have occasion to use u2 = ξ1 ξ1+ξ2 and ξ3 = 1−ξ2 2 ξ2 . ξ ξ ξ For the special case q = 1 we only have one independent cross-ratio since ξ2 →1. Furthermore we have, u2 →v2 = ξ1 ξ1+1 and ξ3 →0. The simpliﬁcation in the case q = 1 is part of a more general phenomena when we consider higher point functions. As discussed in [2, 4], given n bulk points, we can construct the analog of ξ1 and ξ2 for any pair of these points, giving n(n −1) cross ratios in general. However, if p or q is too small compared to n, some of these cross ratios will not be independent, as happens when n = 2 and q = 1. More generally, there will be fewer independent cross ratios if q < n or p + 2 < n. For a lengthier and more detailed discussion of these cross ratios, including when some of the operators live on the defect, see [2]. 2 Conformal ﬁeld theory with a ﬂat defect Our principal interest is in formulating the conformal symmetry constraints on the corre- lation functions of local operators in defect conformal ﬁeld theory. We begin with a brief review of conformal maps preserving the defect and their action on the local operators. In the presence of a ﬂat p-dimensional defect on a d-dimensional spacetime [1], the conformal symmetry is broken to a subgroup, SO(p + 1, 1) × SO(q), where q = d −p is the codimension. Splitting spacetime into Rd = Rp × Rq, the defect is given by D = {(x, 0) ∈ M\|x ∈Rp}. Any points away from the defect (where y ̸= 0 with (x, y) ∈Rd) we call the bulk space B. Likewise, it is also convenient to split the index notation. We use Greek indices which run from 1, . . . , d, Latin indices a, b, c which run from 1, . . . , p, and Latin indices i, j, k which run from p + 1, . . . , d or from 1, . . . , q. Now, we look at the conformal maps that preserve the defect. A conformal map φ preserves the defect if φ(p) ∈D for all p ∈D. In the case of a ﬂat defect deﬁned above, the connected component of the conformal transformations on Rd which preserves the defect – 2 – is given by, is given by, t(x, y) = (x + a, y), rp(x, y) = (Rp · x, y), rq(x, y) = (x, Rq · y), σ(x, y) = (σx, σy), b(x, y) = x + bx2 Ω(x) , y Ω(x) ! . (2.1) (2.1) where x = (x, y), a ∈Rp, Rp ∈SO(p), Rq ∈SO(q), σ ∈R̸=0, b ∈Rp and Ω(x) = 1+2b·x+b2x2. The full symmetry group is SO(p+1, 1)×SO(q) and we will call a theory respecting this symmetry a defect CFT. With a single bulk point, we are not able to form any conformal invariants (cross-ratios) and so the one point function is ﬁxed up to a constant by conformal symmetry [1]. Given two bulk points x = (x, y) and x′ = (x′, y′), we can construct two independent cross-ratios ξa : B × B →R, under the defect conformal maps (2.1), JHEP04(2021)226 ξ1 = s2 4\|y\|\|y′\|, ξ2 = y · y′ \|y\|\|y′\|, s2 = (x −x′)2, \|y\| = q y2 1 + . . . 2.1 O(d) vectors, bi-vectors and rank-2 tensors The action of the defect conformal group (2.1) on the correlation function must obey a Ward identity when the theory is a dCFT. The identity states that for any conformal transformation φ we have the equality, ⟨(φ · OI)(x)(φ · OJ)(x′)⟩= ⟨OI(x)OJ(x′)⟩, (2.3) (2.3) where φ· is the action of the conformal group on primary ﬁelds. Recall that a primary ﬁeld is deﬁned by the transformation property, (φ · OI)(φ(x)) := Ω−∆ φ (x)G J I [Rφ(x)]OJ(x) , where (Rφ)µ ν(x) = (∂νφµ)(x) Ωφ(x) and ∆is the scaling dimension of OI. The I, J are generalised indices that indicate the representation of O(d) under which O transforms. Lastly, G is a matrix acting on the representation space of O(d). – 3 – Independent Tensor Structures Co-Dimension Cross-Ratio Vector Bi-Vector 2-Tensor at x at x′ at x at x′ q > 2 ξ1, ξ2 Ξ(1) µ , Ξ(2) µ Ξ′(1) α , Ξ′(2) α Iµα, J ′ µα δµν, Jµν δαβ, J ′′ αβ q = 2 ξ1, ξ2 Ξ(1) µ , Ξ(2) µ Ξ′(1) α , Ξ′(2) α Iµα δµν δαβ q = 1 ξ1 Ξ(1) µ Ξ′(1) α Iµα δµν δαβ Table 1. List of independent tensor structures used to construct two-point correlation function between any two bulk primaries with arbitrary spin. The deﬁnition of the cross-ratios, vectors, bi-vectors and rank-2 tensors can be found in (2.2), (2.4), (2.6) and (2.8) respectively. Table 1. List of independent tensor structures used to construct two-point correlation function between any two bulk primaries with arbitrary spin. The deﬁnition of the cross-ratios, vectors, bi-vectors and rank-2 tensors can be found in (2.2), (2.4), (2.6) and (2.8) respectively. JHEP04(2021)226 Hence, (2.3) provides constraints on the correlation function arising from the defect conformal group. In table 1, we list a set of independent tensor structures that can be used to construct correlation functions satisfying the Ward identity. Using the cross-ratios (2.2), we can deﬁne two structures Ξ(1) and Ξ(2) which enable us to satisfy the Ward indentity for primary vectors. The structures Ξ(a) can be viewed as O(d) vectors at a point x; they transform as Ξ(a) µ →(Rφ)ν µ(x)Ξ(a) ν under the defect conformal group. Along with Ξ(a), we can deﬁne two vectors Ξ′(n) which transforms as an O(d) vector at x′. 2.1 O(d) vectors, bi-vectors and rank-2 tensors A choice of these are given explicitly in Cartesian coordinates as Ξ′(1) µ (x, x′) = \|y′\| ξ1 ∂ξ1 ∂x′µ = −2\|y′\| s2 sµ −n′ µ, Ξ′(2) µ (x, x′) = \|y′\| ξ2 ∂ξ2 ∂x′µ = nµ ξ2 −n′ µ, (2.4) Ξ(1) µ (x, x′) = \|y\| ξ1 ∂ξ1 ∂xµ = 2\|y\| s2 sµ −nµ, Ξ(2) µ (x, x′) = \|y\| ξ2 ∂ξ2 ∂xµ = n′ µ ξ2 −nµ, Ξ′(1) µ (x, x′) = \|y′\| ξ1 ∂ξ1 ∂x′µ = −2\|y′\| s2 sµ −n′ µ, Ξ′(2) µ (x, x′) = \|y′\| ξ2 ∂ξ2 ∂x′µ = nµ ξ2 −n′ µ, (2.4) Ξ(1) µ (x, x′) = \|y\| ξ1 ∂ξ1 ∂xµ = 2\|y\| s2 sµ −nµ, Ξ(2) µ (x, x′) = \|y\| ξ2 ∂ξ2 ∂xµ = n′ µ ξ2 −nµ, (2.4) where, where, nµ =    0 µ = a, yk \|y\| µ = k, n′ µ =    0 µ = a, y′ k \|y′\| µ = k, (2.5) (2.5) and sµ = xµ −x′ µ. With Ξ(a) and Ξ′(n) in hand, we can construct bi-vectors, which transform as an O(d) vector at x and x′, by taking the product Ξ(a)Ξ′(n). Similarly, we can form rank-2 O(d) tensors at x by taking the product Ξ(a)Ξ(b) and ones at x′ by taking the product Ξ′(n)Ξ′(m). We can also take further derivatives of the vectors (2.4). Since Rφ depends on x or x′, we can only take the derivative w.r.t. x of Ξ′ and w.r.t. x′ of Ξ. Two derivatives w.r.t. the same point results in an object which will not transform correctly. These derivatives yield two additional bi-vectors: Iµν(x, x′) = −2ξ1\|y\| ∂ ∂xµ Ξ′(1) ν = −2ξ1\|y′\| ∂ ∂x′µ Ξ(1) ν , (2.6a) J ′ µν(x, x′) = ξ2\|y\| ∂ ∂xµ Ξ′(2) ν = ξ2\|y′\| ∂ ∂x′ν Ξ(2) µ , (2.6b) (2.6a) (2.6b) – 4 – where Iµν = δµν −2sµsν s2 , is the rotation matrix corresponding to the inversion map [12], Iµν = (Rinv)µν(x −x′) and where Iµν = δµν −2sµsν s2 , is the rotation matrix corresponding to the inversion map [12], Iµν = (Rinv)µν(x −x′) and J ′ µν(x, x′) =    δij −yjy′ i y·y′ µ = i, ν = j 0 otherwise. (2.7) (2.7) The last two independent rank-2 tensors come from contracting two bivectors over the indices which transform at the same point. 2.1 O(d) vectors, bi-vectors and rank-2 tensors The contractions between J ′ and I or J ′ gives a new rank-2 tensor at x and a second at x′: Jµν(x) =    δij −ninj µ = i, ν = j 0 otherwise , J ′′ µν(x′) =    δij −n′ in′ j µ = i, ν = j 0 otherwise . (2.8) JHEP04(2021)226 JHEP04(2021)226 (2.8) The number of primes on J indicates how many times the point x′ is implicated in its transformation properties: no primes means a rank-2 tensor at x, one prime a bivector at x and x′, and two primes a rank-2 tensor at x′. We now demonstrate by explicit computation that the set of tensor structures in table 1 is closed under index contraction. We consider only contractions of indices associated with the point x since the same relations will hold for x′ under the replacement Ξ →Ξ′, J →J ′′. The contractions involving vectors and rank-2 tensors at x are Ξ(1) µ Ξ(1)µ = 1 u2 , Ξ(2) µ Ξ(2)µ = ξ3 ξ2 , Ξ(1) µ Ξ(2)µ = −1 2 ξ3 ξ1 , JµνΞ(1)ν = −ξ2 2ξ1 Ξ(2) µ , JµνΞ(2)ν = Ξ(2) µ , JµαJ α ν = Jµν, J µ µ = q −1. (2.9) JµνΞ(1)ν = −ξ2 2ξ1 Ξ(2) µ , JµνΞ(2)ν = Ξ(2) µ , JµαJ α ν = Jµν, J µ µ = q −1. (2.9) (2.9) The contractions involving the bi-vectors are, The contractions involving the bi-vectors are, The contractions involving the bi-vectors are, The contractions involving the bi-vectors are, IµνΞ(1)ν = X ′ µ, IµνΞ(2)ν = − ξ3Ξ′(1) µ + ξ2Ξ′(2) µ , J ′ µνΞ(1)µ = 1 2ξ1 Ξ′(2) ν , J ′ µνΞ(2)µ = −1 ξ2 Ξ′(2) ν , J ′ µα(J ′) α ν = Jµν + Ξ(2) µ Ξ(2) ν , J ′ µαIα ν = Jµν + Ξ(2) µ Ξ(1) ν , JµνIν α = J ′ µα −Ξ(2) µ X ′ α, J ν µ J ′ να = J ′ µα, IµαIα µ = δµν. (2.10) (2.10) where X ′ µ := ξ2 Ξ′(1) µ −Ξ′(2) µ . where X ′ µ := ξ2 Ξ′(1) µ −Ξ′(2) µ . where X ′ µ := ξ2 Ξ′(1) µ −Ξ′(2) µ . 1For parity odd theories, we must add some Levi-Civita tensors ϵµ1···µd, ϵa1···ap and ϵi1···iq to the construction. 2.1 O(d) vectors, bi-vectors and rank-2 tensors h bl d d h h h b The structures in table 1 are independent in the sense that they cannot be written as a product of lower rank tensor structures. In this language, Ξ(n)Ξ′(m) is not an independent bi-vector even though it is a necessary ingredient in constructing the correlation function of two vector operators. The independent structures are closed under contraction, as seen from (2.9) and (2.10). No new ones can be formed through derivatives or contractions. For a parity even theory, we expect that this set of structures is suﬁcient to construct the correlation function between any two bulk operators of arbitrary spin.1 For bulk For a parity even theory, we expect that this set of structures is suﬁcient to construct the correlation function between any two bulk operators of arbitrary spin.1 For bulk 1For parity odd theories, we must add some Levi-Civita tensors ϵµ1···µd, ϵa1···ap and ϵi1···iq to the t ti – 5 – operators with I1 and I2 indices, we construct all possible terms with I1 and I2 indices using the independent structures in table 1. We then symmetrize or antisymmetrize over the indices and remove traces, as required in order to obtain an object with the right transformation properties under the O(d) × O(d) group acting on the two operators. Indeed, not counting the Kronecker delta function used to remove traces, the num- ber eight of independent structures here is the same as the number of structures used to construct correlation function polynomials in embedding space in [1]. Moreover, as seen in appendix E, the monomial building blocks in embedding space project down to linear combinations of our structures in real space. Therefore, the number of independent terms in any correlation function that we construct must match ref. [1]. We only consider up to rank-2 tensors in this work. However, counting the total number of structures in selected correlators such as ⟨SµνρO⟩, ⟨SµνρVα⟩, ⟨SµνρλO⟩, for symmetric, traceless S, we can match the number obtained from embedding space.2 We also match the number, six, of indepen- dent structures in the correlator ⟨FµνVλ⟩for an antisymmetric operator Fµν (pers. comm. M. Meineri). JHEP04(2021)226 q = 1, 2. When q = 1, we reduce to bCFT. Here Ξ(2) = Ξ′(2) = J = J ′ = J ′′ = 0. 2Eq. (3.19) in of ref. [1] is missing a couple of factors. The correct version should be (pers. com E. Lauria) 2.1 O(d) vectors, bi-vectors and rank-2 tensors Similarly, q = 2 is also a special case because we ﬁnd that J , J ′ and J ′′ are not independent. In this case the following identities hold, J ′ µν = −1 ξ3 Ξ(2) µ Ξ′(2) ν , Jµν = ξ2 ξ3 Ξ(2) µ Ξ(2) ν , J ′′ µν = ξ2 ξ3 Ξ′(2) µ Ξ′(2) ν . (2.11) (2.11) 2.2 Comment about one and higher point functions 2.2 Comment about one and higher point functions The main purpose of this work is to investigate two point functions, but we would like to make a couple of remarks about one point and higher point functions before passing to the main order of business. Curiously, although we found the tensor Jµν through the existence of cross ratios, this structure exists independently of them, and is important for allowing nonzero one-point functions in dCFT. If we have an operator OI in a representation of O(d) such that we can also construct something out of the Jµν and δµν structures in the same representation, then OI is allowed to have a nonzero one point function. Importantly, Jµν does not exist for q = 1 which forbids anything except for scalars developing a nonzero expectation value in bCFT [10]. More generally in dCFT, we see that vectors and anti- symmetric two-forms are also forbidden from having a nonzero expectation value. Having gone through the exercise of constructing two-point correlation functions, the procedure in broad outline is clear for n-point functions as well. Given n bulk points, for each pair (xr, xs) we can construct ξ1 and ξ2 type cross-ratios, calling them ξ(r,s) 1 and ξ(r,s) 2 respectively. Since a total of n(n −1)/2 unique pairs can be formed, we have naively n(n−1) independent cross ratios. For p and q to small compared to n, some of these won’t be independent (see the discussion immediately preceeding section 2.1). 2Eq. (3.19) in of ref. [1] is missing a couple of factors. The correct version should be (pers. comm. E Lauria) min(J1,J2) X k=0 2 Y j=1 Jj −k + 1 − jJj −k 2 k jJj −k 2 k + 1 . – 6 – Given our set of independent cross ratios, we can then construct tensor structures analogous to those in table 1. Fixing a point xr, we can form (n −1) distinct pairs involving xr. Taking derivatives with respect to xr of both ξ(r,s) 1 and ξ(r,s) 2 gives us 2(n−1) independent vectors at the given point xr. Repeating this procedure for all the n points gives a total of 2n(n −1) vectors, Ξ(1)(r,s) µ and Ξ(2)(r,s) µ . By further taking the derivative of Ξ(1)(r,s) and Ξ(2)(r,s) with respect to xs, we can form a bi-vector of type I and type J ′. Let us call them I(r,s) µν and J ′(r,s) µν respectively. 2.2 Comment about one and higher point functions Consequently, at each pair we have 2 independent bi-vectors and hence a total of n(n −1)/2 bi-vectors of type I and J ′ each. Finally, for each point xr we can construct the independent rank-2 tensor J , namely J (r) µν . We then assemble from these constituents objects with the correct O(d) transforma- tion properties — by appropriately antisymmetrizing, symmetrizing, and removing traces. Although we do not check the closure of this set of independent tensor structures under contraction (as done for the two-point case in (2.9) and (2.10)), we do ﬁnd a correspon- dence with the embedding space results in [4]. In particular, the number of vectors matches their K(i) ab and ¯K(i) ab , the number of bi-vectors matches their S(i,j) ab and ¯S(i,j) ab and ﬁnally the number of rank-2 tensors matches their H(i,j) a . JHEP04(2021)226 3 Bulk-bulk two point functions As a warm-up, the two point correlator between two bulk scalar primaries of dimension ∆ and ∆′ is well known to have the form3 ⟨O(x)O′(x′)⟩= \|y′\|∆−∆′ s2∆ f(ξ1, ξ2), (3.1) (3.1) where f(ξ1, ξ2) is an arbitrary function of the cross-ratios. In what follows, we will inves- tigate correlation functions involving a scalar ﬁeld O, a conserved current Jµ, and stress tensor T µν and (in the particular case of four dimensions) a Maxwell ﬁeld strength F µν. Also, when counting the number of PDE constraints arising from conservation or equations of motion (see table 2), we use the argument that taking the divergence reduces the spin of the correlator by 1 and hence the number of independent structures present in the resulting correlator is equal to the number of PDE constraints. For example taking the divergence of ⟨TµνO⟩we see the resulting correlator is of the form ⟨VνO⟩and hence 2 independent PDE constraints are expected. 3.1 ⟨JO⟩ 3Note we can use (2.2) to write \|y\| = s2 4\|y′\|ξ1 . 3.1 ⟨JO⟩ The two point correlator between a bulk vector operator Vµ of dimension ∆and a scalar primary O of dimension ∆′ is ﬁxed up to two functions of two cross ratios: The two point correlator between a bulk vector operator Vµ of dimension ∆and a scalar primary O of dimension ∆′ is ﬁxed up to two functions of two cross ratios: ⟨Vµ(x)O(x′)⟩= 1 \|y\|∆\|y′\|∆′ f1(ξ1, ξ2)Ξ(1) µ + f2(ξ1, ξ2)Ξ(2) µ . (3.2) (3.2) 3Note we can use (2.2) to write \|y\| = s2 4\|y′\|ξ1 . 3Note we can use (2.2) to write \|y\| = s2 4\|y′\|ξ1 . 3Note we can use (2.2) to write \|y\| = s2 4\|y′\|ξ1 . – 7 – Correlators # of functions Conservation q > 2 q = 2 q = 1 q > 2 q = 2 q = 1 ⟨JµO⟩ 2 2 1 1 1 1 ⟨JµJν⟩ 5 4 2 2 2 1 ⟨TµνO⟩ 4 4 1 2 2 1 ⟨TµνJα⟩ 12 8 2 10 8 3 ⟨TµνTαβ⟩ 19 10 3 12 8 2 Correlators, d = 4 # of functions EoM q = 3 q = 2 q = 1 q = 3 q = 2 q = 1 ⟨FµνO⟩ 1 1 0 2 2 0 ⟨FµνFαβ⟩ 10 5 2 6 4 1 JHEP04(2021)226 Table 2. This table contains a list of bulk-bulk correlators which we consider in section 3 and the number of independent structures appearing in the correlators, dependent on the co-dimension q. It also denotes the number of PDE constraints arising from conservation of current ∂µJµ = 0 and stress tensor ∂µTµν = 0, everywhere in the bulk. Correlators involving a Maxwell ﬁeld strength Fµν are also considered, speciﬁcally when d = 4, and we list the number of PDE constraints arising from the bulk equation of motion ∂µFµν = 0. In the special case that V µ = Jµ is a conserved current (with dimension ∆= d−1), we have the constraint ∂µ⟨Jµ(x)O(x′)⟩= 0, satisﬁed everywhere in the bulk. Current conservation leads to the following relation between the functions f1 and f2: (2pξ1 + (d −2)ξ2)f1 + 2ξ2 1 u2 f(1,0) 1 −ξ2ξ3f(0,1) 1 + 2ξ1 2 −q −1 ξ2 2 f2 −ξ1ξ3 f(1,0) 2 −2f(0,1) 2 = 0, (3.3) (3.3) where the identities (A.1) were used in the derivation. where the identities (A.1) were used in the derivation. q = 1. 3.2 ⟨JJ⟩ 3.2 ⟨JJ⟩ The two point correlator between two identical vector ﬁelds of dimension ∆depends on ﬁve arbitrary functions of two cross ratios: The two point correlator between two identical vector ﬁelds of dimension ∆depends on ﬁve arbitrary functions of two cross ratios: ⟨Vµ(x)Vν(x′)⟩= 1 s2∆ f1Ξ(1) µ Ξ′(1) ν + f2Ξ(2) µ Ξ′(2) ν + f3 Ξ(1) µ Ξ′(2) ν + Ξ(2) µ Ξ′(1) ν + f4Iµν + f5J ′ µν , (3.4) ⟨Vµ(x)Vν(x′)⟩= 1 s2∆ f1Ξ(1) µ Ξ′(1) ν + f2Ξ(2) µ Ξ′(2) ν + f3 Ξ(1) µ Ξ′(2) ν + Ξ(2) µ Ξ′(1) ν (3 4) f1Ξ(1) µ Ξ′(1) ν + f2Ξ(2) µ Ξ′(2) ν + f3 Ξ(1) µ Ξ′(2) ν + Ξ(2) µ Ξ′(1) ν + f4Iµν + f5J ′ µν , (3.4) (3.4) + f4Iµν + f5J ′ µν , (3.4) The tensor structure of f3 appears in a symmetric combination because the operators in the correlation function are assumed to be identical. (Conversely, if the operators were distinct, the coeﬃcients of Ξ(1) µ Ξ′(2) ν and Ξ(2) µ Ξ′(1) ν should be independent.) JHEP04(2021)226 Reﬂection positivity places bounds on the bulk-bulk functions appearing in the corre- lator between identical operators. The reﬂection plane is taken to be a hypersurface that intersects the defect at right angles, which ﬁxes ξ2 = 1. The positivity demands that, f4(ξ1, 1) ≥0, (f4 + f5)(ξ1, 1) ≥0, f4 + f1 u2 (ξ1, 1) ≥0. (3.5) (3.5) If V µ = Jµ is a conserved current, then ∆= d−1 and ∂µ⟨Jµ(x)Jν(x′)⟩= 0. This divergence ∂µ⟨Jµ(x)Jν(x′)⟩is the correlation function of a scalar with a vector, and we just saw that it depends generically on two functions of two cross ratios. Thus, conservation will lead to two constraints on the ﬁve fi. Using the identities (A.1) and (A.2), we ﬁnd If V µ = Jµ is a conserved current, then ∆= d−1 and ∂µ⟨Jµ(x)Jν(x′)⟩= 0. This divergence ∂µ⟨Jµ(x)Jν(x′)⟩is the correlation function of a scalar with a vector, and we just saw that it depends generically on two functions of two cross ratios. Thus, conservation will lead to two constraints on the ﬁve fi. 3.1 ⟨JO⟩ In the codimension one case, the structure Ξ(2) is absent and the cross ratios ξ2 →1 and ξ3 →0 degenerate. The constraint (3.3) reduces to, ((d −2) + 2(d −1)ξ1)f1 + 2ξ1(1 + ξ1) df1 dξ1 = 0 which has the simple solution f1(ξ1) = c ξ 1−d 2 1 (1 + ξ1)−d 2 = c ξ1−d 1 vd , where c is an integration constant. This result matches the bCFT result in [12] with appropriate rescaling. where c is an integration constant. This result matches the bCFT result in [12] with appropriate rescaling. – 8 – 3.2 ⟨JJ⟩ 3.2 ⟨JJ⟩ Using the identities (A.1) and (A.2), we ﬁnd 2ξ2 1 u2 f(1,0) 1 −ξ2ξ3f(0,1) 1 −(ξ2(d + 1) + 2ξ1(q −1)) f1 + ξ1ξ3 2f(0,1) 3 −f(1,0) 3 + ξ3d −2ξ1 ξ3 ξ2 + (q −1) f3 + 2ξ2 1ξ2f(1,0) 4 −2ξ1ξ2ξ3f(0,1) 4 = 0, (3.6) (3.6) and ξ2f1 + ξ1ξ3 2f(0,1) 2 −f(1,0) 2 + ξ3(d −1) −2ξ1 ξ3 ξ2 + 1 ξ2 2 + (q −1) f2 + 2ξ2 1 u2 f(1,0) 3 −ξ2ξ3f(0,1) 3 − ξ2(d −1) + 2ξ1(q −1) −1 ξ2 f3 −2ξ2 1ξ2f(1,0) 4 −2ξ1ξ2 2f(0,1) 4 + ξ1 f(1,0) 5 −2f(0,1) 5 + 2ξ1 ξ2 −(d −1) f5 = 0. (3.7) (3.7) q = 1. Here we only have one PDE because (3.7) was obtained by setting the coeﬃcient of Ξ′(2) to zero, which does not exist when q = 1. The PDE constraint for q = 1 is, 2ξ2 1 1 v2 df1 dξ1 + df4 dξ1 = (d + 1)f1. 2ξ2 1 1 v2 df1 dξ1 + df4 dξ1 = (d + 1)f1. Notice that this only involves f1 and f4 since the other tensor structures are zero when q=1. The PDE constraint matches the bCFT result in [12] under appropriate identiﬁcation. Notice that this only involves f1 and f4 since the other tensor structures are zero when q=1. The PDE constraint matches the bCFT result in [12] under appropriate identiﬁcation. q = 2. For this case we take f5 →0 since J ′ is not independent (2.11). – 9 – 3.3 ⟨T O⟩ 3.3 ⟨T O⟩ 3.3 ⟨T O⟩ The two point correlator of a symmetric rank-2 tensor S with dimension ∆and a scalar of dimension ∆′ is given by,4 The two point correlator of a symmetric rank-2 tensor S with dimension ∆and a scalar of dimension ∆′ is given by,4 ⟨Sµν(x)O(x′)⟩= \|y′\|∆−∆′ s2∆ f1Ξ(1) µ Ξ(1) ν + f2Ξ(2) µ Ξ(2) ν + f3Ξ(1) (µ Ξ(2) ν) + f4Jµν + f5δµν . (3.8) (3.8) We need the correlator to be traceless to consider the energy momentum tensor. Taking the trace of the above equation, we obtain a constraint on f5, f5 = − f1 u2d + f2ξ3 ξ2d −f3ξ3 2ξ1d + f4(q −1) d . (3.9) (3.9) JHEP04(2021)226 Now we can write the two point correlator between the energy momentum tensor and a scalar, ⟨Tµν(x)O(x′)⟩= \|y′\|d−∆′ s2d f1 Ξ(1) µ Ξ(1) ν −δµν u2d + f2 Ξ(2) µ Ξ(2) ν −δµνξ3 ξ2d + f3 Ξ(1) (µ Ξ(2) ν) + δµνξ3 2ξ1d + f4 Jµν −q −1 d δµν . (3.10) (3.10) This gives a total of 4 structures matching the number obtained from the embedding space theory [1]. Imposing conservation of T and using the identities (A.4), we obtain two PDE constraints on f1, . . . , f4, 2ξ1(1 −q) −ξ2 d (d + 2)(d −1) f1 + 2ξ2 1 u2d(d −1)f(1,0) 1 −ξ2ξ3f(0,1) 1 + 2ξ1ξ3 ξ2 f2 −2ξ2 1ξ3 ξ2d f(1,0) 2 + ξ3 2d(d + 1)(d −2) −ξ1 ξ3 ξ2 + q −1 f3 + ξ1ξ3 2 −d 2d f(1,0) 3 + f(0,1) 3 + 2(q −1)ξ1f4 −2ξ2 1 q −1 d f(1,0) 4 = 0, (3.11) (3.11) and, ξ2 d (d−2)f1−2ξ1ξ2 u2d f(0,1) 1 + ξ3d−4ξ1 ξ3 ξ2 −1 ξ2 2d + q 2 f2 +ξ1ξ3 2 d(d−1)f(0,1) 2 −f(1,0) 2 + ξ3 2 −qξ1−ξ2 d 1+ d2 2 + 1 ξ2 2 !! f3 + ξ2 1 u2 f(1,0) 3 + ξ2ξ3 2d (2−d)f(0,1) 3 +ξ2df4−ξ1ξ2 f(1,0) 4 +2 q−1 d −1 f(0,1) 4 = 0. (3.12) (3.12) q = 1. For this case f2, . . . , f4 vanish, ξ2 →1 and ξ3 →0, while the second PDE (3.12) (obtained by setting coeﬃcients of Ξ(2) to zero) does not exist. So, the constraint reduces to, q = 1. For this case f2, . . 4We use round brackets on indices to denote symmetrisation and square brackets for antisymmetrisation (including a factor of 1 n!). We use \| to separate indices that are being (anti)symmetrised when not next to each other. 3.4 ⟨T J⟩ The two point correlator between a symmetric rank-2 tensor S of dimension ∆and a vector V of dimension ∆′ is given by, The two point correlator between a symmetric rank-2 tensor S of dimension ∆and a vector V of dimension ∆′ is given by, JHEP04(2021)226 ⟨Sµν(x)Vα(x′)⟩= \|y′\|∆−∆′ s2∆ gnJµνΞ′(n) α + hnδµνΞ′(n) α + FnΞ(n) (µ Iν)α + GnΞ(n) (µ J ′ ν)α + X n≥m,r fmnrΞ(m) (µ Ξ(n) ν) Ξ′(r) α ! , (3.13) ⟨Sµν(x)Vα(x′)⟩= \|y′\|∆−∆′ s2∆ gnJµνΞ′(n) α + hnδµνΞ′(n) α + FnΞ(n) (µ Iν)α + GnΞ(n) (µ J ′ ν)α + X n≥m,r fmnrΞ(m) (µ Ξ(n) ν) Ξ′(r) α ! , (3.13) (3.13) where we restrict the sum over n to avoid double counting and so fmnr has 6 indepen- dent components. There are 14 structures here, but tracelessness of Sµν removes two, constraining h1 and h2, h1 = −1 d g1(q −1) + f111 u2 −f121ξ3 2ξ1 + f221ξ3 ξ2 + F1ξ2 −F2ξ3 , (3.14a) h2 = −1 d g2(q −1) + f112 u2 −f122ξ3 2ξ1 + f222ξ3 ξ2 −F1ξ2 −F2ξ2 + G1 2ξ1 −G2 ξ2 . (3.14b) (3.14a) The embedding space result [1] also involves 12 structures. The embedding space result [1] also involves 12 structures. If the rank-2 tensor Sµν is actually the stress tensor Tµν and the vector Vµ a conserved current Jµ, then we should furthermore set ∆= d, ∆′ = d−1 and impose the conservation conditions. Conservation of Jµ gives four PDE constraints while the conservation of T µν gives six constraints. (We simply count the structures needed to write down ⟨Sµν(x)O(x′)⟩ and ⟨Vµ(x)V ′ ν(x′)⟩, respectively and where S is traceless.) In total, there are 12 functions and 10 PDE relations, which we will spare the reader. q = 1. Here Ξ(2), Ξ′(2), J and J ′ all vanish and the number of structure reduces to 2. The conservation of T µν gives two ODE constraints while the conservation of Jµ gives one ODE constraint. Since there are only 2 functions but 3 constraints, the system is overdetermined, and one might guess the correlation function vanishes. However, some of the constraints are degenerate, and the correlation function is ﬁxed up to a constant (see p 14 of [13]). q = 2. Here J and J ′ are not independent and the number of structures reduces to 8. 3.3 ⟨T O⟩ . , f4 vanish, ξ2 →1 and ξ3 →0, while the second PDE (3.12) (obtained by setting coeﬃcients of Ξ(2) to zero) does not exist. So, the constraint reduces to, 2ξ2 1 v2 df1 dξ1 = (d + 2)f1. 4We use round brackets on indices to denote symmetrisation and square brackets for antisymmetrisation (including a factor of 1 n!). We use \| to separate indices that are being (anti)symmetrised when not next to each other. – 10 – This is simply solved to give, This is simply solved to give, This is simply solved to give, This is simply solved to give, This is simply solved to give, f1(ξ1) = c ξ1 ξ1 + 1 1+ d 2 =⇒f1(v) = cvd+2, which matches the result from [12] with the appropriate rescaling. q = 2. Here, J is not independent and so we set f4 = 0. q = 2. Here, J is not independent and so we set f4 = 0. ⟨T J⟩ 3.5 ⟨T T ⟩ The two point correlator between symmetric rank-2 tensors S and S′ is given by (B.1). There is a total of 36 independent components. Demanding that Sµν is traceless gives 5 constraints and then demanding S′ µν is traceless gives a further 4 constraints. This is a total of 9 constraints for 36 structures, hence reducing the number of independent structures to 27 and so matching the number predicted by the embedding formalism. For ⟨TT⟩we need to further impose symmetry associated with identical operators alongside tracelessness. Symmetrising gives the two point correlation function of T, ⟨Tµν(x)Tαβ(x′)⟩= 1 s2d " 10 X n=1 fn(ξ1, ξ2)T (n) µν;αβ + 9 X m=1 gm(ξ1, ξ2)S(m) µν;αβ + h1δµνδαβ + h2 δµνΞ′(1) α Ξ′(1) β + δαβΞ(1) µ Ξ(1) ν + h3 δµνΞ′(2) α Ξ′(2) β + δαβΞ(2) µ Ξ(2) ν + h4 δµν(Ξ′(1) α Ξ′(2) β + Ξ′(1) β Ξ′(2) α ) + δαβ(Ξ(1) µ Ξ(2) ν + Ξ(1) ν Ξ(2) µ ) + H Jµνδαβ + δµνJ ′′ αβ # , (3.15) JHEP04(2021)226 + H Jµνδαβ + δµνJ ′′ αβ # , (3.15) + H Jµνδαβ + δµνJ ′′ αβ # , (3.15) (3.15) (3.15) where T (n) µν;αβ, S(n) µν;αβ can be found in (B.2) and (B.3) and the functions h1, . . . , h4 and H satisfy the following relations, where T (n) µν;αβ, S(n) µν;αβ can be found in (B.2) and (B.3) and the functions h1, . . . 3.5 ⟨T T ⟩ , h4 and H satisfy the following relations h1 = −2f1 d + 4ξ2 d2 1 u2 + ξ3 2ξ1 f2 −4ξ3 d2 1 −ξ3 2ξ1 f3 −4ξ3 d2 1 + 1 u2 f4 (3.16a) + f5 u4d2 + ξ2 3 ξ2 2d2 f6 + 2ξ3 ξ2u2d2 f7 − 2ξ3 ξ1u2d2 f8 − 2ξ2 3 ξ1ξ2d2 f9 + ξ2 3f10 ξ2 1d2 + 2 d2 (q −1) + ξ3 ξ2 g1 − ξ3 ξ2 1d2 g2 −4ξ3 ξ2 2d2 g3 + 4ξ3 ξ1ξ2d2 g4 + 2(q −1) u2d2 g5 + 2(q −1)ξ3 ξ2d2 g6 −2(q −1)ξ3 ξ1d2 g7 + (q −1)2 d2 g8 + 4 d2 (q −1) −ξ3 2ξ1 g9, h2 = −4ξ2 d f2 + 4ξ3 d f4 −f5 u2d −ξ3 ξ2df7 + ξ3 ξ1df8 −q −1 d g5, (3.16b) h3 = 4ξ2 d f3 + 4ξ2 d f4 −ξ3 ξ2df6 −f7 u2d + ξ3 ξ1df9 −2g1 d + 4g3 ξ2d −2g4 ξ1d −q −1 d g6, (3.16c) h4 = 2ξ2 d f2 + 2ξ3 d f3 −f8 u2d −ξ3 ξ2df9 + ξ3 ξ1df10 −g2 ξ1d + 2g4 ξ2d −q −1 d g7 −2g9 d , (3.16d) and, H = −2g1 −g5 2 −ξ3 g6 + ξ3 g7 −q −1g8 −4g9. 3.4 ⟨T J⟩ The conservation of Jµ now gives three PDE constraints while the conservation of T µν gives ﬁve PDE constraints. This is a system with 8 functions and 8 PDE relations. It would be interesting to see if the system can be solved. – 11 – 3.5 ⟨T T ⟩ 3.5 ⟨T T ⟩ In this case J , J ′, and J ′′ are not independent and so we set all the gm = 0 and H = 0, leaving us with 14 structures. Furthermore, the traceless constraints also reduce down to four and hence ⟨T µν(x)T λρ(x′)⟩has 10 independent structures. These are given by the T (n) structures in (B.2) via (3.15) subject to (3.16) with gm = H = 0. (The S(m) structures vanish.) Conservation of T µν gives eight PDE constraints, and so we have a system of 10 functions with 8 relations. 3.5 ⟨T T ⟩ (3.17) h1 = −2f1 d + 4ξ2 d2 1 u2 + ξ3 2ξ1 f2 −4ξ3 d2 1 −ξ3 2ξ1 f3 −4ξ3 d2 1 + 1 u2 f4 (3.16a) + f5 u4d2 + ξ2 3 ξ2 2d2 f6 + 2ξ3 ξ2u2d2 f7 − 2ξ3 ξ1u2d2 f8 − 2ξ2 3 ξ1ξ2d2 f9 + ξ2 3f10 ξ2 1d2 + 2 d2 (q −1) + ξ3 ξ2 g1 − ξ3 ξ2 1d2 g2 −4ξ3 ξ2 2d2 g3 + 4ξ3 ξ1ξ2d2 g4 + 2(q −1) u2d2 g5 + 2(q −1)ξ3 ξ2d2 g6 −2(q −1)ξ3 ξ1d2 g7 ( 1)2 4 ξ −ξ2 1d2 g2 −ξ2 2d2 g3 + ξ1ξ2d2 g4 + u2d2 g5 + ξ2d2 g6 − ξ1d2 g7 + (q −1)2 d2 g8 + 4 d2 (q −1) −ξ3 2ξ1 g9, h2 = −4ξ2 d f2 + 4ξ3 d f4 −f5 u2d −ξ3 ξ2df7 + ξ3 ξ1df8 −q −1 d g5, (3.16b) 4ξ 4ξ ξ f ξ 2g 4g 2g q 1 h2 = −4ξ2 d f2 + 4ξ3 d f4 −f5 u2d −ξ3 ξ2df7 + ξ3 ξ1df8 −q −1 d g5, (3.16b) h3 = 4ξ2 d f3 + 4ξ2 d f4 −ξ3 ξ2df6 −f7 u2d + ξ3 ξ1df9 −2g1 d + 4g3 ξ2d −2g4 ξ1d −q −1 d g6, (3.16c) h4 = 2ξ2 d f2 + 2ξ3 d f3 −f8 u2d −ξ3 ξ2df9 + ξ3 ξ1df10 −g2 ξ1d + 2g4 ξ2d −q −1 d g7 −2g9 d , (3.16d) and H = −2 dg1 −g5 u2d −ξ3 ξ2dg6 + ξ3 ξ1dg7 −q −1 d g8 −4 dg9. (3.17) (3.17) There are also additional constraints from conservation of T. This gives twelve PDE constraints and so we have a system with 19 functions and 12 PDE relations. q = 1. In this case Ξ(2) = Ξ′(2) = 0 and the independent structures reduce down to 5 with 2 constraints coming from tracelessness. This leaves a total of 3 independent structures appearing in ⟨T µν(x)T λρ(x′)⟩. Conservation of T µν gives two ODE constraints and so we have a system of 3 functions with 2 ODE relations, reducing the number of independent functions to 1, as discussed long ago in [12]. – 12 – q = 2. 5Although we don’t know what to make of it, it is interesting to note that one can impose a massless free ﬁeld equation on the scalar ﬁeld as well, provided q = 3 and ∆′ = 1. 3.6 Constraints from Maxwell’s equation in d = 4 on ⟨F O⟩and ⟨F F ⟩ When q = 3 we ﬁnd that, q = 3. When q = 3 we ﬁnd that, J ′ µ[α\|J ′ ν\|β] = −2 ξ3 Ξ(2) [µ J ′ ν][βΞ′(2) α] , (3.24) (3.24) and the number of independent structures reduces to nine. and the number of independent structures reduces to nine. and the number of independent structures reduces to nine. q = 2. When q = 2 we can set f6, . . . , f10 to zero due to (2.11). So, focusing on the p = q = 2 case and applying the equation of motion gives four PDE constraints for f1, . . . , f5, JHEP04(2021)226 JHEP04(2021)226 2ξ2 1ξ2f(1,0) 1 −2ξ1ξ2ξ3f(0,1) 1 −2(ξ1 + 2ξ2)f2 + 2ξ2 1 u2 f(1,0) 2 −ξ2ξ3f(0,1) 2 + −2ξ1 ξ2 2 + 3ξ3 f3 −ξ1ξ3 f(1,0) 3 −2f(0,1) 3 = 0, (3.25) (3.25) ξ2 −2ξ2 1ξ2f(1,0) 1 −2ξ1ξ2 2f(0,1) 1 + ξ2f2 + (ξ3 −ξ2 −2ξ1)f3 + 2ξ2 1 u2 f(1,0) 3 −ξ2ξ3f(0,1) 3 −2 2ξ1 ξ2 2 −ξ3 f4 −ξ1ξ3f(1,0) 4 + 2ξ1ξ3f(0,1) 4 = 0, (3.26) (3.26) 2 −2ξ2 1ξ2f(1,0) 2 −2ξ1ξ2 2f(0,1) 2 −2ξ1 ξ2 (1 + ξ2 2)f3 −2ξ2 1ξ2f(1,0) 3 + 2ξ1ξ2ξ3f(0,1) 3 + 2ξ1ξ3f4 −4 ξ1 ξ2 2 −ξ3 f5 −ξ1ξ3 f(1,0) 5 −2f(0,1) 5 = 0, (3.27) 2ξ1ξ2 2 ξ3 f2 + 2ξ1ξ2 2 ξ3 f3 −2ξ2 1ξ2f(1,0) 3 −2ξ1ξ2 2f(0,1) 3 −4ξ1 ξ2 f4 −2ξ2 1ξ2f(1,0) 4 + 2ξ1ξ2ξ3f(0,1) 4 + 2(ξ2 −ξ3)f5 −2ξ2 1 u2 f(1,0) 5 + ξ2ξ3f(0,1) 5 = 0. (3.28) −2ξ2 1ξ2f(1,0) 2 −2ξ1ξ2 2f(0,1) 2 −2ξ1 ξ2 (1 + ξ2 2)f3 −2ξ2 1ξ2f(1,0) 3 + 2ξ1ξ2ξ3f(0,1) 3 + 2ξ1ξ3f4 −4 ξ1 ξ2 2 −ξ3 f5 −ξ1ξ3 f(1,0) 5 −2f(0,1) 5 = 0, (3.27) 2 2 (3.27) 2ξ1ξ2 2 ξ3 f2 + 2ξ1ξ2 2 ξ3 f3 −2ξ2 1ξ2f(1,0) 3 −2ξ1ξ2 2f(0,1) 3 −4ξ1 ξ2 f4 −2ξ2 1ξ2f(1,0) 4 + 2ξ1ξ2ξ3f(0,1) 4 2 (3.2 2ξ1ξ2 2 ξ3 f2 + 2ξ1ξ2 2 ξ3 f3 −2ξ2 1ξ2f(1,0) 3 −2ξ1ξ2 2f(0,1) 3 −4ξ1 ξ2 f4 −2ξ2 1ξ2f(1,0) 4 + 2ξ1ξ2ξ3f(0,1) 4 + 2(ξ2 −ξ3)f5 −2ξ2 1 u2 f(1,0) 5 + ξ2ξ3f(0,1) 5 = 0. (3.28) (3.28) q = 1. 3.6 Constraints from Maxwell’s equation in d = 4 on ⟨F O⟩and ⟨F F ⟩ F O⟩. The correlation function between a Maxwell ﬁeld strength and a scalar is given by, JHEP04(2021)226 ⟨Fµν(x)O(x′)⟩= \|y′\|2−∆′ (s2)2 f Ξ(1) µ Ξ(2) ν −Ξ(1) ν Ξ(2) µ . (3.18) (3.18) Imposing the equation of motion ∂µF µν = 0 and using the identity on (A.3), we obtain two PDE constraints for f, −3ξ3 + 2ξ1 ξ3 ξ2 + q −1 f + ξ1ξ3 f(1,0) −2f(0,1) = 0, (3.19) (3.19) and, (2ξ1(2 −q) + ξ3 −2ξ2) f + 2ξ2 1 u2 f(1,0) −ξ2ξ3f(0,1) = 0. (3.20) (3.20) The PDEs can be solved to ﬁnd,5 The PDEs can be solved to ﬁnd,5 f(ξ1, ξ2) = cξ3 1ξ2 (1 −ξ2 2) q−1 2 (−1 + (2ξ1 + ξ2)2) p+1 2 , (3.21) (3.21) where c is an integration constant, p = d −q and the solution is valid for p = 1, q = 3 and p = 2, q = 2. The combination −1 + (2ξ1 + ξ2)2 in the denominator of the expression diverges at the defect and goes to zero in the coincident limit. (For the case q = 1, Ξ(2) vanishes and the ⟨FO⟩correlator is automatically zero.) ⟨F F ⟩. The correlation function between two Maxwell ﬁeld strength tensors is given by, ⟨F F ⟩. The correlation function between two Maxwell ﬁeld strength tensors is given by, ⟨Fµν(x)Fαβ(x′)⟩= 4 (s2)2 f1 2 Iµ[αIβ]ν+f2Ξ(1) [ν Iµ][αΞ′(1) β] +f3 Ξ(1) [ν Iµ][αΞ′(2) β] +Ξ(2) [ν Iµ][αΞ′(1) β] +f4Ξ(2) [ν Iµ][αΞ′(2) β] +f5Ξ(1) [µ Ξ(2) ν] Ξ′(1) [α Ξ′(2) β] + f6 2 J ′ µ[α\|J ′ ν\|β]+f7Ξ(1) [µ J ′ ν][βΞ′(1) α] +f8 Ξ(1) [µ J ′ ν][βΞ′(2) α] +Ξ(2) [µ J ′ ν][βΞ′(1) α] +f9Ξ(2) [µ J ′ ν][βΞ′(2) α] +f10J ′ [µ\|[αIβ]\|ν] . (3.22) (3.22) Reﬂection positivity demands that, Reﬂection positivity demands that, Reﬂection positivity demands that, 2(f1 + f10)(ξ1, 1) ≥0, 2(f1 + 2f10 + f6)(ξ1, 1) ≥0, 2 f1 + f2 u2 (ξ1, 1) ≥0, 2 f1 + f10 + f2 + f7 u2 (ξ1, 1) ≥0. (3.2 (3.23) 5Although we don’t know what to make of it, it is interesting to note that one can impose a massless free ﬁeld equation on the scalar ﬁeld as well, provided q = 3 and ∆′ = 1. – 13 – – 13 – q = 3. 4 Bulk-defect two point functions One reason to study bulk-defect two point functions is the fact that any bulk primary operator can be expressed as a sum over operators on the defect — the defect OPE. The defect OPE of any bulk primary OI(x) with dimension ∆O can be written as, OI(x, y) = X ˆφJ X ˆ∆ˆφ 1 \|y\|∆O−ˆ∆ˆφ DJ I (⃗∂, y, c(n) O ˆφ, ˆcˆφˆφ)ˆφJ(x), (4.1) (4.1) where ˆφJ is a defect primary with dimension ˆ∆ˆφ and DJ I is a parallel derivative operator depending on the bulk-to-defect coeﬃcients c(n) O ˆφ and the two point coeﬃcient ˆcˆφˆφ of ˆφ. In particular, if the bulk-to-defect coeﬃcients between O and some ˆφ are vanishing, then ˆφ cannot appear in defect OPE of O. To derive the contribution of a particular ˆφK to the defect OPE, we simply take the product with (4.1) and calculate the correlation function, JHEP04(2021)226 1 \|y\|∆O−ˆ∆ˆφ DJ I ⟨ˆφJ(x)ˆφK(x′)⟩= ⟨OI(x)ˆφK(x′)⟩= c(n) O ˆφ \|y\|∆O−ˆ∆ˆφ T(n)IK (s2 + \|y\|2) ˆ∆, (4.2) (4.2) where T(n) are the appropriate tensor structures. By matching the expansion on both sides as y →0, one can in principle determine the DJ I , although our interest is not in these operators.6 Instead, we focus on the precise form of bulk-defect two point functions and the tensor structures T(n). 3.6 Constraints from Maxwell’s equation in d = 4 on ⟨F O⟩and ⟨F F ⟩ When q = 1, only f1 and f2 are present with the usual simpliﬁcations and there is only one ODE constraint, 2ξ2 1 f′ 1 + f′ 2 v2 = 4f2. (3.29) (3.29) We may make this ODE look simpler by changing basis f2 →v2g2 and by changing variables from ξ1 →v. This results in the ODE, v d dv(f1 + g2) = 2g2. Since the coeﬃcient of the derivative terms are equal, we may change basis again to f1 → g1 −g2, which simpliﬁes the ODE further, g2 = v 2 dg1 dv . (3.30) (3.30) A free Maxwell theory in the bulk has only one independent structure for q = 1. A free Maxwell theory in the bulk has only one independent structure for q = 1. – 14 – 6See [10] for the q = 1 case and [1] for q > 1. 4.1 Tensor structures for the defect limit A defect of our formalism, which is not shared by the embedding space method, is that in the absence of a cross ratio, we do not have a procedure for constructing relevant tensor quantities. We saw this issue in the case of the one-point function, which must be constructed out of products of the rank-2 tensor Jµν. We found the tensor Jµν in the process of constraining the two-point functions. The structure Jµν occurs in the contraction of J ′ µα with itself, and the bivector J ′ µα in turn arises as a mixed derivative of the cross ratio ξ2. Despite the fact that there is no cross ratio for the one-point function, having found Jµν, we are free to use it in the construction of one-point functions. The situation in the case of bulk-to-defect two point functions is similar. We have no cross ratio, but again there is a workaround. We can study the defect limit of bulk-bulk two point functions. From this procedure, we recover all of the relevant tensor structures necessary for constructing a general bulk-to-defect two-point function from scratch and the independent structures are given in table 3. We make this claim because there is a one-to- one match of the structures we ﬁnd here to the structures required in embedding space [1]. Here we analyse the limit where y′ = 0. An immediate issue is that the cross ratio ξ2 and several of the tensor structures have an ill behaved y′ →0 limit. We remedy this problem by taking various linear combinations of the cross ratios and multiplying by 6See [10] for the q = 1 case and [1] for q > 1. – 15 – Independent Tensor Structures for Bulk-to-Defect Vectors Bivector 2-Tensor at x at x′ at x at x′ spin = a i (µ, a) (µ, i) (a,b) (a,i) (i,a) (i,j) ˆΞ(1) µ 0 ˆ X ′ i ˆIµa ˆIµi Jµν, δµν δab 0 0 δij Table 3. List of independent tensor structures used to construct bulk-to-defect two point corre- lation functions. The deﬁnition of these structures can be found in (4.5) where we use a hat on the bulk tensor structure to indicate it being evaluated at y′ = 0. For a bulk-to-defect two point correlation function, the tensor structures at x can have bulk Lorentz spin while the structures at x′ can either have parallel and/or orthogonal spin. 4.1 Tensor structures for the defect limit Likewise, a bivector at (x, x′) will have one bulk spin and one parallel or orthogonal spin. Hence we separate the spin combinations in this table. JHEP04(2021)226 appropriate powers of ξ1 and ξ2. A basis with well deﬁned limit as y′ →0 is, appropriate powers of ξ1 and ξ2. A basis with well deﬁned limit as y′ →0 is, Ξ(1) µ , ξ2 ξ1 Ξ(2) µ & X ′ α, ξ2 ξ2 1 Ξ′(2) α , n Iµα, ¯ J ′ µα o , {δµν, Jµν} & n δαβ, ¯ J ′′ αβ o , (4.3) (4.3) for vectors at x and x′, bivectors, and rank two tensors at x and x′ respectively and where X ′ α := ξ2(Ξ′(1) α −Ξ′(2) α ), Iµα := Iµα −Ξ(1) µ X ′ α, ¯ J ′ µα := J ′ µα −Ξ(2) µ X ′ α and ¯ J ′′αβ := J ′′ αβ + Ξ′(1) α Ξ′(1) β . In fact, several of these structures simply vanish or are not independent in the defect limit as y′ →0, ξ2 ξ1 Ξ(2) µ →0 , ξ2 ξ2 1 Ξ′(2) α →0 , X ′ a →0 , ¯ J ′ µa →0 , ¯ J ′ µi →Iµi , ¯ J ′′ A →0 , ¯ J ′′ ij →δij (4.4) where A represents the choices of spin (a, b), (a, i) and (i, a). As in the one-point function case, Jµν is not aﬀected by the limit. Not counting the Kronecker delta’s, the remaining structures reduce to, where A represents the choices of spin (a, b), (a, i) and (i, a). As in the one-point function case, Jµν is not aﬀected by the limit. Not counting the Kronecker delta’s, the remaining structures reduce to, Ξ(1) µ y′=0 =    2\|y\| s2+\|y\|2 sa µ = a, 2\|y\|2 s2+\|y\|2 −1 ni µ = i, X ′ i y′=0 = −ni, Iµb y′=0 =    δab − 2sasb s2+\|y\|2 µ = a, −2\|y\|nisb s2+\|y\|2 µ = i, Iµj y′=0 =    0 µ = a, δij −ninj µ = i. (4.5) (4.5) We represent the bulk-to-defect tensor structures (see table 3) with a hat, e.g. ˆΞ(1) µ = Ξ(1) µ \|y′=0 etc. 7We continue to use this convention throughout this section. 4.1 Tensor structures for the defect limit The ﬁrst constraint is required for a bulk conserved current while the second implies if the dimension of the defect scalar ˆ∆̸= p then cV ˆ O = 0 when V is conserved. In particular, the defect OPE of a bulk conserved current Jµ(x) can only have scalars of dimension p. This reduces to the bCFT result when q = 1 and hence p = d −1. Conserved current. When ∂µV µ(x) = 0, then we get the constraints ∆= d −1 and ˆ∆= d−q = p. The ﬁrst constraint is required for a bulk conserved current while the second implies if the dimension of the defect scalar ˆ∆̸= p then cV ˆ O = 0 when V is conserved. In particular, the defect OPE of a bulk conserved current Jµ(x) can only have scalars of dimension p. This reduces to the bCFT result when q = 1 and hence p = d −1. Bulk limit. We consider the special case where the defect OPE of a bulk operator O(x′, y′) expanded around y →0 has a ﬁnite leading order contribution from only one defect scalar primary ˆO, i.e. O(x, 0) = ˆO(x). For such a case, we can obtain cV ˆ O through the function f1 present in the bulk-bulk correlator ⟨Vµ(x)O(x′)⟩in (3.2). The relation is simply the boundary condition, lim ξ1→∞(4ξ1)∆′f1(ξ1, ξ2) = cV ˆ O, lim ξ1→∞ξ∆′+1 1 ξ−1 2 f2(ξ1, ξ2) = ﬁnite . (4.8) (4.8) Note the limit ξ1 →∞needs to exist independent of ξ2 since ξ2 has an undeﬁned limit as y′ →0. That the structure ξ2ξ−1 1 Ξ(2) µ vanishes in the defect limit places a ﬁniteness constraint on f2. Note the limit ξ1 →∞needs to exist independent of ξ2 since ξ2 has an undeﬁned limit as y′ →0. That the structure ξ2ξ−1 1 Ξ(2) µ vanishes in the defect limit places a ﬁniteness constraint on f2. 4.3 ⟨V ˆV ⟩ 4.1 Tensor structures for the defect limit Similar to the bulk tensor structures (see (2.9) and (2.10)), the set of in- dependent bulk-to-defect tensor structures are also closed under contraction. Leaving out the trivial contractions involving the δ’s we have, We represent the bulk-to-defect tensor structures (see table 3) with a hat, e.g. ˆΞ(1) µ = Ξ(1) µ \|y′=0 etc. Similar to the bulk tensor structures (see (2.9) and (2.10)), the set of in- dependent bulk-to-defect tensor structures are also closed under contraction. Leaving out the trivial contractions involving the δ’s we have, ˆΞ(1) µ ˆΞ(1)µ = ˆ X ′ i ˆ X ′i = 1, ˆΞ(1) µ ˆIµ a = ˆΞ(1) µ ˆIµ i = 0, ˆ X ′iˆIµ i = Jµν ˆIµ a = 0, ˆΞ(1) µ J µ ν = ˆIµiˆIµ a = 0, Jµν ˆIµ i = ˆIνi, ˆIµaˆIµ b = δab, ˆIµiˆI i ν = Jµν, ˆIµiˆIµ j = δij −ˆ X ′ i ˆ X ′ j = ˆIij, ˆIµaˆI a ν = δµν −ˆΞ(1) µ ˆΞ(1) ν −Jµν. (4.6) (4.6) – 16 – We believe we have found all the relevant structures from which to construct the bulk-to- defect correlation function between operators of arbitrary spin. Indeed, in embedding space, ref. [1] construct these same bulk-to-defect two point functions from the ﬁve monomials Qi BD, i = 0, . . . , 4. In our language, Q0 BD maps to ˆIµa, Q1 BD to ˆ X ′ i, Q2 BD to ˆΞ(1) µ , Q3 BD to ˆIµi, and Q4 BD to Jµν. Given this mapping, we can see from a combinatorial point of view that exactly the same set of two-point functions should arise in both cases. In appendix C, we write a selection of bulk two point functions using the basis (4.3) in order to explore certain special cases where the bulk-to-defect coeﬃcient can be obtained from the functions appearing in the bulk correlator. 4.2 ⟨V ˆ O⟩ JHEP04(2021)226 4.2 ⟨V ˆ O⟩ wo point correlation function between any bulk vector and any defect scalar is given by, The two point correlation function between any bulk vector and any defect scalar is g ⟨Vµ(x) ˆO(x′)⟩= cV ˆ O s2 ˆ∆\|y\|∆−ˆ∆ ˆΞ(1) µ (4.7) (4.7) where7 s = x −x′ and s2 = s2 + \|y\|2. Conserved current. When ∂µV µ(x) = 0, then we get the constraints ∆= d −1 and ˆ∆= d−q = p. 4.3 ⟨V ˆV ⟩ If V ′ i (x′, 0) = ˆWi(x′), we have the following boundary conditions on the functions appearing in (C.2), lim ξ1→∞g1(ξ1, ξ2) = cV ˆ W , lim ξ1→∞gr(ξ1, ξ2) = ﬁnite, lim ξ1→∞(g5 + g6)(ξ1, ξ2) = c′ V ˆ W , (4.1 lim ξ1→∞g1(ξ1, ξ2) = cV ˆ W , lim ξ1→∞gr(ξ1, ξ2) = ﬁnite, lim ξ1→∞(g5 + g6)(ξ1, ξ2) = c′ V ˆ W , (4.12) where r ∈{2, 3, 4}. An example can be found in section 5.1. ξ1→∞ ξ1→∞ ξ1→∞ where r ∈{2, 3, 4}. An example can be found in section 5.1. where r ∈{2, 3, 4}. An example can be found in section 5.1. 4 ⟨S ˆ O⟩ 8This result corrects a typo in (2.38) of [1]. 4.3 ⟨V ˆV ⟩ The two point correlation function between any bulk vector and any defect vector with parallel spin is given by, The two point correlation function between any bulk vector and any defect vector with parallel spin is given by, ⟨Vµ(x) ˆVa(x′)⟩= cV ˆV s2 ˆ∆\|y\|∆−ˆ∆ ˆIµa . (4.9) (4.9) Conserved current. When ∂µV µ(x) = 0, we simply get one condition ∆= d −1 independent of ˆ∆. If ˆV is a conserved defect current and hence has ˆ∆= p −1, the only way to satisfy the Ward identity ∂a ˆV a(x) = 0 is to set cV ˆV = 0. In other words, a conserved defect current cannot appear in the defect OPE of any bulk vector. – 17 – Bulk limit. If there exists a ˆV such that V ′ a(x′, 0) = ˆVa(x′) we have the following bound- ary conditions on the functions appearing in (C.2), lim ξ1→∞gs(ξ1, ξ2) = ﬁnite, lim ξ1→∞g5(ξ1, ξ2) = cV ˆV , (4.10) (4.10) where s ∈{1, 2, 3, 4, 6}. To reiterate, ﬁniteness of the basis (4.3) as y′ →0 enables us to put a ﬁniteness condition on the bulk correlation functions directly, for this type of special case. The two point correlation function between a bulk vector and a defect vector with orthogonal spin can also be obtained from (C.2):8 here s ∈{1, 2, 3, 4, 6}. To reiterate, ﬁniteness of the basis (4.3) as y′ →0 enables us to put ﬁniteness condition on the bulk correlation functions directly, for this type of special case. where s ∈{1, 2, 3, 4, 6}. To reiterate, ﬁniteness of the basis (4.3) as y′ →0 enables us to put a ﬁniteness condition on the bulk correlation functions directly, for this type of special case. The two point correlation function between a bulk vector and a defect vector with orthogonal spin can also be obtained from (C.2):8 The two point correlation function between a bulk vector and a defect vector wit orthogonal spin can also be obtained from (C.2):8 ⟨Vµ(x) ˆWj(x′)⟩= 1 s2 ˆ∆\|y\|∆−ˆ∆ cV ˆ W Ξ(1) µ ˆ X ′ j + c′ V ˆ W ˆIµj (4.11) JHEP04(2021)226 (4.11) Conserved current. When ∂µV µ = 0, we get the constraints ∆= d −1 and cV ˆ W ( ˆ∆− p) = c′ V ˆ W (q −1), reducing to one independent coeﬃcient. 4.3 ⟨V ˆV ⟩ In particular when ˆ∆= p and q ̸= 1, we have c′ V ˆ W = 0. When q = 1, the c′ V ˆ W term vanishes since δij −ninj →0, and conservation states cV ˆ W = 0 unless ˆ∆= p = d −1; as expected, this is the same condition as for ⟨J ˆO⟩. Conversely, we see when ˆ∆= ∆= d −1 and q ̸= 1, the Ward identity simply reduces to cV ˆ W = c′ V ˆ W . Conserved current. When ∂µV µ = 0, we get the constraints ∆= d −1 and cV ˆ W ( ˆ∆− p) = c′ V ˆ W (q −1), reducing to one independent coeﬃcient. In particular when ˆ∆= p and q ̸= 1, we have c′ V ˆ W = 0. When q = 1, the c′ V ˆ W term vanishes since δij −ninj →0, and conservation states cV ˆ W = 0 unless ˆ∆= p = d −1; as expected, this is the same condition as for ⟨J ˆO⟩. Conversely, we see when ˆ∆= ∆= d −1 and q ̸= 1, the Ward identity simply reduces to cV ˆ W = c′ V ˆ W . Conserved current. When ∂µV µ = 0, we get the constraints ∆= d −1 and cV ˆ W ( ˆ∆− p) = c′ V ˆ W (q −1), reducing to one independent coeﬃcient. In particular when ˆ∆= p and q ̸= 1, we have c′ V ˆ W = 0. When q = 1, the c′ V ˆ W term vanishes since δij −ninj →0, and conservation states cV ˆ W = 0 unless ˆ∆= p = d −1; as expected, this is the same condition as for ⟨J ˆO⟩. Conversely, we see when ˆ∆= ∆= d −1 and q ̸= 1, the Ward identity simply reduces to cV ˆ W = c′ V ˆ W . Bulk limit. If V ′ i (x′, 0) = ˆWi(x′), we have the following boundary conditions on the functions appearing in (C.2), Bulk limit. 4.4 ⟨S ˆ O⟩ The correlation function between a bulk symmetric and traceless tensor with a defect scalar is given by, The correlation function between a bulk symmetric and traceless tensor with a defect scalar is given by, ⟨Sµν(x) ˆO(x′)⟩= 1 s2 ˆ∆\|y\|∆−ˆ∆ cS ˆO ˆΞ(1) µ ˆΞ(1) ν −1 dδµν + c′ S ˆO Jµν −q −1 d δµν (4.13) (4.13) Energy momentum tensor. When Sµν = Tµν and hence ∂µT µ ν = 0 everywhere in the bulk, we obtain the usual constraint ∆= d and also the relation, Energy momentum tensor. When Sµν = Tµν and hence ∂µT µ ν = 0 everywhere in the bulk, we obtain the usual constraint ∆= d and also the relation, ˆ∆ d (q −1)c′ S ˆ O = − ˆ∆ d (1 −d) + p ! cS ˆ O, (4.14) (4.14) and so we only have one independent coeﬃcient. Similar to the ⟨V ˆV ⟩case we just consid- ered, when q = 1, the structure multiplying c′ S ˆ O is absent and ˆ∆= d (otherwise cS ˆ O = 0) meaning that the defect OPE of Tµν can only have scalars with dimension d (i.e. the bCFT result). Likewise, when ˆ∆= d and q ̸= 1 the Ward identity simply reduces to cS ˆ O = c′ S ˆ O. – 18 – Bulk limit. When O(x′, 0) = ˆO(x′), we have the following boundary conditions on the functions appearing in (C.3), Bulk limit. When O(x′, 0) = ˆO(x′), we have the following boundary conditions on the functions appearing in (C.3), lim ξ1→∞g1(ξ1, ξ2) = cS ˆ O, lim ξ1→∞gn(ξ1, ξ2) = ﬁnite, lim ξ1→∞g4(ξ1, ξ2) = c′ S ˆ O, (4.15) (4.15) where n ∈{2, 3}. We will see a counter example in section 5.1. 4.5 ⟨F ˆV ⟩ reduces to the statement of Coulomb’s Law. Equation of motion. When ∂µF µ ν = 0 in the bulk (i.e. when the Maxwell ﬁeld is free), we get the constraints ∆= d −2 and ˆ∆= p −1 and hence the defect vector has to be a conserved current meaning that the defect OPE of Fµν which is free in the bulk can only contain defect vectors which are conserved. Furthermore, for a Maxwell theory we have ∆= d/2 and we ﬁnd that the equation of motion can only be applied when d = 4. When ˆV is a conserved current for any Fµν, we obtain the simple condition ˆ∆= p −1. Bulk limit. When V ′ a(x′, 0) = ˆVa(x′) we have the following boundary conditions on the functions appearing in (C.4), lim ξ1→∞gm(ξ1, ξ2) = ﬁnite, lim ξ1→∞g3(ξ1, ξ2) = cF ˆV , (4.17) (4.17) where m ∈{1, 2, 4, 5, 6}. where m ∈{1, 2, 4, 5, 6}. ˆ F ⟩ 4.6 ⟨F ˆ F ⟩ 4.6 ⟨F ˆ F ⟩ 4.5 ⟨F ˆV ⟩ The correlation function between a bulk anti-symmetric tensor and a parallel spin defect vector is given by, JHEP04(2021)226 ⟨Fµν(x) ˆVa(x′)⟩= 2cF ˆV s2 ˆ∆\|y\|∆−ˆ∆Ξ(1) [µ ˆIν]a (4.16) (4.16) We observe that for a line defect p = 1 in four dimensions, where we additionally assume ˆVt = ˆJt is a conserved charge, we can interpret ˆJt as the insertion of a charge on the defect. Then the correlation function Then the correlation function ⟨FitJt⟩∼ni \|y\|2 reduces to the statement of Coulomb’s Law. reduces to the statement of Coulomb’s Law. 4.6 ⟨F ˆ F ⟩ Here are some more correlators which we list without further analysis, Here are some more correlators which we list without further analysis, ⟨O(x, y) ˆWj(x′)⟩= − cO ˆ W (s2) ˆ∆\|y\|∆−ˆ∆nj, (4.21a) ⟨O(x, y) ˆSij(x′)⟩= cO ˆS (s2) ˆ∆\|y\|∆−ˆ∆ ninj −δij q , (4.21b) (4.21a) where ˆSij is a traceless and symmetric defect tensor. where ˆSij is a traceless and symmetric defect tensor. 5 Free ﬁeld defect CFT We will now focus on speciﬁc examples of free theories symmetric under the restricted conformal group (2.1). The purpose of looking at a free theory is mostly to study a simple example which has a defect symmetry allowing us to verify the general results in sections 3 and 4. 5.1 Free scalar theory on Rp × (Rq/Z2) 4.6 ⟨F ˆ F ⟩ The correlation function between a bulk anti-symmetric tensor and a defect anti-symmetric orthogonal tensor is given by, The correlation function between a bulk anti-symmetric tensor and a defect anti-symmetric orthogonal tensor is given by, ⟨Fµν(x) ˆFij(x′)⟩= 2 s2 ˆ∆\|y\|∆−ˆ∆ h cF ˆF (ˆIµiˆIνj −ˆIµj ˆIνi) + c′ F ˆF ˆ X ′ i ˆΞ(1) [µ ˆIν]j −ˆ X ′ j ˆΞ(1) [µ ˆIν]i i (4.18) ⟨Fµν(x) ˆFij(x′)⟩= 2 s2 ˆ∆\|y\|∆−ˆ∆ h cF ˆF (ˆIµiˆIνj −ˆIµj ˆIνi) + c′ F ˆF ˆ X ′ i ˆΞ(1) [µ ˆIν]j −ˆ X ′ j ˆΞ(1) [µ ˆIν]i i (4.18) (4.18) – 19 – – 19 – Equation of motion. When ∂µF µ ν = 0 in the bulk, we obtain the condition ∆= d −2 and the relation Equation of motion. When ∂µF µ ν = 0 in the bulk, we obtain the condition ∆= d −2 and the relation, (4.19) c′ F ˆF ( ˆ∆−p) = −2(2 −q)cF ˆF , (4.19) c′ F ˆF ( ˆ∆−p) = −2(2 −q)cF ˆF , leaving only one independent coeﬃcient. When q = 2 and ˆ∆̸= p we see that c′ F ˆF = 0 and also the structure involving cF ˆF vanishes. Hence the correlation function is zero. If instead ˆ∆= p and q ̸= 2, we see that cF ˆF = 0. In the special case when both q = 2 and ˆ∆= p = d −2 = ∆the relation is automatically satisﬁed and we seem to have two independent coeﬃcients. However, since for q = 2 the structures corresponding to cF ˆF vanishes, we only have c′ F ˆF . Finally, when ˆ∆= ∆= d −2, irrespective of q and p, the Ward identity simply reduces to c′ F ˆF = 2cF ˆF . JHEP04(2021)226 Bulk limit. When F ′ ij(x′, 0) = ˆFij(x′), we have the following boundary conditions on the functions appearing in (C.5), lim ξ1→∞gm = ﬁnite, lim ξ1→∞ g1 + g7 2 + g12 = cF ˆF , lim ξ1→∞(g2 + g8) = c′ F ˆF , (4.20) (4.20) where m ∈{3, 4, 5, 6, 9, 10, 11}. An example can be found in section 5.3. where m ∈{3, 4, 5, 6, 9, 10, 11}. An example can be found in section 5.3. where m ∈{3, 4, 5, 6, 9, 10, 11}. An example can be found in section 5.3. 5.1 Free scalar theory on Rp × (Rq/Z2) The parent theory is N massless scalar ﬁelds in d dimensions with the free propagator δAB (s2)∆, where ∆= d 2 −1. We then take an orbifold, where we identify directions normal to the defect y ∼−y. The spacetime becomes Rp × (Rq/Z2), and we are faced with a choice what to do to φA under the orbifold action. Two natural choices are to send φA →±φA. The method of images then produces the propagator ⟨φA(x)φB(x′)⟩= δAB 1 (s2)∆+ λ (˜s2)∆ , (5.1) (5.1) – 20 – where ˜s2 = (x −x′)2 + (y + y′)2 and λ = ±1. In the codimension one case, these choices correspond to more familiar Neumann and Dirichlet boundary conditions. In fact, letting λ ∈R be arbitrary, the correlators have the appropriate symmetry for a dCFT with a p- dimensional defect.9 However, we will see shortly that λ > 0 violates an energy condition. This theory exhibits a global O(N) symmetry giving rise to the bulk conserved current, JAB µ = φA∂µφB −φB∂µφA, (5.2) (5.2) and from translation invariance (in the bulk) we have an improved energy-momentum tensor, JHEP04(2021)226 Tµν = ∂µφA∂νφA −1 2δµν∂αφA∂αφA − d −2 4(d −1)(∂µ∂ν −δµν∂α∂α)φ2, (5.3) (5.3) which has a non-zero one point function respecting the conformal symmetry given by, ⟨Tµν(x)⟩= −λN (2y)d d(d −2) (d −1) Jµν −q −1 d δµν . (5.4) (5.4) Curiously, positive λ is not consistent with the Average Null Energy Condition (ANEC) when q > 1. The ANEC states that the integral of the tangential-tangential component of the stress-tensor along a light-like trajectory must be positive. Appropriately Wick rotating our result to Lorentzian signature, that integral in this case is proportional to λ [14]. Of course in the codimension one case, ⟨Tµν⟩itself vanishes and there is no such constraint. The ANEC was proven to hold assuming a Rd−1,1 space-time [15, 16], and it is not clear that the theorem should hold in this more general orbifolded context. 9At least perturbatively, one can access more general values of λ by including degrees of freedom on the boundary that interact with the scalar [13]. It can be shown that these functions satisfy the conservation PDEs (3.6) and (3.7). 5.1 Free scalar theory on Rp × (Rq/Z2) Using (5.1) we ﬁnd that the two point correlator between the conserved current respects the defect conformal symmetry, ⟨JAB µ (x)JCD ν (x′)⟩= 2 (s2)d−1 f1Ξ(1) µ Ξ′(1) ν + f2Ξ(2) µ Ξ′(2) ν + f3 Ξ(1) µ Ξ′(2) ν + Ξ(2) µ Ξ′(1) ν + f4Iµν + f5J ′ µν (δACδBD −δADδBC), (5.5) (5.5) where, where, f1 = f2 = −f3 = −2λ(d −2)ξ2(u2) d 2 1 + λ(u2) d 2 + d 2(u2 −1) , f4 = (d −2) 1 + λ(u2) d 2 −1 1 + λ(u2) d 2 , f5 = −2λ(d −2) 1 + λ(u2) d 2 −1 (u2) d 2 . (5.6) (5.6) It can be shown that these functions satisfy the conservation PDEs (3.6) and (3.7). 9At least perturbatively, one can access more general values of λ by including degrees of freedom on the boundary that interact with the scalar [13]. – 21 – Defect limit: y′ →0. If we change the basis to the one corresponding to (C.2), the functions gi are given by, Defect limit: y′ →0. If we change the basis to the one corresponding to (C.2), the functions gi are given by, g1 = (d −2) 1 −λ(u2) d 2 −1(u2 −1)(1 + du2) −λ2(u2)d−1(−1 + 2u2) , g4 = −λ(d −2)(u2) d 2 du2 + 2λ(u2) d 2 , g5 = (d −2) 1 + λ(u2) d 2 −1 1 + λ(u2) d 2 , g6 = −2λ(d −2) 1 + λ(u2) d 2 −1 (u2) d 2 , g2 = g3 = 0. (5.7) (5.7) g2 = g3 = 0. JHEP04(2021)226 We see that the gi’s are all ﬁnite as ξ1 →0. This implies the existence of a defect primary ˆWi(x) such that the bulk-to-defect coeﬃcients for ⟨Jµ ˆWi⟩are given by (4.12), cV ˆ W = c′ V ˆ W = (d −2)(1 −λ2). (5.8) (5.8) The Ward identity is also automatically satisﬁed by setting ˆ∆= d −1. So, we see that for this free theory JAB i (x, 0) is a defect primary. Furthermore, reﬂection positivity (3.5) demands that c′ V ˆ W ≥0 and hence we have a bound on λ, that \|λ\| ≤1. V W For this theory, φ2 = φAφA is a bulk scalar primary. 5.1 Free scalar theory on Rp × (Rq/Z2) The one point and two point functions of φ2 obey the defect conformal symmetry and are given by, ⟨φ2(x)⟩= λN (2\|y\|)d−2 , (5.9) (5.9) and and ⟨φ2(x)φ2(x′)⟩= 1 (s2)d−2 h λ2N 2ξd−2 1 + 2N(1 + λud−2)2i . (5.10) (5.10) From this we can read oﬀthe scaling dimension of φ2 to be ∆φ2 = d−2. Using that φ2 is a primary, we can then look at its two point correlation function with the energy-momentum tensor. This two point function is consistent with conformal symmetry and is given by, ⟨Tµν(x)φ2(x′)⟩= −\|y′\|2 s2d g1 Ξ(1) µ Ξ(1) ν −δµν u2d + g2 Ξ(2) µ Ξ(2) ν −δµνξ3 ξ2d + g3 2Ξ(1) (µ Ξ(2) ν) + δµνξ3 ξ1d + g4 Jµν −q −1 d δµν , (5.11) (5.11) where, where, g1 = g2 = −g3 = 4λN d(d −2)2 (d −1) (u2) d 2 +1ξ2 2 , g4 = 4λ2N 2 d(d −2) (d −1) ξd 1. (5.12) (5.12) It can be checked that these functions satisfy the conservation PDEs (3.11) and (3.12) for ⟨TO⟩. This correlator provides a counter example to the relations on (4.15) since g4 diverges. This is due to the presence of the identity in the defect OPE of φ2 whose contribution to the OPE is singular as y →0. Note that the two point correlator with a single JAB will always be zero since the AB indices are antisymmetric. – 22 – 5.2 Free Maxwell theory on a wedge 5.2 Free Maxwell theory on a wedge Next, we consider a free U(1) gauge theory in four dimensions with a two dimensional orbifold defect. The orbifold in this case is obtained by identifying the transverse R2 under rotations by 2π/N for N ≥2 an integer. We parametrise the space R2 × (R2/ZN) by x = (x, y1, y2). In the unorbifolded space, the free propagator in Feynman gauge is ⟨Aµ(x)Aν(x′)⟩= δµν s2 . (5.13) (5.13) We obtain the propagator on the orbifolded space by using the method of images. The sum is complicated to evaluate for general values of N for arbitrary points x and x′. General results for the sum looking at special points exist for the free scalar and spinor ﬁeld theory on a wedge [17, 18], but not to our knowledge for the Maxwell ﬁeld. We will content ourselves by showing this Maxwell theory on a wedge is a (4,2) defect CFT in just two examples, N = 2 and N = 4. JHEP04(2021)226 Unlike the scalar case, the Maxwell ﬁeld has a space-time index on which the rotation acts in a nontrivial fashion. Like the scalar case, we have to decide further if the rotation gives an extra ±1 phase when acting on the Maxwell ﬁeld (the analog of absolute and relative boundary conditions in the q = 1 case). The action of the rotation on the space- time index is straightforward to work out in polar coordinates, ˆx = (x, r, θ). The extra phase we incorporate by introducing real parameters λ and λ′. In fact, we will see that the correlator has the correct dCFT form for general λ and λ′ and not just for the special values ±1. Wedge with N = 2. When N = 2, we are working with a special case, R2 × (R2/Z2), of the orbifold used for the scalar theory above. The propagator on the wedge is given by, Gλ µν(x, x′) = δµν s2 + λMµν ˜s2 , (5.14) (5.14) where Mµν = diag{1, 1, −1, −1}. The correlation function ⟨FF⟩is now speciﬁed by the functions, where Mµν = diag{1, 1, −1, −1}. The correlation function ⟨FF⟩is now speciﬁed by the functions, f1 = 4(1 + λu4), f2 = −8λu6ξ2, f3 = 8λu6ξ2, f4 = −8λu6ξ2 + 8λu4 ξ3 , f5 = −16λu6ξ2 ξ3 , (5.15) (5.15) on (3.22). 5.2 Free Maxwell theory on a wedge (5.19b) (5.19a) (5.19b) The stress tensor one point function takes the usual form The stress tensor one point function takes the usual form ⟨Tµν(x)⟩= −λ + 8λ′ y4 Jµν −1 4δµν . (5.20) (5.20) Consistency with the ANEC would require the weaker constraint λ + 8λ′ ≤0, which eliminates the particular choice λ = −1 and λ′ = 1. Although we don’t explicitly calculate ⟨FF⟩for more general N, we expect this wedge theory to obey the conformal constraints for any positive integer N. The particular func- tions f1, . . . , f5 in (5.15) and (5.18) for the wedge case above can also be shown to satisfy the four PDE constraints in section 3.6. ⟨Tµν(x)Tρσ(x′)⟩. Given (for any N) a free Maxwell theory on a wedge that is conformal, the connected correlation function between two energy momentum tensors can be written using just the functions f1, . . . , f5 appearing in ⟨FF⟩. The result is (D.2). 5.3 Maxwell theory on a R × (R3/Z2) 5.2 Free Maxwell theory on a wedge The free Maxwell theory has a bulk stress tensor, Tµν(x) = F γ µ Fνγ −δµν 4 FαβF αβ, which has a non-zero one point function, on (3.22). The free Maxwell theory has a bulk stress tensor, Tµν(x) = F γ µ Fνγ −δµν 4 FαβF αβ, which has a non-zero one point function, ⟨Tµν(x)⟩= −λ y4 Jµν −1 4δµν . (5.16) (5.16) As for the scalar, this result is consistent with the ANEC only for λ ≤0. As for the scalar, this result is consistent with the ANEC only for λ ≤0. – 23 – Wedge with N = 4. The propagator in this case on R2 × (R2/Z4) is given by the matrix, Wedge with N = 4. The propagator in this case on R2 × (R2/Z4) is given by the matrix, G4 µν =         1 s2 + λ ˜s2 + λ′ s2 + + λ′ s2 − 0 0 0 0 1 s2 + λ ˜s2 + λ′ s2 + + λ′ s2 − 0 0 0 0 1 s2 −λ ˜s2 λ′ s2 + −λ′ s2 − 0 0 −λ′ s2 + + λ′ s2 − 1 s2 −λ ˜s2         , (5.17) (5.17) where s2 + = s2 +(y1 −y′ 2)2 +(y2 +y′ 1)2 and s2 −= s2 +(y1 +y′ 2)2 +(y2 −y′ 1)2. We also deﬁne new functions of the cross ratios, ξ± 3 = 1 2(ξ2 ± q 1 −ξ2 2) and u2 ± = ξ1 ξ1+ξ± 3 . Calculating ⟨FF⟩ we ﬁnd that it again matches the general result on (3.22) with the functions f1, . . . , f5 given by, JHEP04(2021)226 f1 = 4(1 + λu4 + λ′u4 + + λ′u4 −), f2 = −8(λu6ξ2 + λ′u6 +ξ+ 3 + λ′u6 −ξ− 3 ), f3 = −f2 + λ′g(u2 + + u2 −)3, f4 = −f3 + 8λu4 ξ3 + λ′h(u2 + + u2 −)3, f5 = 2f2 ξ3 −2λ′gξ2(u2 + + u2 −)3. (5.18) (5.18) here, where, g = 2 6ξ2 1 + 6ξ1ξ2 + ξ2 2 + 1 (2ξ1 + ξ2)3 , (5.19a) h = −ξ2(ξ1 + ξ2) 4ξ2 1 + 4ξ1ξ2 −2ξ2 2 + 3 ξ1(ξ2 2 −1)(2ξ1 + ξ2)2 . 5.3 Maxwell theory on a R × (R3/Z2) Another way to generalise the propagator (5.14) is to set p = 1 and q = 3. In doing so, we arrive at δ ¯ ¯G2 µν = δµν s2 + λ ¯ Mµν ˜s2 , (5.21) (5.21) – 24 – where ¯ Mµν = diag{1, −1, −1, −1} and our spacetime points are x = (x1, y1, y2, y3). Cal- culating the correlator ⟨FF⟩, we ﬁnd that it respects conformal symmetry and is given by (3.22), where the functions are, where ¯ Mµν = diag{1, −1, −1, −1} and our spacetime points are x = (x1, y1, y2, y3). Cal- culating the correlator ⟨FF⟩, we ﬁnd that it respects conformal symmetry and is given by (3.22), where the functions are, f1 = 4(1 + λu4), f2 = f4 = −f3 = −8λu6ξ2, f5 = f9 = 0, f6 = 16λu4(1 −u2ξ2ξ3), f7 = −f8 = 16λu6ξ2, f10 = −8λu4. (5.22) (5.22) Note that since q = 3, we used (3.24) to remove f9 leaving us with only nine independent structures instead of ten. In this case ⟨Tµν(x)⟩= 0 and so there is no constraint on λ from the ANEC. Note that since q = 3, we used (3.24) to remove f9 leaving us with only nine independent structures instead of ten. In this case ⟨Tµν(x)⟩= 0 and so there is no constraint on λ from the ANEC. JHEP04(2021)226 Defect limit: y′ →0. If we change the basis to the one corresponding to (C.5), the functions gi are given by, g1 = 2(1 + λu4), g2 = 4[1 + λu4(1 −2u2)], g4 = −8λu6, g3 = g5 = g6 = 0, g7 = 16λu4, g8 = 8λu4(2u2 −1), g10 = 16λu6, g12 = −8λu4, g9 = g11 = 0. (5.23) (5.23) g3 = g5 = g6 = 0, We see that the gi’s are ﬁnite as ξ1 →∞. This implies the existence of a defect primary ˆFij(x) such that the bulk-to-defect coeﬃcients for ⟨Fµν ˆFij⟩are given by (4.20), We see that the gi’s are ﬁnite as ξ1 →∞. This implies the existence of a defect primary ˆFij(x) such that the bulk-to-defect coeﬃcients for ⟨Fµν ˆFij⟩are given by (4.20), cF ˆF = 2(1 + λ), c′ F ˆF = 4(1 + λ). 5.3 Maxwell theory on a R × (R3/Z2) (5.24) (5.24) The Ward identity (4.19) is also satisﬁed as it reduces to c′ F ˆF = 2cF ˆF when ∆′ = 2, p = 1 and q = 3. So, for this theory Fij(x, 0) is a defect primary. Reﬂection positivity demands that both cF ˆF and c′ F ˆF are greater than zero, placing a lower bound λ ≥−1. 6 Conclusion and further discussion In this paper we provide the necessary tensor structures, in conﬁguration space, required to construct bulk two-point correlation functions for a conformal ﬁeld theory with a ﬂat defect. In doing so, we ﬁnd the appropriate tensor structures required for constructing any one-point and bulk-to-defect two point function as well. We further examine the conservation constraints on correlators involving a conserved current and the stress tensor. We also looked at the free ﬁeld constraints on correlators involving a Maxwell ﬁeld. These constraints are summarized in table 2. While we did not provide detailed constructions, we believe, based on the discussion in section 2.2, that it is straightforward to extend our result to higher point correlation functions. One simply duplicates the tensor structures in table 1 for each pair of points and uses these as building blocks for the multi-point functions. It would be interesting to explore these higher point cases further, and as a starting point to check that the relevant structures form a complete set, which we did in the two-point case but not in general. – 25 – Given the generally large number of undetermined functions required to specify our two-point functions, the particularly harsh constraints on a couple of our correlation func- tions call out for further analysis. In analyzing ⟨Fµν(x)O(x′)⟩, we found that applying a free ﬁeld constraint to Fµν meant that the correlation function was determined up to a constant for theories with d = 4 and q = 2 or 3. We would like to ﬁnd an example where such a correlation function can be calculated and the result is nontrivial, i.e. not zero. We also found that in codimension q = 2 and for ⟨Tµν(x)Jλ(x′)⟩where Tµν(x) is the stress tensor and Jλ(x′) a conserved current, the correlation function is ﬁxed up to eight functions of two cross ratios that furthermore satisﬁed eight partial diﬀerential equations. In other words, if the correlation function is speciﬁed along a particular slice in cross ratio space, it should generically be deﬁned everywhere through the conservation equations. JHEP04(2021)226 A couple of other results are worthy of remark. We analyzed the constraints of reﬂec- tion positivity on the ⟨Jµ(x)Jν(x′)⟩and ⟨Fµν(x)Fλρ(x′)⟩correlation functions, ﬁnding (3.5) and (3.23). 6 Conclusion and further discussion (The structure of the ⟨Tµν(x)Tλρ(x′)⟩correlator is complicated enough that we leave an analysis of reﬂection postivity of this structure for the future.) We also found that some of our orbifold theories failed to satisfy the ANEC. As the ANEC was proven only for Lorentz invariant theories [15, 16], the result is intriguing but not in violation of the theorem. We have a particular interest in defect theories that are free in the bulk and have interactions conﬁned to the defect. In this set, perhaps the simplest are theories with only a free scalar in the bulk, one example of which we looked at in section 5. The subset of such free scalar theories appears to be very constrained [19, 20], with essentially only the codimension q = 1 case leading to defect theories which are not “trivial”. Equally if not more interesting are defect theories with a free Maxwell ﬁeld in the bulk. Considerable research has been conducted on a codimension q = 1 theory with a free photon in the bulk and charged fermionic matter on the boundary. This theory is sometimes called mixed dimensional or reduced QED and has been used as an “ultra-relativistic limit” of graphene (see for example [21]). In section 5, we looked at a q = 2 and q = 3 “wedge” theory as a prelude to looking at higher codimension theories with charged matter on the defect. Literature suggests that the q = 2 theory with charged matter on the defect is problematic [22, 23] because the eﬀective photon propagator experienced by the matter has a logarithm in it and requires a scale to be well deﬁned. We would like to explore what happens in dimensional regularization, moving slightly away from the q = 2 limit whether conformal defect constraints can be applied. The q = 3 case is also very interesting, for example in the study of Wilson and ’t Hooft lines. A Useful identities for ⟨JO⟩, ⟨JJ⟩, ⟨T O⟩, and ⟨F O⟩ Here we list some identities used in deriving the constraints arising from the conservation equations. ⟨JO⟩. ∂µ Ξ(1)µ (s2)d−1 ! = −2y′ s2d (2ξ1(q −1) + ξ2d), (A.1a) ∂µ Ξ(2)µ (s2)d−1 ! = 2y′ s2d (ξ3(d −1) −2ξ1(ξ3/ξ2 + q −1)). (A.1b) ∂µ Ξ(1)µ (s2)d−1 ! = −2y′ s2d (2ξ1(q −1) + ξ2d), (A.1a) (A.1a) ∂µ Ξ(2)µ (s2)d−1 ! = 2y′ s2d (ξ3(d −1) −2ξ1(ξ3/ξ2 + q −1)). (A.1b) JHEP04(2021)226 ⟨JJ⟩. ⟨JJ⟩. ∂µΞ′(2) ν = 2y′ s2 2ξ1/ξ2J′ µν , ∂µΞ′(1) ν = −2y′ s2 Iµν, ∂µ J′µ ν (s2)d−1 = 2y′ s2d (2ξ1/ξ2 −(d −1)) Ξ′(2) ν , ∂µ Iµ ν (s2)d−1 = 0, (A.2) (A.2) s ∂µΞ′(1) ν = −2y′ s2 Iµν, ( ) ∂µ Iµ ν (s2)d−1 = 0, (A.2) ⟨F O⟩. ∂µ 2 s4 Ξ(1) [µ Ξ(2) ν] = 2\|y′\| s6 −3ξ3 + 2ξ1 ξ3 ξ2 + q −1 Ξ(1) ν + (2ξ1(2 −q) + ξ3 −2ξ2) Ξ(2) ν , (A.3) ⟨F O⟩. ⟨F O⟩. O⟩. ∂µ 2 s4 Ξ(1) [µ Ξ(2) ν] = 2\|y′\| s6 −3ξ3 + 2ξ1 ξ3 ξ2 + q −1 Ξ(1) ν + (2ξ1(2 −q) + ξ3 −2ξ2) Ξ(2) ν , (A.3) (A.3) ⟨T O⟩. ∂µ 1 s2d Ξ(1)µΞ(1) ν −δµ ν u2d = 2\|y′\| s2(d+1) 2ξ1(1 −q) −ξ2 d (d + 2)(d −1) Ξ(1) ν + ξ2 d (d −2) Ξ(2) ν , ∂µ 1 s2d Ξ(2)µΞ(2) ν −δµ ν ξ3 ξ2d = 2\|y′\| s2(d+1) 2ξ1ξ3 ξ2 Ξ(1) ν + ξ3d −4ξ1 ξ3 ξ2 − 1 ξ2 2d + q 2 Ξ(2) ν , ∂µ 1 s2d Ξ(1) (µ Ξ(2) ν) −δµ ν ξ3 2ξ1d = 2\|y′\| s2(d+1) + ξ3 2d(d + 1)(d −2) −ξ1 ξ3 ξ2 + q −1 Ξ(1) ν + ξ3 2 −ξ1q −ξ2 d 1 + d2 2 + 1 ξ2 2 !! Ξ(2) ν ∂µ 1 s2d Jµ ν −(q −1) d δµ ν = 2\|y′\| s2(d+1) 2ξ1(q −1)Ξ(1) ν + ξ2dΞ(2) ν , (A.4) ⟨T O⟩. Acknowledgments We thank K. Ray and V. Schaub for discussion. We also thank E. Lauria, M. Meineri, and E. Trevisani for correspondence. This research was supported in part by the U.K. Science & Technology Facilities Council Grants ST/P000258/1 and ST/T000759/1. C.H. would like to acknowledge a Wolfson Fellowship from the Royal Society. – 26 – A Useful identities for ⟨JO⟩, ⟨JJ⟩, ⟨T O⟩, and ⟨F O⟩ A Useful identities for ⟨JO⟩, ⟨JJ⟩, ⟨T O⟩, and ⟨F O⟩ A Useful identities for ⟨JO⟩, ⟨JJ⟩, ⟨T O⟩, and ⟨F O⟩ ∂µ 1 s2d Ξ(1)µΞ(1) ν −δµ ν u2d = 2\|y′\| s2(d+1) 2ξ1(1 −q) −ξ2 d (d + 2)(d −1) Ξ(1) ν + ξ2 d (d −2) Ξ(2) ν , ∂µ 1 s2d Ξ(2)µΞ(2) ν −δµ ν ξ3 ξ2d = 2\|y′\| s2(d+1) 2ξ1ξ3 ξ2 Ξ(1) ν + ξ3d −4ξ1 ξ3 ξ2 − 1 ξ2 2d + q 2 Ξ(2) ν , (A 4) (A.4) = 2\|y′\| s2(d+1) (A.4) ∂µ 1 s2d Ξ(1) (µ Ξ(2) ν) −δµ ν ξ3 2ξ1d = 2\|y \| s2(d+1) + ξ3 2d(d + 1)(d −2) −ξ1 ξ3 ξ2 + q −1 Ξ(1) ν + ξ3 2 −ξ1q −ξ2 d 1 + d2 2 + 1 ξ2 2 !! Ξ(2) ν ∂µ 1 s2d Jµ ν −(q −1) d δµ ν = 2\|y′\| s2(d+1) 2ξ1(q −1)Ξ(1) ν + ξ2dΞ(2) ν , + ξ3 2d(d + 1)(d −2) −ξ1 ξ3 ξ2 + q −1 Ξ(1) ν + ξ3 2 −ξ1q −ξ2 d 1 + d2 2 + 1 ξ2 2 !! Ξ(2) ν = 2\|y′\| s2(d+1) 2ξ1(q −1)Ξ(1) ν + ξ2dΞ(2) ν , – 27 – B Tensor structures for ⟨T T ⟩ (B.2) S(1) µν;αβ = 2J ′ µ(α\|J ′ ν\|β), S(2) µν;αβ = 4Ξ(1) (µ J ′ ν)(αΞ′(1) β) , S(3) µν;αβ = 4Ξ(2) (µ J ′ ν)(αΞ′(2) β) , S(9) µν;αβ = 4I(µ\|(α\|J ′ \|ν)\|β), S(4) µν;αβ = 4Ξ(1) (µ J ′ ν)(αΞ′(2) β) + 4Ξ(2) (µ J ′ ν)(αΞ′(1) β) , S(5) µν;αβ = Ξ(1) µ Ξ(1) ν J ′′ αβ + JµνΞ′(1) α Ξ′(1) β , S(6) µν;αβ = Ξ(2) µ Ξ(2) ν J ′′ αβ + JµνΞ′(2) α Ξ′(2) β , S(7) µν;αβ = 2Ξ(1) (µ Ξ(2) ν) J ′′ αβ + 2JµνΞ′(1) (α Ξ′(2) β) , S(8) µν;αβ = JµνJ ′′ αβ. (B.3) C Taking the defect limit of ⟨V O⟩, ⟨V V ′⟩, ⟨SO⟩, ⟨F V ′⟩and ⟨F F ′⟩ T (1) µν;αβ = 2Iµ(αIβ)ν, T (2) µν;αβ = 4Ξ(1) (µ Iν)(αΞ′(1) β) , T (3) µν;αβ = 4Ξ(2) (µ Iν)(αΞ′(2) β) , T (5) µν;αβ = Ξ(1) µ Ξ(1) ν Ξ′(1) α Ξ′(1) β , T (6) µν;αβ = Ξ(2) µ Ξ(2) ν Ξ′(2) α Ξ′(2) β , T (4) µν;αβ = 4Ξ(1) (µ Iν)(αΞ′(2) β) + 4Ξ(2) (µ Iν)(αΞ′(1) β) T (7) µν;αβ = Ξ(1) µ Ξ(1) ν Ξ′(2) α Ξ′(2) β + Ξ(2) µ Ξ(2) ν Ξ′(1) α Ξ′(1) β , T (8) µν;αβ = 2Ξ(1) µ Ξ(1) ν Ξ′(1) (α Ξ′(2) β) + 2Ξ(1) (µ Ξ(2) ν) Ξ′(1) α Ξ′(1) β , T (9) µν;αβ = 2Ξ(2) µ Ξ(2) ν Ξ′(1) (α Ξ′(2) β) + 2Ξ(1) (µ Ξ(2) ν) Ξ′(2) α Ξ′(2) β , T (10) µν;αβ = 4Ξ(1) (µ Ξ(2) ν) Ξ′(1) (α Ξ′(2) β) . (B.2) S(1) µν;αβ = 2J ′ µ(α\|J ′ ν\|β), S(2) µν;αβ = 4Ξ(1) (µ J ′ ν)(αΞ′(1) β) , S(3) µν;αβ = 4Ξ(2) (µ J ′ ν)(αΞ′(2) β) , S(9) µν;αβ = 4I(µ\|(α\|J ′ \|ν)\|β), S(4) µν;αβ = 4Ξ(1) (µ J ′ ν)(αΞ′(2) β) + 4Ξ(2) (µ J ′ ν)(αΞ′(1) β) , S(5) µν;αβ = Ξ(1) µ Ξ(1) ν J ′′ αβ + JµνΞ′(1) α Ξ′(1) β , S(6) µν;αβ = Ξ(2) µ Ξ(2) ν J ′′ αβ + JµνΞ′(2) α Ξ′(2) β , S(7) µν;αβ = 2Ξ(1) (µ Ξ(2) ν) J ′′ αβ + 2JµνΞ′(1) (α Ξ′(2) β) , S(8) µν;αβ = JµνJ ′′ αβ. B Tensor structures for ⟨T T ⟩ (B.3) C Taking the defect limit of ⟨V O⟩, ⟨V V ′⟩, ⟨SO⟩, ⟨F V ′⟩and ⟨F F ′⟩ T (4) µν;αβ = 4Ξ(1) (µ Iν)(αΞ′(2) β) + 4Ξ(2) (µ Iν)(αΞ′(1) β) T (7) µν;αβ = Ξ(1) µ Ξ(1) ν Ξ′(2) α Ξ′(2) β + Ξ(2) µ Ξ(2) ν Ξ′(1) α Ξ′(1) β , T (8) µν;αβ = 2Ξ(1) µ Ξ(1) ν Ξ′(1) (α Ξ′(2) β) + 2Ξ(1) (µ Ξ(2) ν) Ξ′(1) α Ξ′(1) β , T (9) µν;αβ = 2Ξ(2) µ Ξ(2) ν Ξ′(1) (α Ξ′(2) β) + 2Ξ(1) (µ Ξ(2) ν) Ξ′(2) α Ξ′(2) β , T (10) µν;αβ = 4Ξ(1) (µ Ξ(2) ν) Ξ′(1) (α Ξ′(2) β) . (B.2) (B.2) S(4) µν;αβ = 4Ξ(1) (µ J ′ ν)(αΞ′(2) β) + 4Ξ(2) (µ J ′ ν)(αΞ′(1) β) , S(5) µν;αβ = Ξ(1) µ Ξ(1) ν J ′′ αβ + JµνΞ′(1) α Ξ′(1) β , S(6) µν;αβ = Ξ(2) µ Ξ(2) ν J ′′ αβ + JµνΞ′(2) α Ξ′(2) β , S(7) µν;αβ = 2Ξ(1) (µ Ξ(2) ν) J ′′ αβ + 2JµνΞ′(1) (α Ξ′(2) β) , S(8) µν;αβ = JµνJ ′′ αβ. (B.3) (B.3) B Tensor structures for ⟨T T ⟩ B Tensor structures for ⟨T T ⟩ The correlation function between two diﬀerent symmetric rank-2 tensors is given by, The correlation function between two diﬀerent symmetric rank-2 tensors is given by, ⟨Sµν(x)S′ αβ(x′)⟩= \|y′\|∆−∆′ s2∆ f1I(µ\|(αIβ)\|ν) + hJµνδαβ + h′′J ′′ αβδµν + Hδµνδαβ + f2[IµαJ ′ νβ + IµβJ ′ να + IναJ ′ µβ + IνβJ ′ µα] + X n≥m;s≥r fnmrsΞ(m) (µ Ξ(n) ν) Ξ′(r) (α Ξ′(s) β) + fmrΞ(m) (µ Iν)(αΞ′(r) β) + FmrΞ(m) (µ J ′ ν)(αΞ′(r) β) + GJµνJ ′′ αβ + X s≥r h′ rsΞ′(r) (α Ξ′(s) β) δµν + X n≥m gmnΞ(m) (µ Ξ(n) ν) J ′′ αβ + X s≥r g′ rsΞ′(r) (α Ξ′(s) β) Jµν + X n≥m hmnΞ(m) (µ Ξ(n) ν) δαβ + fJ ′ (µ(α\|J ′ ν)\|β) ! , (B (B.1) JHEP04(2021)226 where we use the summation convention except when explicitly restricting the sums so that fmnrs has 9 components, gmn, g′ rs, hmn and h′ rs each have 3 components. This is a total of 36 structures. The tensor structures appearing on the correlation function between two stress ten- sors (3.15) are the following, T (1) µν;αβ = 2Iµ(αIβ)ν, T (2) µν;αβ = 4Ξ(1) (µ Iν)(αΞ′(1) β) , T (3) µν;αβ = 4Ξ(2) (µ Iν)(αΞ′(2) β) , T (5) µν;αβ = Ξ(1) µ Ξ(1) ν Ξ′(1) α Ξ′(1) β , T (6) µν;αβ = Ξ(2) µ Ξ(2) ν Ξ′(2) α Ξ′(2) β , T (4) µν;αβ = 4Ξ(1) (µ Iν)(αΞ′(2) β) + 4Ξ(2) (µ Iν)(αΞ′(1) β) T (7) µν;αβ = Ξ(1) µ Ξ(1) ν Ξ′(2) α Ξ′(2) β + Ξ(2) µ Ξ(2) ν Ξ′(1) α Ξ′(1) β , T (8) µν;αβ = 2Ξ(1) µ Ξ(1) ν Ξ′(1) (α Ξ′(2) β) + 2Ξ(1) (µ Ξ(2) ν) Ξ′(1) α Ξ′(1) β , T (9) µν;αβ = 2Ξ(2) µ Ξ(2) ν Ξ′(1) (α Ξ′(2) β) + 2Ξ(1) (µ Ξ(2) ν) Ξ′(2) α Ξ′(2) β , T (10) µν;αβ = 4Ξ(1) (µ Ξ(2) ν) Ξ′(1) (α Ξ′(2) β) . C Taking the defect limit of ⟨V O⟩, ⟨V V ′⟩, ⟨SO⟩, ⟨F V ′⟩and ⟨F F ′⟩ C Taking the defect limit of ⟨V O⟩, ⟨V V ′⟩, ⟨SO⟩, ⟨F V ′⟩and ⟨F F ′⟩ Here we list some of the bulk-bulk correlation functions used for obtaining the bulk-defect correlations function. The only diﬀerence here compared to section 3 is the choice of overall normalisation and the tensor structure basis as mentioned in section 4. – 28 – ⟨V O⟩. The correlation function between any vector (no need to be conserved) and a scalar is given by, ⟨Vµ(x)O(x′)⟩= \|y\|∆′−∆ (s2)∆′ g1Ξ(1) µ + g2 ξ2 ξ1 Ξ(2) µ . (C.1) (C.1) ⟨V V ′⟩. The correlation function between any two distinct vectors is given by, ⟨V V ′⟩. The correlation function between any two distinct vectors is given by, ⟨Vµ(x)V ′ ν(x′)⟩= \|y\|∆′−∆ (s2)∆′ g1Ξ(1) µ X ′ ν + g2 ξ2 2 ξ3 1 Ξ(2) µ Ξ′(2) ν + g3 ξ2 ξ2 1 Ξ(1) µ Ξ′(2) ν + g4 ξ2 ξ1 Ξ(2) µ X ′ ν + g5Iµα + g6 ¯ J ′ µν . (C.2) JHEP04(2021)226 JHEP04(2021)226 (C.2) ⟨SO⟩. The correlation function between any symmetric, traceless tensor and a scalar is given by, ⟨SO⟩. The correlation function between any symmetric, traceless tensor and a scalar is given by, ⟨Sµν(x)O(x′)⟩= \|y\|∆′−∆ (s2)∆′ " g1 Ξ(1) µ Ξ(1) ν −δµν u2d + g2 ξ2 2 ξ2 1 Ξ(2) µ Ξ(2) ν −δµνξ2ξ3 ξ2 1d ! +g3 ξ2 ξ1 Ξ(1) (µ Ξ(2) ν) + δµνξ2ξ3 2ξ2 1d + g4 Jµν −q −1 d δµν # . (C.3) (C.3) ⟨F V ′⟩. The correlation function between any antisymmetric tensor and a vector is given by, ⟨F V ′⟩. The correlation function between any antisymmetric tensor and a vector is given by, ⟨Fµν(x)V ′ α(x′)⟩= \|y\|∆′−∆ (s2)∆′ g1 ξ2 ξ1 Ξ(1) [µ Ξ(2) ν] X ′ α + g2 ξ2 2 ξ3 1 Ξ(1) [µ Ξ(2) ν] Ξ′(2) α + 2g3Ξ(1) [µ Iν]α + g4 ξ2 ξ1 Ξ(2) [µ Iν]α + g5Ξ(1) [µ ¯ J ′ ν]α + g6 ξ2 ξ1 Ξ(2) [µ ¯ J ′ ν]α ! . (C.4) (C.4) ⟨F F ′⟩. The correlation function between any two diﬀerent antisymmetric tensors is given by, ⟨F F ′⟩. C Taking the defect limit of ⟨V O⟩, ⟨V V ′⟩, ⟨SO⟩, ⟨F V ′⟩and ⟨F F ′⟩ The correlation function between any two diﬀerent antisymmetric tensors is given by, ⟨Fµν(x)F ′ αβ(x′)⟩= 4\|y\|∆′−∆ (s2)∆′ " g1Iµ[α\|Iν\|β] + g2Ξ(1) [ν Iµ][αX ′ β] + g3 ξ2 ξ2 1 Ξ(1) [ν Iµ][αΞ′(2) β] + g4 ξ2 ξ1 Ξ(2) [ν Iµ][αX ′ β] + g5 ξ2 2 ξ3 1 Ξ(2) [ν Iµ][αΞ′(2) β] + g6 ξ2 2 ξ3 1 Ξ(1) [µ Ξ(2) ν] X ′ [αΞ′(2) β] + g7 2 ¯ J ′ µ[α\| ¯ J ′ ν\|β] + g8Ξ(1) [µ ¯ J ′ ν][βX ′ α] + g9 ξ2 ξ2 1 Ξ(1) [µ ¯ J ′ ν][βΞ′(2) α] + g10 ξ2 ξ1 Ξ(2) [µ ¯ J ′ ν][βX ′ α] + g11 ξ2 2 ξ3 1 Ξ(2) [µ ¯ J ′ ν][βΞ′(2) α] + g12 ¯ J ′ [µ[α\|Iν]\|β] # . (C.5) (C.5) – 29 – D ⟨T T ⟩in the free Maxwell theory for d = 4, q = 2 D ⟨T T ⟩in the free Maxwell theory for d = 4, q = 2 g5 = 2(d −4 + 2ξ2 2)f2 2 + 2ξ2 3 ξ2 2 f2 5 + 4ξ2 3f2 3 −4ξ2 3f1f5 −2ξ3 ξ3 ξ1 + 2 f2f5 g5 = 2(d −4 + 2ξ2 2)f2 2 + 2ξ2 3 ξ2 2 f2 5 + 4ξ2 3f2 3 −4ξ2 3f1f5 −2ξ3 ξ3 ξ1 + 2 f2f5 −8ξ2ξ3f2f3 + 8ξ2 3 ξ2 f3f5, 2 g6 = 2f2 5 u4 + 2(d −4 + 2ξ2 2)f2 4 + 4ξ2 2f2 3 −4ξ2 2f1f5 + 2ξ2 2 u2 −ξ3 ξ1 f4f5 + 8ξ2 2f3f4 + 8ξ2 u2 f3f5, + 8ξ2 2f3f4 + ξ2 u2 f3f5, g7 = 2ξ3 ξ2u2 f2 5 + 2(d −4 + 2ξ2ξ3)f2 3 −4ξ2 2f1f5 −4ξ2 2f2f4 −4ξ2 u2 f2f5 + 4ξ3f4f5 −4ξ2 2f2f3 + 4ξ2ξ3f3f4 + 4ξ3 1 + ξ2 2ξ1 f3f5, g8 = −2ξ2 2f2 2 + ξ2 3 ξ1ξ2 f2 5 −4ξ2ξ3f1f5 −2ξ2ξ3f2f4 −3ξ2ξ3 ξ1 f2f5 + 2ξ2 3 ξ2 f4f5 + 2ξ2 3f3f4 + 2(d −3 −ξ2 2)f2f3 + 2ξ3 1 + ξ3 2ξ1 f3f5, g9 = ξ3 ξ1u2 f2 5 + 2ξ2ξ3f2 4 + 4ξ2 2f1f5 + 2ξ2 2f2f4 + 2ξ2 u2 f2f5 + 3ξ2ξ3 ξ1 f4f5 + 2ξ2 2f2f3 + 2(d −3 −ξ2 2)f3f4 + 2ξ2 ξ3 2ξ1 −1 u2 f3f5, g10 = ξ2 2f2 2 + ξ2 3 2ξ2 1 f2 5 + ξ2 3f2 4 + (4 −d)f2 3 −2ξ2(ξ2 −ξ3)f1f5 + (d −4 + 2ξ2ξ3)f2f4 + ξ2 3ξ3 2ξ1 −1 u2 f2f5 + ξ3 3ξ3 2ξ1 + 1 f4f5 + ξ3 ξ2 ξ1 −2 u2 f3f5, (D g7 = 2ξ3 ξ2u2 f2 5 + 2(d −4 + 2ξ2ξ3)f2 3 −4ξ2 2f1f5 −4ξ2 2f2f4 −4ξ2 u2 f2f5 + 4ξ3f4f5 −4ξ2 2f2f3 + 4ξ2ξ3f3f4 + 4ξ3 1 + ξ2 2ξ1 f3f5, JHEP04(2021)226 (D.3) and the functions h1, . . . , h4 are obtained from the traceless condition (3.16). E Embedding space to physical space D ⟨T T ⟩in the free Maxwell theory for d = 4, q = 2 Given that the two point correlation function of the ﬁeld strength Fµν obeys the defect conformal symmetry (3.22), ⟨Fµν(x)Fαβ(x′)⟩= 4 (s2)2 f1I[µ\|[αIβ]\|ν] + f2Ξ(1) [ν Iµ][αΞ′(1) β] +f3 Ξ(1) [ν Iµ][αΞ′(2) β] + Ξ(2) [ν Iµ][αΞ′(1) β] + f4Ξ(2) [ν Iµ][αΞ′(2) β] + f5Ξ(1) [ν Ξ(2) µ] Ξ′(1) [α Ξ′(2) β] , (D.1) (D.1) JHEP04(2021)226 and given the bulk Maxwell energy momentum tensor Tµν(x) = F γ µ Fνγ −δµν 4 FαβF αβ, we ﬁnd the two point correlation function of Tµν is given by (3.15), and given the bulk Maxwell energy momentum tensor Tµν(x) = F γ µ Fνγ −δµν 4 FαβF αβ, we ﬁnd the two point correlation function of Tµν is given by (3.15), ⟨Tµν(x)Tαβ(x′)⟩= 1 s8 " 10 X n=1 gn(ξ1,ξ2)T (n) µν;αβ+h1δµνδαβ +h2 δµνΞ′(1) α Ξ′(1) β +δαβΞ(1) µ Ξ(1) ν +h3 δµνΞ′(2) α Ξ′(2) β +δαβΞ(2) µ Ξ(2) ν +h4 δµν(Ξ′(1) α Ξ′(2) β +Ξ′(1) β Ξ′(2) α )+δαβ(Ξ(1) µ Ξ(2) ν +Ξ(1) ν Ξ(2) µ ) # , (D.2) (D.2) where, g1 = (d −2)f2 1 + f2 2 u4 + ξ2 3 ξ2 2 f2 4 + ξ3 2 ξ2u2 + ξ3 2ξ2 1 f2 3 + 2ξ2 1 u2 + ξ3 2ξ1 f1f2 + 2ξ3 ξ3 2ξ1 −1 f1f4 + ξ2 3 2ξ2 1 f2f4 −4ξ3 ξ2 2ξ1 + 1 f1f3 −2ξ3 ξ1u2 f2f3 −2ξ2 3 ξ1ξ2 f3f4 g2 = ξ2 ξ3 2ξ1 −1 u2 f2 2 −ξ3 2 + ξ3 ξ1 f2 3 + (d −3 −ξ2 2)f1f2 −ξ2 3f1f4 + ξ3 ξ3 2ξ1 −1 f1f5 −ξ3 1 + ξ3 2ξ1 f2f4 + ξ2 3 4ξ2 1 f2f5 + ξ2 3 ξ2 2 f4f5 + 2ξ2ξ3f1f3 + 3ξ2ξ3 ξ1 f2f3 + 2ξ2 3 ξ2 f3f4 −ξ2 3 ξ1ξ2 f3f5, g3 = ξ3 ξ3 2ξ1 + 1 f2 4 + ξ2 2 u2 −ξ3 ξ1 f2 3 −ξ2 2f1f2 + (d −3 −ξ2 2)f1f4 + ξ2 1 u2 + ξ3 2ξ1 f1f5 + ξ2 1 u2 −ξ3 2ξ1 f2f4 + f2f5 u4 + ξ2 3 4ξ2 1 f4f5 −2ξ2 2f1f3 + 2ξ2 u2 f2f3 −3ξ2ξ3 ξ1 f3f4 − ξ3 ξ1u2 f3f5, g4 = ξ2 u2 f2 2 + ξ2 3 ξ2 f2 4 + ξ2 2f1f2 −ξ2ξ3f1f4 + ξ3 ξ2 2ξ1 + 1 f1f5 + ξ3 2ξ1u2 f2f5 + ξ2 3 2ξ1ξ2 f4f5 + (d −3 + ξ2 2 −ξ2ξ3)f1f3 + ξ2 1 u2 −ξ3 2ξ1 f2f3 −ξ3 1 + ξ3 2ξ1 f3f4 −ξ3 ξ3 4ξ2 1 + 1 ξ2u2 f3f5, g1 = (d −2)f2 1 + f2 2 u4 + ξ2 3 ξ2 2 f2 4 + ξ3 2 ξ2u2 + ξ3 2ξ2 1 f2 3 + 2ξ2 1 u2 + ξ3 2ξ1 f1f2 + 2ξ3 ξ3 2ξ1 −1 f1f4 + ξ2 3 2ξ2 1 f2f4 −4ξ3 ξ2 2ξ1 + 1 f1f3 −2ξ3 ξ1u2 f2f3 −2ξ2 3 ξ1ξ2 f3f4, g2 = ξ2 ξ3 2ξ1 −1 u2 f2 2 −ξ3 2 + ξ3 ξ1 f2 3 + (d −3 −ξ2 2)f1f2 −ξ2 3f1f4 + ξ3 ξ3 2ξ1 −1 f1f5 −ξ3 1 + ξ3 2ξ1 f2f4 + ξ2 3 4ξ2 1 f2f5 + ξ2 3 ξ2 2 f4f5 + 2ξ2ξ3f1f3 2 2 g3 = ξ3 ξ3 2ξ1 + 1 f2 4 + ξ2 2 u2 −ξ3 ξ1 f2 3 −ξ2 2f1f2 + (d −3 −ξ2 2)f1f4 + ξ2 1 u2 + ξ3 2ξ1 f1f5 + ξ2 1 u2 −ξ3 2ξ1 f2f4 + f2f5 u4 + ξ2 3 4ξ2 1 f4f5 −2ξ2 2f1f3 + 2ξ2 u2 f2f3 −3ξ2ξ3 ξ1 f3f4 − ξ3 ξ1u2 f3f5, g4 = ξ2 u2 f2 2 + ξ2 3 ξ2 f2 4 + ξ2 2f1f2 −ξ2ξ3f1f4 + ξ3 ξ2 2ξ1 + 1 f1f5 + ξ3 2ξ1u2 f2f5 + ξ2 3 2ξ1ξ2 f4f5 + (d −3 + ξ2 2 −ξ2ξ3)f1f3 + ξ2 1 u2 −ξ3 2ξ1 f2f3 −ξ3 1 + ξ3 2ξ1 f3f4 −ξ3 ξ3 4ξ2 1 + 1 ξ2u2 f3f5, – 30 – g5 = 2(d −4 + 2ξ2 2)f2 2 + 2ξ2 3 ξ2 2 f2 5 + 4ξ2 3f2 3 −4ξ2 3f1f5 −2ξ3 ξ3 ξ1 + 2 f2f5 −8ξ2ξ3f2f3 + 8ξ2 3 ξ2 f3f5, g6 = 2f2 5 u4 + 2(d −4 + 2ξ2 2)f2 4 + 4ξ2 2f2 3 −4ξ2 2f1f5 + 2ξ2 2 u2 −ξ3 ξ1 f4f5 + 8ξ2 2f3f4 + 8ξ2 u2 f3f5, g7 = 2ξ3 ξ2u2 f2 5 + 2(d −4 + 2ξ2ξ3)f2 3 −4ξ2 2f1f5 −4ξ2 2f2f4 −4ξ2 u2 f2f5 + 4ξ3f4f5 −4ξ2 2f2f3 + 4ξ2ξ3f3f4 + 4ξ3 1 + ξ2 2ξ1 f3f5, g8 = −2ξ2 2f2 2 + ξ2 3 ξ1ξ2 f2 5 −4ξ2ξ3f1f5 −2ξ2ξ3f2f4 −3ξ2ξ3 ξ1 f2f5 + 2ξ2 3 ξ2 f4f5 + 2ξ2 3f3f4 + 2(d −3 −ξ2 2)f2f3 + 2ξ3 1 + ξ3 2ξ1 f3f5, g9 = ξ3 ξ1u2 f2 5 + 2ξ2ξ3f2 4 + 4ξ2 2f1f5 + 2ξ2 2f2f4 + 2ξ2 u2 f2f5 + 3ξ2ξ3 ξ1 f4f5 + 2ξ2 2f2f3 + 2(d −3 −ξ2 2)f3f4 + 2ξ2 ξ3 2ξ1 −1 u2 f3f5, g10 = ξ2 2f2 2 + ξ2 3 2ξ2 1 f2 5 + ξ2 3f2 4 + (4 −d)f2 3 −2ξ2(ξ2 −ξ3)f1f5 + (d −4 + 2ξ2ξ3)f2f4 + ξ2 3ξ3 2ξ1 −1 u2 f2f5 + ξ3 3ξ3 2ξ1 + 1 f4f5 + ξ3 ξ2 ξ1 −2 u2 f3f5, (D. E Embedding space to physical space We project the embedding space building blocks for symmetric traceless bulk-bulk two point functions to physical space, Q1 BB →ξ2Ξ(2) µ , Q2 BB →−2ξ1ξ2 Ξ(1) µ −Ξ(2) µ , Q3 BB →−2ξ1Ξ′(1) α −ξ2Ξ′(2) α , Q4 BB →−2ξ1ξ2 Ξ′(1) α −Ξ′(2) α , Q5 BB →J ′ µα + ξ2Ξ(2) µ Ξ′(2) α , Q6 BB →ξ2 Iµα −J ′ µα −2ξ1ξ2 Ξ(1) µ −Ξ(2) µ Ξ′(1) α , Q7 BB → q −1 d δµν −Jµν , Q8 BB → q −1 d δαβ −J ′′ αβ . (E.1) (E.1) The Qk BB’s are given by equation (3.18) of [1]. The Qk BB’s are given by equation (3.18) of [1]. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. – 31 – References [1] M. Billò, V. Gonçalves, E. Lauria and M. Meineri, Defects in conformal ﬁeld theory, JHEP 04 (2016) 091 [arXiv:1601.02883] [INSPIRE]. [1] M. Billò, V. Gonçalves, E. Lauria and M. Meineri, Defects in conformal ﬁeld theory, JHEP 04 (2016) 091 [arXiv:1601.02883] [INSPIRE]. [2] E. Lauria, M. Meineri and E. Trevisani, Radial coordinates for defect CFTs, JHEP 11 (2018) 148 [arXiv:1712.07668] [INSPIRE]. [2] E. Lauria, M. Meineri and E. Trevisani, Radial coordinates for defect CFTs, JHEP 11 (2018) 148 [arXiv:1712.07668] [INSPIRE]. [3] N. Kobayashi and T. Nishioka, Spinning conformal defects, JHEP 09 (2018) 134 [arXiv:1805.05967] [INSPIRE]. [4] S. Guha and B. Nagaraj, Correlators of Mixed Symmetry Operators in Defect CFTs, JHEP 10 (2018) 198 [arXiv:1805.12341] [INSPIRE]. [4] S. Guha and B. Nagaraj, Correlators of Mixed Symmetry Operators in Defect CFTs, JHEP 10 (2018) 198 [arXiv:1805.12341] [INSPIRE]. JHEP04(2021)226 [5] E. Lauria, M. Meineri and E. Trevisani, Spinning operators and defects in conformal ﬁeld theory, JHEP 08 (2019) 066 [arXiv:1807.02522] [INSPIRE]. [6] P.A.M. Dirac, Wave equations in conformal space, Annals Math. 37 (1936) 429 [INSPIRE]. [7] S. Weinberg, Six-dimensional Methods for Four-dimensional Conformal Field Theories, Phys. Rev. D 82 (2010) 045031 [arXiv:1006.3480] [INSPIRE]. [8] M.S. Costa, J. Penedones, D. Poland and S. Rychkov, Spinning Conformal Correlators, JHEP 11 (2011) 071 [arXiv:1107.3554] [INSPIRE]. [9] M.S. Costa and T. Hansen, Conformal correlators of mixed-symmetry tensors, JHEP 02 (2015) 151 [arXiv:1411.7351] [INSPIRE]. [10] D.M. McAvity and H. Osborn, Conformal ﬁeld theories near a boundary in general dimensions, Nucl. Phys. B 455 (1995) 522 [cond-mat/9505127] [INSPIRE]. [11] Mathematica 12.1, Wolfram Inc. (2020). [11] Mathematica 12.1, Wolfram Inc. (2020). [12] D.M. McAvity and H. Osborn, Energy momentum tensor in conformal ﬁeld theories near a boundary, Nucl. Phys. B 406 (1993) 655 [hep-th/9302068] [INSPIRE]. [13] C.P. Herzog and K.-W. Huang, Boundary Conformal Field Theory and a Boundary Central Charge, JHEP 10 (2017) 189 [arXiv:1707.06224] [INSPIRE]. [14] K. Jensen, A. O’Bannon, B. Robinson and R. Rodgers, From the Weyl Anomaly to Entropy of Two-Dimensional Boundaries and Defects, Phys. Rev. Lett. 122 (2019) 241602 [arXiv:1812.08745] [INSPIRE]. [15] T. Faulkner, R.G. Leigh, O. Parrikar and H. Wang, Modular Hamiltonians for Deformed Half-Spaces and the Averaged Null Energy Condition, JHEP 09 (2016) 038 [arXiv:1605.08072] [INSPIRE]. [16] T. Hartman, S. Kundu and A. Tajdini, Averaged Null Energy Condition from Causality, JHEP 07 (2017) 066 [arXiv:1610.05308] [INSPIRE]. [17] C.P. Herzog and J. [21] A.V. Kotikov and S. Teber, Two-loop fermion self-energy in reduced quantum electrodynamics and application to the ultrarelativistic limit of graphene, Phys. Rev. D 89 (2014) 065038 [arXiv:1312.2430] [INSPIRE]. [22] E.V. Gorbar, V.P. Gusynin and V.A. Miransky, Dynamical chiral symmetry breaking on a brane in reduced QED, Phys. Rev. D 64 (2001) 105028 [hep-ph/0105059] [INSPIRE]. References Nian, Thermal corrections to Rényi entropies for conformal ﬁeld theories, JHEP 06 (2015) 009 [arXiv:1411.6505] [INSPIRE]. [18] C.P. Herzog and M. Spillane, Thermal corrections to Rényi entropies for free fermions, JHEP 04 (2016) 124 [arXiv:1506.06757] [INSPIRE]. [19] E. Lauria, P. Liendo, B.C. Van Rees and X. Zhao, Line and surface defects for the free scalar ﬁeld, JHEP 01 (2021) 060 [arXiv:2005.02413] [INSPIRE]. [20] C. Behan, L. Di Pietro, E. Lauria and B.C. Van Rees, Bootstrapping boundary-localized interactions, JHEP 12 (2020) 182 [arXiv:2009.03336] [INSPIRE]. – 32 – [21] A.V. Kotikov and S. Teber, Two-loop fermion self-energy in reduced quantum electrodynamics and application to the ultrarelativistic limit of graphene, Phys. Rev. D 89 (2014) 065038 [arXiv:1312.2430] [INSPIRE]. [22] E.V. Gorbar, V.P. Gusynin and V.A. Miransky, Dynamical chiral symmetry breaking on a brane in reduced QED, Phys. Rev. D 64 (2001) 105028 [hep-ph/0105059] [INSPIRE]. [23] M. Heydeman, C.B. Jepsen, Z. Ji and A. Yarom, Renormalization and conformal invariance of non-local quantum electrodynamics, JHEP 08 (2020) 007 [arXiv:2003.07895] [INSPIRE]. JHEP04(2021)226 – 33 –
https://openalex.org/W3034055129	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0233981&type=printable	English	null	Intra-aneurysmal pressure changes during stent-assisted coiling	PloS one	2,020	cc-by	5,483	Piotr Piasecki, Piotr Ziecina, Krzysztof Brzozowski, Marek Wierzbicki, Jerzy NarlochID* D t t f I t ti l R di l Milit I tit t f M di i W P l d Department of Interventional Radiology, Military Institute of Medicine, Warsaw, Poland * jerzy.narloch@gmail.com PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Piasecki P, Ziecina P, Brzozowski K, Wierzbicki M, Narloch J (2020) Intra-aneurysmal pressure changes during stent-assisted coiling. PLoS ONE 15(6): e0233981. https://doi.org/ 10.1371/journal.pone.0233981 Citation: Piasecki P, Ziecina P, Brzozowski K, Wierzbicki M, Narloch J (2020) Intra-aneurysmal pressure changes during stent-assisted coiling. PLoS ONE 15(6): e0233981. https://doi.org/ 10.1371/journal.pone.0233981 Editor: Raffaele Serra, University Magna Graecia of Catanzaro, ITALY Editor: Raffaele Serra, University Magna Graecia of Catanzaro, ITALY Received: January 7, 2020 Accepted: May 16, 2020 Published: June 4, 2020 Received: January 7, 2020 Accepted: May 16, 2020 Published: June 4, 2020 Received: January 7, 2020 Accepted: May 16, 2020 Published: June 4, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0233981 Copyright: © 2020 Piasecki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract We aimed to examine aneurysm hemodynamics with intra-saccular pressure measurement, and compare the effects of coiling, stenting and stent-assisted coiling in proximal segments of intracranial circulation. A cohort of 45 patients underwent elective endovascular coil embolization (with or without stent) for intracranial aneurysm at our department. Arterial pressure transducer was used for all measurements. It was attached to proximal end of the microcatheter. Measurements were taken in the parent artery before and after embolization, at the aneurysm dome before embolization, after stent implantation, and after embolization. Stent-assisted coiling was performed with 4 different stents: LVIS and LVIS Jr (Microven- tion, Tustin, CA, USA), Leo (Balt, Montmorency, France), Barrel VRD (Medtronic/ Covidien, Irvine, CA, USA). Presence of the stent showed significant reverse correlation with intra- aneurysmal pressure–both systolic and diastolic—after its implantation (r = -0.70 and r = -0.75, respectively), which was further supported by correlations with stent cell size–r = 0.72 and r = 0.71, respectively (P<0.05). Stent implantation resulted in significant decrease in diastolic intra-aneurysmal pressure (p = 0.046). Systolic or mean intra-aneurysmal pressure did not differ significantly. Embolization did not significantly change the intra-aneurysmal pressure in matched pairs, regardless of the use of stent (p>0.05). In conclusion, low-profile braided stents show a potential to divert blood flow, there was significant decrease in dia- stolic pressure after stent placement. Flow-diverting properties were related to stent poros- ity. Coiling does not significantly change the intra-aneurysmal pressure, regardless of packing density. PLOS ONE PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling Piotr Piasecki, Piotr Ziecina, Krzysztof Brzozowski, Marek Wierzbicki, Jerzy NarlochID* Introduction Endovascular coiling has an established position in treatment of intracranial aneurysms. [1] Self-expandable stents extended the indications for endovascular therapy for wide-necked and complex aneurysms, which were not amenable to coiling [2, 3]. Placement of the stents across the aneurysm neck provides the scaffold for the coils, stabilizing their position and preventing the protrusion into the parent artery. Stent struts cover the aneurysm orifice, and modify the inflow of blood into the sac. This effect is thought to be dependent on strut density and the degree of blood inflow impairment [4–6]. Recently, low-profile, self-expandable, braided Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. 1 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. intracranial stents (LEO Baby (Balt, Montmorency, France) and LVIS Jr. (MicroVention, Tus- tin, California)) have been used in monotherapy of distally located small aneurysms [7, 8]. There is also a limited number of case series on the application of stent monotherapy in treat- ment of aneurysms located proximal to the circle of Willis [9–12]. Hemodynamic effects were assessed by angiography alone by computational fluid dynamics simulations (CFD) [7–12]. In this study, we aimed to examine aneurysm hemodynamics with intra-saccular pressure measurement, and compare the effects of coiling, stenting and stent-assisted coiling in proxi- mal segments of intracranial circulation. Materials and methods A cohort of 45 patients underwent elective endovascular coil embolization (with or without stent) for intracranial aneurysm at our department. Patient data regarding demographics, angiography and hemodynamics were evaluated retrospectively. Informed consent was obtained from all patients. The study was approved by Institutional Review Board—Bioethical Committee of Military Institute of Medicine (decision 43/WIM/2011). Arterial pressure transducer was used for all measurements. It was attached to proximal end of the microcatheter. Before the measurement the transducer was zeroed at the level of the right atrium. The microcatheter was carefully positioned under fluoroscopic guidance to avoid adherence to or damage to the vessel wall; measurements were taken in the parent artery before and after embolization, at the aneurysm dome before embolization, after stent implan- tation, and after embolization. Measurements were recorded, as soon as the read values stabi- lized (i.e. average of 30 seconds). Simultaneous recording of systemic blood pressure and heart rate were taken. Stent-assisted coiling was performed with 4 different stents: LVIS and LVIS Jr (Microvention, Tustin, CA, USA), Leo (Balt, Montmorency, France), Barrel VRD (Medtronic/ Covidien, Irvine, CA, USA). During the procedure, all patients were under general anesthesia with mean systemic blood pressure maintained between 65 and 100 mmHg. Systemic heparinization was achieved by weight-adjusted intravenous bolus and maintenance dose of 1000 IU of unfractioned heparin/ 1 hour. A combination of 8-Fr introducer sheath and a 6-Fr guiding catheter, or a 7 -Fr long sheath with a 5- Fr guiding catheter were used. A standard 1.7-Fr coiling microcatheter was used for aneurysm coil embolization and pressure monitoring (Headway 17 (Microvention, Tustin, CA, USA) or Echelon 10 (Medtronic, Dublin Ireland)). A stent implantation was performed with a dedicated 2 Fr microcatheter–it was not utilized for pressure monitoring. Descriptive statistics of all variables are presented. Wilcoxon signed-rank test was used to compare differences between matched samples. For unmatched samples Mann-Whitney U test was used. Alpha<0.05 was considered significant. All relevant data are within the manu- script and its S1 File. Competing interests: The authors have declared that no competing interests exist. ICA–internal carotid artery, MCA–middle cerebral artery, BA–basilar artery, ACA–anterior cerebral artery, Dmax–length of aneurysm sac, delta angle–angle formed between the axis of proximal part of parent artery and Dmax https://doi.org/10.1371/journal.pone.0233981.t002 between the axis of proximal part of parent artery and Dmax https://doi.org/10.1371/journal.pone.0233981.t002 ICA–internal carotid artery, MCA–middle cerebral artery, BA–basilar artery, ACA–anterior cerebral artery, Dmax–length of aneurysm sac, delta angle–angle formed between the axis of proximal part of parent artery and Dmax https://doi.org/10.1371/journal.pone.0233981.t002 Results Intra-aneurysmal pressure measurements were performed in 45 patients (4 men and 41 women, aged 67.3±4.6 years and 58.2±13.6 years, respectively). Descriptive statistics regarding intra-aneurysmal pressure and aneurysm morphology were collected in Tables 1 and 2. For schematic morphological measurements please refer to Fig 1. Mean systemic blood pressure was 105.7±15.4 mmHg in systole and 62.7±10.5 mmHg in diastole; mean heart rate was 66±8/ min. Locations of treated aneurysms were: (1) internal carotid artery (ICA)– 17 cases of right ICA and 17 cases of left ICA, total 76%; (2) basilar artery (BA)– 6 cases, 13%; (3) anterior cere- bral artery (ACA)– 3 cases, 6%; and (4) middle cerebral artery (MCA)– 2 cases, 5%. 2 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling Table 1. Descriptive statistics regarding intra-aneurysmal pressure in subgroups with and without stenting. POSITION VARIABLE MEAN SD WITH STENT parent artery systolic pressure (mmHg) 72 12.7 diastolic pressure (mmHg) 64 11.9 aneurysm dome before embolization systolic pressure (mmHg) 71.3 8.6 diastolic pressure (mmHg) 65.3 7.8 aneurysm dome after stent implantation systolic pressure (mmHg) 68.3 15 diastolic pressure (mmHg) 62.2 14.4 aneurysm dome after embolization systolic pressure (mmHg) 72.1 12.5 diastolic pressure (mmHg) 64.4 9.4 WITHOUT STENT parent artery systolic pressure (mmHg) 75.1 18.2 diastolic pressure (mmHg) 67.5 17.9 aneurysm dome before embolization systolic pressure (mmHg) 75.5 18.4 diastolic pressure (mmHg) 69.3 17.5 aneurysm dome after embolization systolic pressure (mmHg) 73.3 15 diastolic pressure (mmHg) 67.1 12.5 ANEURYSM VOLUME aneurysm volume (mm3) 371.1 840.7 STENT + packing (%) 26.4 19.8 coil volume (mm3) 38.3 30.3 - packing (%) 12.2 12.2 coil volume (mm3) 46.1 91.1 https://doi.org/10.1371/journal.pone.0233981.t001 https://doi.org/10.1371/journal.pone.0233981.t001 In 32/45 cases, coil embolization was assisted with a stent, for details please refer to Fig 2. Aneurysms embolized with stent assistance were significantly more densely coiled, compared to those where stent was not used–p = 0.015, yet the total coil volume did not differ signifi- cantly–p = 0.10 –Fig 3. Accordingly, Spearman Rank Correlation Coefficient showed a signifi- cant association between the presence of a stent and packing density–r = 0.36, p<0.05. In 32/45 cases, coil embolization was assisted with a stent, for details please refer to Fig 2. Aneurysms embolized with stent assistance were significantly more densely coiled, compared to those where stent was not used–p = 0.015, yet the total coil volume did not differ signifi- cantly–p = 0.10 –Fig 3. Results 2 –diameter of efferent parent artery; Dist. 4 –width of the sac; Dist. 6 –length of the sac (Dmax); Dist. 10 –height of the sac; Ang. 13 –delta angle. https://doi.org/10.1371/journal.pone.0233981.g001 Embolization did not significantly change the intra-aneurysmal pressure in matched pairs, regardless of the use of stent. In cases of stent-assisted coiling p values were 0.69, 0.81, and 0.91 for systolic, diastolic and mean intra-aneurysmal pressure; respectively. In a subgroup with no stenting, the respective p values were 0.31, 0.21, and 0.21. Coil packing density was not signifi- cantly associated with intra-aneurysmal pressure after embolization The effect on intra-aneurysmal pressures between stents was analyzed. Both non-normal- ized and normalized measurements were compared (respective to systemic values as a ratio to account for potential variations in systemic parameters). Values were presented Table 3. Among aneurysm morphology characteristics, significant association was confirmed between the diameter of the aneurysm neck and the need to use a stent–r = 0.32, p<0.05. Simi- larly, the presence of more than two arteries originating around the aneurysm neck rendered the use of a stent necessary–r = 0.43, p<0.05. None of the measured intra-saccular pressures were significantly correlated with aneurysm morphology (p>0.05). Results Accordingly, Spearman Rank Correlation Coefficient showed a signifi- cant association between the presence of a stent and packing density–r = 0.36, p<0.05. Presence of the stent showed significant reverse correlation with intra-aneurysmal pres- sure–both systolic and diastolic—after its implantation (r = -0.70 and r = -0.75, respectively), which was further supported by correlations with stent cell size–r = 0.72 and r = 0.71, respec- tively (P<0.05). Stent implantation resulted in significant decrease in diastolic intra-aneurys- mal pressure (p = 0.046) (Fig 4). Systolic or mean intra-aneurysmal pressure did not differ significantly. Presence of the stent showed significant reverse correlation with intra-aneurysmal pres- sure–both systolic and diastolic—after its implantation (r = -0.70 and r = -0.75, respectively), which was further supported by correlations with stent cell size–r = 0.72 and r = 0.71, respec- tively (P<0.05). Stent implantation resulted in significant decrease in diastolic intra-aneurys- mal pressure (p = 0.046) (Fig 4). Systolic or mean intra-aneurysmal pressure did not differ significantly. Table 2. Descriptive statistics of treated aneurysms. Table 2. Descriptive statistics of treated aneurysms. LOCATION DMAX (mm) WIDTH (mm) HEIGHT (mm) NECK (mm) DELTA ANGLE (˚) STENTING (%) ICA 6.8±4.2 7.8±3.9 5.9±3.7 5.3±1.7 112.8±32.4 77 MCA 13.9±14.1 9.8±6.9 12.5±14.2 3.7±1.8 140.5±36.1 0 BA 6.6±2.9 6.4±3.4 5.9±2.9 5.5±3.1 139.3±37.3 83 ACA 3.4 ±0.4 4.3±0.8 2.9±0.4 3.1±0.7 156.8±36.8 33 ICA–internal carotid artery, MCA–middle cerebral artery, BA–basilar artery, ACA–anterior cerebral artery, Dmax–length of aneurysm sac, delta angle–angle formed between the axis of proximal part of parent artery and Dmax PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 3 / 11 PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling Fig 1. Schematic morphological measurements on 3D angiography. Please refer to caption for Table 2. Dist. 1 –diameter of afferent parent artery; Dist. 2 –diameter of efferent parent artery; Dist. 4 –width of the sac; Dist. 6 –length of the sac (Dmax); Dist. 10 –height of the sac; Ang. 13 –delta angle. https://doi org/10 1371/journal pone 0233981 g001 Fig 1. Schematic morphological measurements on 3D angiography. Please refer to caption for Table 2. Dist. 1 –diameter of afferent parent artery; Dist. 2 –diameter of efferent parent artery; Dist. 4 –width of the sac; Dist. 6 –length of the sac (Dmax); Dist. 10 –height of the sac; Ang. 13 –delta angle. Fig 1. Schematic morphological measurements on 3D angiography. Please refer to caption for Table 2. Dist. 1 –diameter of afferent parent artery; Dist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 Discussion Intra-aneurysmal pressure would theoretically change in two situations: (1) replacing the blood in the aneurysm sac with embolization material, (2) changing the amount of blood flow- ing into the sac from the parent vessel. Both pathways were investigated in our study. Endovascular coiling aims at preventing blood flow into the aneurysm by partially filling the aneurysm sac. Even though, coils alone cannot fill the entire aneurysm, they provoke intra- aneurysmal thrombosis, which eventually leads to complete occlusion. There are mixed data on the effect of aneurysm embolization on intra-saccular pressure. We found embolization did not significantly change the intra-aneurysmal pressure in matched pairs, regardless of the use of stent, which is in accordance with previous experimental and numerical-based research 4 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling Fig 2. Number of devices used for stent-assisted coiling. https://doi.org/10.1371/journal.pone.0233981.g002 Fig 2. Number of devices used for stent-assisted coiling. https://doi.org/10.1371/journal.pone.0233981.g002 [13–16]. Coil packing density showed no significant association with post-embolization pres- sure measurements, even though coil packing density averaged 23%, with 3 cases of over 60%. Similarly, Groden et al. found no change in the pressure inside the aneurysm sac after up to 20% of coil packing [15]. In vitro observations led to similar conclusions where packing densi- ties reached 93% [14]. Most of in-vivo-based published research focused on the differences between systemic, parent artery and intra-saccular pressure alone [17–20]. Despite the angiographic success of coiling, the flow in the aneurysm sac remains not affected enough to render significant pressure change. In about one-third of cases, coiling does not lead to adequate thrombus formation; these emphasize the role of hemodynamics in the process. As a consequence, recanalization and aneurysm recurrence can ensue. The potential is especially visible in wide-neck and large aneurysms [21]. Blood stagnation promoting thrombus formation in the aneurysm sac could be a result of decreased velocity of blood flow. Its reduction depends on coil packing density; when the packing density is low, coil configura- tion plays an important role [16, 22]. Coil orientation and packing density at the neck are directly responsible for flow velocity inside the aneurysm [23]. Effective packing density could 5 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling Fig 3. Differences in coil use between stent-assisted and not assisted embolization. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 Discussion https://doi org/10 1371/journal pone 0233981 g003 Fig 3. Differences in coil use between stent-assisted and not assisted embolization. https://doi.org/10.1371/journal.pone.0233981.g003 https://doi.org/10.1371/journal.pone.0233981.g003 be especially challenging in case of the wide neck aneurysm, when there is a risk of coil hernia- tion into the parent vessel. Stents can stabilize the position of the coils. Indeed, we found coil packing density significantly increased when the stent was utilized. It could be explained by stent struts facilitating a more stable position of the microcatheter inside the aneurysm sac. Stents themselves could promote aneurysm thrombosis by facilitating endothelialization of the aneurysmal orifice and diverting the blood flow away from the aneurysm [24]. These effects are considered to be related to stent porosity. In vitro studies indicate that high porosity —laser-cut—stents tend to have minimal effects on intra-aneurysmal flow [25]. Seshadhri et al. studied different wire densities and showed the flow-diverter with the highest wire den- sity induced the most significant hemodynamic change [26]. There are inconsistent reports on the relationship of intra-aneurysmal pressure after stent implantation, done predominantly on flow-diverters [26–29]. Paper published by Corriveau et al. has shown in in-vivo measurements that flow-diverter stent implantation results in an increase of the intra-saccular pressure inside large or giant intracranial aneurysm [30]. They theorized the phenomenon is the result of outflow obstruction, which in consequence could lead to delayed saccular rupture in angiographically successful cases. Schneiders et al. per- formed in-vivo measurements before and after flow-diverter placement, in a giant aneurysm; 6 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling they found transient decrease in intra-saccular pressure, which was restored to original within minutes [31]. All stents in this study, except one, were braided self-expanding closed-cell stents–Leo, LVIS and LVIS Jr. Each of them has characteristic cell size, innate to its design— 0.9mm, 1mm, and 1.5mm; respectively. Although they were not originally intended for use as Fig 4. Differences in systolic and diastolic intra-aneurysmal pressure between stent-assisted and not assisted embolization. https://doi.org/10.1371/journal.pone.0233981.g004 Fig 4. Differences in systolic and diastolic intra-aneurysmal pressure between stent-assisted and not assisted embolization. https://doi.org/10.1371/journal.pone.0233981.g004 4. Differences in systolic and diastolic intra-aneurysmal pressure between stent-assisted and not assisted embolization. ps://doi.org/10.1371/journal.pone.0233981.g004 they found transient decrease in intra-saccular pressure, which was restored to original within minutes [31] All stents in this study except one were braided self expanding closed cell Fig 4. PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling Table 3. Comparison of intra-aneurysmal pressure after stent implantation and after embolization between different devices. POSITION VARIABLE LVIS vs LEO LVIS vs LVIS Jr LEO vs LVIS Jr aneurysm dome after stent implantation non-normalized systolic pressure 0.15 0.22 0.007 non-normalized diastolic pressure 0.33 0.15 0.02 normalized mean pressure 0.97 0.77 0.72 aneurysm dome after embolization non-normalized systolic pressure 0.36 0.81 0.64 non-normalized diastolic pressure 0.91 0.73 0.76 normalized mean pressure 0.48 0.62 0.72 Values represent p-values. https //doi org/10 1371/jo rnal pone 0233981 t003 neurysmal pressure after stent implantation and after embolization between different devices. a flow-diverter they have shown potential to occlude small aneurysms in monotherapy, espe- cially when telescoping stenting technique was used [32, 33]. We showed the implantation of the stent leads to significant decrease in diastolic pressure within the aneurysm sac (p = 0.046). Systolic or mean intra-aneurysmal pressure did not differ significantly. Given the significant reverse correlation of stenting with intra-aneurysmal pressure, further supported by similar relationship with stent cell size, we decided to compare the effect between stents. The differences in intra-saccular pressure after stent placement reached significance, when Leo and LVIS Jr devices were compared. These observations were not reproduced when aneurysms were coiled. It could be attributable to non-uniform coil packing density between the aneurysms. In view of all observations, any significant change of intra-saccular pressure may lead to delayed aneurysm rupture by: (a) outflow obstruction–higher pressure, or (b) generating local drop of wall shear stress (WSS)–i.e. stasis at the periphery of the sac, and secondary inflamma- tion–lower pressure. Taking clinical perspective into consideration, it might be reasonable to promote uniform stasis throughout the aneurysmal sac, by coiling the aneurysm, regardless of type of stent–flow-diverter or non-flow-diverter. Although in our study series, we found no association between morphological parameters of the aneurysm sac and pressure measurements, CFD simulations found aneurysm and par- ent artery geometry to be relevant to their hemodynamics [34–36]. Aneurysm volume, sac depth, neck maximum width and neck area showed inverse relationship with wall shear stress, i.e. increase in the former resulted in decrease of the latter. Decrease in WSS promoted local blood stasis, as mentioned already, which might lead to rupture of the aneurysm–innate due to large aneurysm sac, or secondary due to suboptimal disruption of the flow inside the sac. Discussion Differences in systolic and diastolic intra-aneurysmal pressure between stent-assisted and not assisted embolization. https://doi.org/10.1371/journal.pone.0233981.g004 Fig 4. Differences in systolic and diastolic intra-aneurysmal pressure between stent-assisted and not assisted embolization. https://doi.org/10.1371/journal.pone.0233981.g004 https://doi.org/10.1371/journal.pone.0233981.g004 they found transient decrease in intra-saccular pressure, which was restored to original within minutes [31]. All stents in this study, except one, were braided self-expanding closed-cell stents–Leo, LVIS and LVIS Jr. Each of them has characteristic cell size, innate to its design— 0.9mm, 1mm, and 1.5mm; respectively. Although they were not originally intended for use as PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 7 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 Author Contributions Conceptualization: Piotr Piasecki, Jerzy Narloch. Conceptualization: Piotr Piasecki, Jerzy Narloch. Data curation: Piotr Piasecki, Piotr Ziecina, Krzysztof Brzozowski, Marek Wierzbicki, Jerzy Narloch. Data curation: Piotr Piasecki, Piotr Ziecina, Krzysztof Brzozowski, Marek Wierzbicki, Jerzy Narloch. Formal analysis: Piotr Piasecki, Krzysztof Brzozowski, Jerzy Narloch. Investigation: Piotr Piasecki, Piotr Ziecina, Marek Wierzbicki. Methodology: Piotr Piasecki, Piotr Ziecina, Krzysztof Brzozowski, Marek Wierzbicki, Jerzy Narloch. Software: Piotr Ziecina, Krzysztof Brzozowski, Marek Wierzbicki, Jerzy Narloch. S i i J N l h Software: Piotr Ziecina, Krzysztof Brzozowski, Marek Wierzbicki, Jerzy Narloch. Supervision: Jerzy Narloch. Validation: Piotr Piasecki, Krzysztof Brzozowski. Validation: Piotr Piasecki, Krzysztof Brzozowski. Validation: Piotr Piasecki, Krzysztof Brzozowski. Visualization: Piotr Piasecki. Visualization: Piotr Piasecki. Writing – original draft: Piotr Piasecki, Jerzy Narloch. Writing – original draft: Piotr Piasecki, Jerzy Narloch. Writing – review & editing: Piotr Piasecki, Jerzy Narloch. Writing – review & editing: Piotr Piasecki, Jerzy Narloch. Writing – review & editing: Piotr Piasecki, Jerzy Narloch. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE The hemodynamic effect of stenting was mostly affected by aneurysm morphology, less by its posi- tion or orientation relative to parent vessel [34]. Meng et al. demonstrated the higher the par- ent vessel curvature the lower effect does stenting have on intra-aneurysmal flow reduction [35].Impact of stents on inflow rate and mean velocity is more effective in narrow-necked cerebral aneurysms [36]. In view of our data, combined with the latest reports, there is added value to the utility of low-profile braided stents in proximal segments of cerebral vasculature. Not only do the struts support the coils, which could be more densely packed, but also impede the inflow of blood into the aneurysm sac–especially in lower curvature or side-wall aneurysms. Our study has limitation inherent to single-center cohort. Elective treatment of non-rup- tured aneurysms was performed. Location and morphology of the aneurysm were not uni- form, although special care was taken to center the microcatheter position for all pressure readings. Measurements taken through long microcatheter bear the risk of substantial imped- ance and destructive interference effect on the accuracy of measurements, yet these were inde- pendently validated in published reports [17–20]. 8 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling Finally, thrombus formation does not depend on hemodynamics and embolic materials alone. The process is complex, dependent also on a variety of hematologic factors such rheo- logical properties and platelet function (medication-driven included) [27, 29, 37]. Substantial reduction of flow might limit the supply of prothrombotic factors to secure stable thrombus formation. In conclusion, low-profile braided stents show a potential to divert blood flow–a property shown in our study to be related to their porosity–as reflected in significant decrease in dia- stolic pressure after stent placement. Coiling does not significantly change the intra-aneurys- mal pressure, regardless of packing density. References 1. Molyneux AJ, Kerr RS, Yu LM, Clarke M, Sneade M, Yarnold JA, et al. International subarachnoid aneu- rysm trial (ISAT) of neurosurgical clipping versus endovascular coiling in 2143 patients with ruptured intracranial aneurysms: a randomised comparison of effects on survival, dependency, seizures, rebleeding, subgroups, and aneurysm occlusion. Lancet. 2005; 366(9488):809–17. https://doi.org/10. 1016/S0140-6736(05)67214-5 PMID: 16139655 1. Molyneux AJ, Kerr RS, Yu LM, Clarke M, Sneade M, Yarnold JA, et al. International subarachnoid aneu- rysm trial (ISAT) of neurosurgical clipping versus endovascular coiling in 2143 patients with ruptured intracranial aneurysms: a randomised comparison of effects on survival, dependency, seizures, rebleeding, subgroups, and aneurysm occlusion. Lancet. 2005; 366(9488):809–17. https://doi.org/10. 1016/S0140-6736(05)67214-5 PMID: 16139655 2. Akpek S, Arat A, Morsi H, Klucznick RP, Strother CM, Mawad ME. Self-expandable stent-assisted coil- ing of wide-necked intracranial aneurysms: a single-center experience. AJNR Am J Neuroradiol. 2005; 26(5):1223–31. PMID: 15891189 2. Akpek S, Arat A, Morsi H, Klucznick RP, Strother CM, Mawad ME. Self-expandable stent-assisted coil- ing of wide-necked intracranial aneurysms: a single-center experience. AJNR Am J Neuroradiol. 2005; 26(5):1223–31. PMID: 15891189 3. Wanke I, Doerfler A, Schoch B, Stolke D, Forsting M. Treatment of wide-necked intracranial aneurysms with a self-expanding stent system: initial clinical experience. AJNR Am J Neuroradiol. 2003; 24 (6):1192–9. PMID: 12812954 3. Wanke I, Doerfler A, Schoch B, Stolke D, Forsting M. Treatment of wide-necked intracranial aneurysms with a self-expanding stent system: initial clinical experience. AJNR Am J Neuroradiol. 2003; 24 (6):1192–9. PMID: 12812954 4. Shobayashi Y, Tateshima S, Kakizaki R, Sudo R, Tanishita K, Viñuela F. Intra-aneurysmal hemody- namic alterations by a self-expandable intracranial stent and flow diversion stent: high intra-aneurysmal pressure remains regardless of flow velocity reduction. J Neurointerv Surg. 2013; 5 Suppl 3:iii38–42. 4. Shobayashi Y, Tateshima S, Kakizaki R, Sudo R, Tanishita K, Viñuela F. Intra-aneurysmal hemody- namic alterations by a self-expandable intracranial stent and flow diversion stent: high intra-aneurysmal pressure remains regardless of flow velocity reduction. J Neurointerv Surg. 2013; 5 Suppl 3:iii38–42. 9 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling 5. Tateshima S, Tanishita K, Hakata Y, Tanoue SY, Viñuela F. Alteration of intraaneurysmal hemodynam- ics by placement of a self-expandable stent. Laboratory investigation. J Neurosurg. 2009; 111(1):22–7. https://doi.org/10.3171/2009.2.JNS081324 PMID: 19267529 6. Wanke I, Forsting M. Stents for intracranial wide-necked aneurysms: more than mechanical protection. Neuroradiology. 2008; 50(12):991–8. https://doi.org/10.1007/s00234-008-0460-0 PMID: 18807024 7. References Behme D, Weber A, Kowoll A, Berlis A, Burke TH, Weber W. Low-profile Visualized Intraluminal Sup- port device (LVIS Jr) as a novel tool in the treatment of wide-necked intracranial aneurysms: initial expe- rience in 32 cases. J Neurointerv Surg. 2015; 7(4):281–5. https://doi.org/10.1136/neurintsurg-2014- 011157 PMID: 24699567 8. Cohen JE, Melamed I, Itshayek E. X-microstenting and transmesh coiling in the management of wide- necked tent-like anterior communicating artery aneurysms. J Clin Neurosci. 2014; 21(4):664–7. https:// doi.org/10.1016/j.jocn.2013.09.003 PMID: 24291480 9. Fiorella D, Albuquerque FC, Deshmukh VR, Woo HH, Rasmussen PA, Masaryk TJ, et al. Endovascular reconstruction with the Neuroform stent as monotherapy for the treatment of uncoilable intradural pseu- doaneurysms. Neurosurgery. 2006; 59(2):291–300; discussion 291–300. https://doi.org/10.1227/01. NEU.0000223650.11954.6C PMID: 16823325 10. Kim YJ, Ko JH. Sole stenting with large cell stents for very small ruptured intracranial aneurysms. Interv Neuroradiol. 2014; 20(1):45–53. https://doi.org/10.15274/INR-2014-10007 PMID: 24556299 11. Song J, Oh S, Kim MJ, Chung J, Lim YC, Kim BS, et al. Endovascular treatment of ruptured blood blis- ter-like aneurysms with multiple (3) overlapping Enterprise stents and coiling. Acta Neurochir (Wien). 2016; 158(4):803–9. 12. Zenteno MA, Santos-Franco JA, Freitas-Modenesi JM, Go´mez C, Murillo-Bonilla L, Aburto-Murrieta Y, et al. Use of the sole stenting technique for the management of aneurysms in the posterior circulation in a prospective series of 20 patients. J Neurosurg. 2008; 108(6):1104–18. https://doi.org/10.3171/JNS/ 2008/108/6/1104 PMID: 18518712 13. Boecher-Schwarz HG, Ringel K, Kopacz L, Heimann A, Kempski O. Ex vivo study of the physical effect of coils on pressure and flow dynamics in experimental aneurysms. AJNR Am J Neuroradiol. 2000; 21 (8):1532–6. PMID: 11003291 14. Canto´n G, Levy DI, Lasheras JC. Changes in the intraaneurysmal pressure due to HydroCoil emboliza- tion. AJNR Am J Neuroradiol. 2005; 26(4):904–7. PMID: 15814942 15. Groden C, Laudan J, Gatchell S, Zeumer H. Three-dimensional pulsatile flow simulation before and after endovascular coil embolization of a terminal cerebral aneurysm. J Cereb Blood Flow Metab. 2001; 21(12):1464–71. https://doi.org/10.1097/00004647-200112000-00011 PMID: 11740208 16. Schirmer CM, Malek AM. Critical influence of framing coil orientation on intra-aneurysmal and neck region hemodynamics in a sidewall aneurysm model. Neurosurgery. 2010; 67(6):1692–702; discussion 702. https://doi.org/10.1227/NEU.0b013e3181f9a93b PMID: 21107200 17. Duong DH, Young WL, Vang MC, Sciacca RR, Mast H, Koennecke HC, et al. Feeding artery pressure and venous drainage pattern are primary determinants of hemorrhage from cerebral arteriovenous mal- formations. Stroke. 1998; 29(6):1167–76. https://doi.org/10.1161/01.str.29.6.1167 PMID: 9626290 18. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 References Fleischer LH, Young WL, Pile-Spellman J, terPenning B, Kader A, Stein BM, et al. Relationship of tran- scranial Doppler flow velocities and arteriovenous malformation feeding artery pressures. Stroke. 1993; 24(12):1897–902. https://doi.org/10.1161/01.str.24.12.1897 PMID: 8248974 19. Fogarty-Mack P, Pile-Spellman J, Hacein-Bey L, Osipov A, DeMeritt J, Jackson EC, et al. The effect of arteriovenous malformations on the distribution of intracerebral arterial pressures. AJNR Am J Neurora- diol. 1996; 17(8):1443–9. PMID: 8883639 20. Li Y, Corriveau M, Aagaard-Kienitz B, Ahmed A, Niemann D. Differences in Pressure Within the Sac of Human Ruptured and Nonruptured Cerebral Aneurysms. Neurosurgery. 2019; 84(6):1261–8. https:// doi.org/10.1093/neuros/nyy182 PMID: 29741656 21. Hayakawa M, Murayama Y, Duckwiler GR, Gobin YP, Guglielmi G, Viñuela F. Natural history of the neck remnant of a cerebral aneurysm treated with the Guglielmi detachable coil system. J Neurosurg. 2000; 93(4):561–8. https://doi.org/10.3171/jns.2000.93.4.0561 PMID: 11014533 22. Morales HG, Kim M, Vivas EE, Villa-Uriol MC, Larrabide I, Sola T, et al. How do coil configuration and packing density influence intra-aneurysmal hemodynamics? AJNR Am J Neuroradiol. 2011; 32 (10):1935–41. https://doi.org/10.3174/ajnr.A2635 PMID: 21885712 23. Nguyen TN, Hoh BL, Amin-Hanjani S, Pryor JC, Ogilvy CS. Comparison of ruptured vs unruptured aneurysms in recanalization after coil embolization. Surg Neurol. 2007; 68(1):19–23. https://doi.org/10. 1016/j.surneu.2006.10.021 PMID: 17586214 10 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 PLOS ONE Intra-aneurysmal pressure changes during stent-assisted coiling 24. Lylyk P, Cohen JE, Ceratto R, Ferrario A, Miranda C. Endovascular reconstruction of intracranial arter- ies by stent placement and combined techniques. J Neurosurg. 2002; 97(6):1306–13. https://doi.org/ 10.3171/jns.2002.97.6.1306 PMID: 12507128 25. Babiker MH, Gonzalez LF, Albuquerque F, Collins D, Elvikis A, Zwart C, et al. An in vitro study of pulsa- tile fluid dynamics in intracranial aneurysm models treated with embolic coils and flow diverters. IEEE Trans Biomed Eng. 2013; 60(4):1150–9. https://doi.org/10.1109/TBME.2012.2228002 PMID: 23192467 26. Seshadhri S, Janiga G, Beuing O, Skalej M, The´venin D. Impact of stents and flow diverters on hemody- namics in idealized aneurysm models. J Biomech Eng. 2011; 133(7):071005. https://doi.org/10.1115/1. 4004410 PMID: 21823744 27. Darsaut TE, Rayner-Hartley E, Makoyeva A, Salazkin I, Berthelet F, Raymond J. Aneurysm rupture after endovascular flow diversion: the possible role of persistent flows through the transition zone asso- ciated with device deformation. Interv Neuroradiol. 2013; 19(2):180–5. https://doi.org/10.1177/ 159101991301900206 PMID: 23693041 28. Roszelle BN, Gonzalez LF, Babiker MH, Ryan J, Albuquerque FC, Frakes DH. Flow diverter effect on cerebral aneurysm hemodynamics: an in vitro comparison of telescoping stents and the Pipeline. PLOS ONE \| https://doi.org/10.1371/journal.pone.0233981 June 4, 2020 References Neuroradiology. 2013; 55(6):751–8. https://doi.org/10.1007/s00234-013-1169-2 PMID: 23515661 29. Zanaty M, Jabbour PM, Bou Sader R, Chalouhi N, Tjoumakaris S, Rosenwasser RH, et al. Intra-aneu- rysmal thrombus modification after flow-diversion. J Clin Neurosci. 2015; 22(1):105–10. https://doi.org/ 10.1016/j.jocn.2014.05.033 PMID: 25192591 30. Corriveau M, Ahmed A, Dawkins D, Kienitz BA, Niemann D, Li Y. The effect of flow diverting stents on in vivo intrasaccular aneurysm pressure. J Clin Neurosci. 2019; 59:339–41. https://doi.org/10.1016/j.jocn. 2018.10.093 PMID: 30446369 31. Schneiders JJ, VanBavel E, Majoie CB, Ferns SP, van den Berg R. A flow-diverting stent is not a pres- sure-diverting stent. AJNR Am J Neuroradiol. 2013; 34(1):E1–4. https://doi.org/10.3174/ajnr.A2613 PMID: 21852372 32. Aydin K, Barburoglu M, Sencer S, Berdikhojayev M, Coskun B, Akpek S. Flow Diversion with Low-Pro- file Braided Stents for the Treatment of Very Small or Uncoilable Intracranial Aneurysms at or Distal to the Circle of Willis. AJNR Am J Neuroradiol. 2017; 38(11):2131–7. https://doi.org/10.3174/ajnr.A5362 PMID: 28882859 33. Feng Z, Li Q, Zhao R, Zhang P, Chen L, Xu Y, et al. Endovascular Treatment of Middle Cerebral Artery Aneurysm with the LVIS Junior Stent. J Stroke Cerebrovasc Dis. 2015; 24(6):1357–62. https://doi.org/ 10.1016/j.jstrokecerebrovasdis.2015.02.016 PMID: 25851343 34. Larrabide I, Geers AJ, Morales HG, Aguilar ML, Ru¨fenacht DA. Effect of aneurysm and ICA morphology on hemodynamics before and after flow diverter treatment. J Neurointerv Surg. 2015; 7(4):272–80. https://doi.org/10.1136/neurintsurg-2014-011171 PMID: 24692666 35. Meng H, Wang Z, Kim M, Ecker RD, Hopkins LN. Saccular aneurysms on straight and curved vessels are subject to different hemodynamics: implications of intravascular stenting. AJNR Am J Neuroradiol. 2006; 27(9):1861–5. PMID: 17032857 36. Wu YF, Yang PF, Shen J, Huang QH, Zhang X, Qian Y, et al. A comparison of the hemodynamic effects of flow diverters on wide-necked and narrow-necked cerebral aneurysms. J Clin Neurosci. 2012; 19 (11):1520–4. https://doi.org/10.1016/j.jocn.2011.10.015 PMID: 22704947 37. Fox B, Humphries WE, Doss VT, Hoit D, Elijovich L, Arthur AS. Rupture of giant vertebrobasilar aneu- rysm following flow diversion: mechanical stretch as a potential mechanism for early aneurysm rupture. J Neurointerv Surg. 2015; 7(11):e37. https://doi.org/10.1136/neurintsurg-2014-011325.rep PMID: 25361560 11 / 11
https://openalex.org/W3111677531	https://www.shs-conferences.org/articles/shsconf/pdf/2020/15/shsconf_ictp2020_00056.pdf	English	null	Preparation of future teachers for tourism and local history activities	SHS web of conferences	2,020	cc-by	5,858	Preparation of future teachers for tourism and local history activities T.A. Maslova¹, E.A. Akimova², and V.A. Romanov³ ¹ V.I. Kaluga State University named after K.E. Tsiolkovsky, Kaluga, Russia ² V.I. Kaluga State University named after K.E. Tsiolkovsky, Kaluga, Russia ³ V.I. Tula State Pedagogical University named after L.N. Tolstoy, Tula, Russia ¹ V.I. Kaluga State University named after K.E. Tsiolkovsky, Kaluga, Russia ² V.I. Kaluga State University named after K.E. Tsiolkovsky, Kaluga, Russia ³ V.I. Tula State Pedagogical University named after L.N. Tolstoy, Tula, Russia Abstract. The article deals with the process of preparing students for tourism and local history activities. The need to develop students ' skills in tourism and local history activities is justified by the need to develop components of universal competence specified in the Federal State Educational Standard of Higher Education (FSES HE). The pedagogical conditions necessary for preparing future teachers for tourism and local history activities in higher education are determined. The skills that students should master in the process of preparing for the implementation of tourist and local history activities are highlighted. 1 A problem statement The current situation in higher education requires new, modern approaches to the educational training of teachers and their activities. These processes form the prerequisites for creating individually directed educational strategies, for adapting the content of educational programs and manuals to the real needs and capabilities of students, and for moving to the flexible nature of the pedagogical process. Higher school pedagogy today focuses on the development of means in order to intensify the professional development of the individual. The educational result of becoming a specialist is represented by readiness for professional activity in the logic of the competence approach. In pedagogy, readiness for professional activity is considered as a system of components, personal qualities that ensure the performance of professional functions for a future specialist. This issue is reflected in the works of V.A. Slastenin [1], his scientific school, the works of O.V. Voronova, E.P. Grishina, N.M. Izoria [2], S.V. Kalinina and other young scientists. y g The Russian state and society have set a goal for the pedagogical community - to create and implement optimal conditions for the education and development of modern youth. Article 2, paragraph 1 of the Federal law of the Russian Federation" On Education "defines the concept of education:" 1. Education - a single purposeful process of education and training, which is a socially significant good and is carried out in the interests of a person, family, society, state, as well as a set of acquired knowledge, skills, values, © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). SHS Web of Conferences 87, 00056 (2020) ICTP 2020 SHS Web of Conferences 87, 00056 (2020) ICTP 2020 https://doi.org/10.1051/shsconf/20208700056 SHS Web of Conferences 87, 00056 (2020) SHS Web of Conferences 87, 00056 (2020) Corresponding author: ipcs-profped@yandex.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). SHS Web of Conferences 87, 00056 (2020) SHS Web of Conferences 87, 00056 (2020) ICTP 2020 https://doi.org/10.1051/shsconf/20208700056 experience and competence of a certain volume and complexity for the purpose of intellectual, spiritual, moral, creative, physical and (or) professional development of a person, for satisfaction of his educational needs and interests" [3]. In the Federal state educational standard of higher education (bachelor's program), the requirements for the results of mastering the bachelor's program specify the following competencies: UC-6. Able to manage their time, build and implement a self-development trajectory based on the principles of education throughout life. UC-7. Able to maintain a proper level of physical fitness to ensure a full-fledged social and professional activity. UC- 8. Able to create and maintain safe living conditions, including in the event of emergencies [4]. Tourist and local history activities provide such an opportunity. 1.1 The objective of the work Scientific development of issues of tourism and local lore activity is based on the concept of continuity of tourism and local history (A.A. Ostapets-Sveshnikov, I.L. Ivanov, A.D. Soldatenkov, Yu.S. Konstantinov, etc.) [5]. Pedagogical aspects of local history are studied by a number of leading experts in this field (S.O. Schmidt, A.E. Seinensky, V.E. Tumanov, N.I. Reshetnikov [6-7], I. N. beskorovayny, etc.). The educational role of this activity is revealed in the works of Ya.A. Komensky, I.G. Pestalozzi, V.N. Tatishchev, M.V. Lomonosov, S.M. Golitsyn, Yu.S. Konstantinov, V.M. Kulikov, A.A. Ostapts-Sveshnikov and others. In the Soviet period, the role of children and youth tourism as a means of Communist education of the individual is revealed in the works of I.M. Grev, N.K. Krupskaya, A.E. Fersman, D.A. Shadrikov, S.T. Shatsky, and others. I.V. Zorin analyzed the problems of forming the content of professional tourism education from the position of a systematic approach. He developed the theoretical basis for designing the content of professional tourism education, and presented a method for forming a didactic complex for training tourism specialists [8]. Among the works dedicated to tourist and local history activity of students at the present stage important are the studies of I.A. Drugova, Y.S. Konstantinova [9], A.G. Maslova, G.S. Usyskina et al. Some aspects are revealed in dissertation research, in which tourist and local history activities are presented as a means of professional orientation of students (D.V. Smirnov, etc.); the form of organization of the educational process in school (S.M. Gubanenkov, etc.), education of environmental culture (G.V. bukovskaya [10], V.P. Fomina, etc.), training of personnel for tourist and local history activities (Z. I. Gordeeva) [11], the importance of tourism in the spiritual and Patriotic education of secondary school students (M.A. Gorbova and D.S. Senyuk). The educational value of tourism and local history activities is considered by Yu.T. Borodkina, I.N. Pilat, P.I. Istomin [12-13], M.N. Yamnitsky, V.G. Tsygankov. In their works, the model of formation of young people readiness for responsible interaction by means of sports tourism is tested. Criteria for identifying readiness for conscious, responsible interaction are given. The Fundamentals of the state youth policy of the Russian Federation for the period up to 25 years reflect proposals for youth involvement: "in the active work of search, archaeological, military-historical, local history, student groups and youth associations" [14]. 1.1 The objective of the work At the present stage, there are a number of contradictions between: - the high need of society for a person who is ready to consciously serve the Motherland and the insufficient attention of the Institute of education to the problems of Patriotic education during the active growing up of the younger generation; - the need for scientific and methodological support for Patriotic education of higher education institutions and insufficient knowledge of the specifics of Patriotic education organization in its, institutions; 2 SHS Web of Conferences 87, 00056 (2020) ICTP 2020 SHS Web of Conferences 87, 00056 (2020) https://doi.org/10.1051/shsconf/20208700056 - students ' need for social self-determination and low level of parity of social and personal values. The development of youth tourism and local history is the most important social order of modern society. The role of the teacher in the formation of a socially active modern personality through tourism and local history activities should not be underestimated. Modern pedagogical science and practice have proved that the training of teachers in the tourist and local history movement is a highly effective means of teaching and educating the younger generation. Training teachers of tourism and local history activities for higher education is not only a personnel issue, but a whole system, a way of life that gives knowledge, skills, and ideology in the education and upbringing of the future generation. It is very important for preserving the identity of the native land, the inadmissibility of falsifying history, and preserving the common history of our homeland. Among the factors hindering the development of tourism and local history activities in the regions of the Russian Federation, there is a shortage of qualified personnel. Since tourism and local history are not included in the database of General education subjects, such as mathematics or history, etc., they are mostly subjecting of additional education. However, the requirements for tourism and local history activities teachers are generally accepted, and they include all levels of competence and readiness for professional activity. 2 Materials and the results of the research By tourism and local history activities, we will understand tourism and local history activities organized by the public, educational institutions, in order to ensure the pedagogically appropriate use of educational and leisure time, which is an optimized means of harmonizing the training, education and development of young people in the process of tourism and local history. The purpose of our research is to identify the pedagogical conditions necessary for preparing future teachers for tourism and local history activities in higher education. 1. Consider the current trends in tourism and local history. 2. Consider the psychological and pedagogical foundations of tourism and local history activities. 3. Identify the features of tourism and local history activities formation in higher education. 4. Develop a set of classes on formation, for higher education. We have determined the pedagogical conditions necessary for preparing future teachers for tourism and local history activities in higher education: - implementation of the directed formation of professionally significant qualities in tourist and local history activities, through the inclusion of mechanisms of emotional and volitional regulation; - inclusion of students in reflexive evaluation activities and co-reflection in situations of professional and moral choice and awareness of professional duty; - creation of a multi-faceted emotionally rich space for interaction between subjects of the educational process, which contributes to the development of skills in tourism and local history; - inclusion of students in tourist and local history activities: expeditions, exhibitions, hikes, excursions, gatherings, etc., contributing to the formation of spiritual and moral values; - using tourism and local history activities as a form of additional education for students in the form of tour clubs, youth organizations, museums, educational institutions, etc. 3 3 SHS Web of Conferences 87, 00056 (2020) ICTP 2020 https://doi.org/10.1051/shsconf/20208700056 Components of a student's readiness to organize tourism and local history activities: cognitive, practical, communicative, value, reflexive, motivational, personal (Table 1). Components of a student's readiness to organize tourism and local history activities: cognitive, practical, communicative, value, reflexive, motivational, personal (Table 1). cognitive, practical, communicative, value, reflexive, motivational, personal (Table 1). Table 1. Indicators of students ' readiness for tourist and local history activities components readiness indicators measurement tools points Cognitive Knowledge of the legal framework of the tourist and local history activities. Know Federal and regional Programs, Instructions, classifications, regulations, and Laws that regulate tourist and local history activities in the region. Russia and abroad. 2 Materials and the results of the research Have an idea of the structure of interaction between organizations that implement tourist and local history activities. Know The Training Programs. Knowledge of geographical tourist and local history centers of your region and regions of Russia. Survey. Analysis of students ' activities. Practical Ability to organize various forms of tourist and local history activities (hikes, expeditions, excursions, gatherings, competitions). Ability to conduct practical exercises (setting up a tent, organizing a bivouac, lighting a fire, etc.). Ability to take responsibility. Ability to build productive relationships with fellow students, be an organizer and organize for the sake of achieving a common goal. Ability to be in demand in the team, to perform tasks set in the classroom. Ability to be disciplined and resolve conflicts. Getting experience of independent activity, in the process of working out turnarounds on the ground. Pedagogical supervision. Analysis of students ' activities. Communicative Ability to communicate effectively and to organize the educational process, practical activities in the tourist and local history activities. Involvement of diverse specialists in the preparation of events in the shopping center. Initial fundraising skills (raising funds for organizing events in the tourist and local history activities.) Ability to interact with classmates in the process of organizing tourist and local history activities. classes. Pedagogical supervision. Conversation. Analysis of students ' activities Value Value attitude to the tourist and local history activities, formation of a value attitude to social reality - a tourist trip, local history research, tourist club. Pedagogical supervision. Conversation. Analysis of students ' activities. Reflexive Adequate general and professional assessment and self-assessment. Objective assessment of the level of self- development as a result of tourist and local history activities classes. Ability to process and interpret basic knowledge of Pedagogical observation. Conversation. Analysis of students ' activities. SHS Web of Conferences 87, 00056 (2020) ICTP 2020 https://doi.org/10.1051/shsconf/20208700056 components readiness indicators measurement tools points tourist and local history activities and apply it in the organization and implementation of tourist and local history activities (hikes, clubs, conferences, clubs, excursions, etc.) Ability to analyze scientific and technical, specialized information in the organization of events, classes (study of special literature, modern equipment, technical devices, etc.) and apply them in their activities. Empathy, self- organization, social reflection. Motivational Desire to acquire knowledge on tourist and local history activities, the possibility of their use in professional activities. Desire to work in the field of tourism and local lore. After analyzing the indicators of students ' readiness for tourism and local history activities, we can identify the following levels of readiness: 2 Materials and the results of the research Desire to become a high-class specialist. Desire to get an interesting job. Desire to work with people. Pedagogical supervision. Conversation. Analysis of students ' activities. Personal Ability to independently find and use sources of information on tourism and local history. Ability to organize work, take management decisions in the process of tourist and local history activities (in the mode of emergencies, unforeseen circumstances, etc.), qualities: independence, responsibility, organization, tolerance. Pedagogical supervision. Conversation. Analysis of students ' activities. The result of professional training of future teachers will be the " readiness assessment " of students to organize tourist and local history activities. The evaluation took into account the indicators of the criteria for the readiness of future teachers to organize tourist and local history activities in educational institutions (Table 2). High level - from 4 to 5 points. High level - from 4 to 5 points. Average level - from 3 to 4 points Low level-less than 3 points Up to 50% - three points From 50% to 75% - four points From 75% to 100% - five points High level - from 4 to 5 points. Average level - from 3 to 4 points Low level-less than 3 points Up to 50% - three points From 50% to 75% - four points From 75% to 100% - five points Table 2. The level of students ' performance in tourist and local history activities classes Table 2. The level of students ' performance in tourist and local history activities classes p y Names of the criterion Level of performance The original % Total in points Cognitive 60 4 Practical 79 5 Communication 78 5 Value 70 4 Reflexive 80 5 Motivational 95 5 Personal 90 5 After analyzing the indicators of students ' readiness for tourism and local history activities, we can identify the following levels of readiness: 5 SHS Web of Conferences 87, 00056 (2020) ICTP 2020 https://doi.org/10.1051/shsconf/20208700056 1. System-modulating (high) - ability to form a system of knowledge, skills and abilities on the tourism and local history activities as a whole. 1. System-modulating (high) - ability to form a system of knowledge, skills and abilities on the tourism and local history activities as a whole. y 2. Local-modulating (average) - knowledge and skills transfer skills for individual sections and topics of the tourism and local history activities. 2. 2 Materials and the results of the research Local-modulating (average) - knowledge and skills transfer skills for individual sections and topics of the tourism and local history activities. 3. Reproductive, adaptive (low) - is the ability to tell what he knows about the tourism and local history activities, ability to adapt your knowledge to the characteristics of the audience. 3. Reproductive, adaptive (low) - is the ability to tell what he knows about the tourism and local history activities, ability to adapt your knowledge to the characteristics of the audience. The results of the students ' assessment of their own training allowed them to evaluate the levels according to the 5-point system. At the first, high level, we can say about the pedagogical giftedness of students, which is manifested in the ability to subordinate their interests to the achievement of a goal: in our case, autonomous survival in the wild. At a high level special abilities of students are distinguished: stress resistance to unforeseen situations, the ability to organize the process of self-survival in extreme conditions, and the ability to organize a group and offer their own solutions to the situation. They used mostly professional terminology: "I can organize a hike, I can organize and hold a conference, I can conduct practical classes on organizing a bivouac, lighting a fire, setting up a tent, I independently develop training programs and conduct training, etc». Second-and middle-level students mainly used general concepts: "I can teach the technique of setting up a tent, the technique of lighting a fire, the technique of organizing a bivouac; ...I mastered the methods of organizing excursions, methods of collecting local history material". Middle-level students showed practical, communicative reflexive abilities to reproduce the worked-out process of self-survival in extreme conditions and to organize a group. However, they offered standard solutions to the situation. Middle-level students had a general characteristic of tourist and local history activities. Students of the third low level do not have conscious strategies of tourist and local history activities, they carried it out mainly due to their knowledge and intellectual qualities, under the guidance of a teacher, receiving clear instructions in the process of work. They avoided a direct answer and did not take the initiative. Low-level students had the terminology: "I can hold classes, I can set up a tent, light a fire, organize a bivouac». 2 Materials and the results of the research Name the Reserves of the Kaluga region. 14. What is ecoduct, and where were built the first Russian ecoducts? 15. Name the National parks of the Kaluga region. 16. Are you interested in knowledge about the Kaluga region? Underline your answer. Yes No 17. What new things did you learn in the tourist and local history activities class? 18. What important event is our country celebrating in may 2020? 19. What do you know about the Museum at KSU named after K. E. Tsiolkovsky? 20. What forms of work on tourist and local history activities would you choose as a teacher? 21. Your Small Homeland. Tell your little story about it. 22. What do you personally mean by the concept of Patriotism? 23. Do to date in higher education, tourist and local lore activity, and why? 24. Do you think it is necessary to introduce such a form of additional educatio as a tourist circle or tour club? Or write your proposal. 25. What form would you choose: a hike, an excursion, an expedition (local history, rism, ethnography)? g p y) 26. What safety rules do you know when hiking in the woods? 27. In what form of tourist and local history activities would your independent activity and your personality be more clearly manifested? 28. In what kind of tourist and local history activities would you get a versatile experience? 29. Would you like to use the development of practical classes on tourist and local history activities as future teachers and why? 30. Do you think it is necessary to introduce tourist and local history activities in the educational process of higher education as a subject and why? 31. What are your suggestions for improving the teaching of tourist and local history activities. The development of a set of practical exercises on tourist and local history activities was carried out in practice. The development of a set of practical exercises on tourist and local history activities was carried out in practice. Lesson №1 Lesson №1 2 Materials and the results of the research The assessment of independent activity of students is adequate, allowing to dete the level of their own competence and readiness to apply it in teaching activities. Only a thinking, competent, adaptive specialist who is able to organize the conditions for achieving results in the labor code will be competitive and in demand in the labor market. He will be able to provide knowledge and skills that are simply necessary in modern life. To date, we see that we are not protected from emergencies, when threats to the life of the population appear suddenly and unexpectedly, knowledge of tourist and local history activities becomes especially relevant. And higher education should not be left out of it. Knowing the methods of tourist and local history activities, self-survival techniques we can successfully organize our safety, our survival not only in the wild, but also in megacities and in general at any point of residence and in any situation. Before and after the practical training, we conducted a written survey by means of a questionnaire among the students of Kaluga State University (KSU) named after K.E. Tsiolkovsky. Questionnaire Questionnaire 1. What do you know about tourism and local history? 2. Give your definition: Tourism is - 3. What do you understand by local history? Continue with the phrase local history is - 6 SHS Web of Conferences 87, 00056 (2020) ICTP 2020 https://doi.org/10.1051/shsconf/20208700056 4. Do you think we need teacher training for the tourist and local history activities. Underline the right answer. derline the right answer. Yes No 5 Wh t d t f t t d k ? 5. What modern types of tents do you know? 6. What is the main factor of autonomous survival in the natural environment in the event of an emergency? g y 7. What tourist equipment would you take with you in case of an emergency? 8. What do you know about the Department of tourism at KSU? 9. What public associations dealing with tourist and local history activities are there in KSU? 10. Where would you use and apply your knowledge of tourist and local history activities as a teacher? 11. Would you expand your opportunities as a teacher when applying practical skills in tourist and local history activities in teaching activities and why? 12. What are the main rivers of the Kaluga region? 13. Name the Reserves of the Kaluga region. 13. Lesson №1 Practical lesson "Technique of autonomous survival in the wild" Lesson №1 Practical lesson "Technique of autonomous survival in the wild" Practical lesson "Technique of autonomous survival in the wild" Practical lesson "Technique of autonomous survival in the wild" Practical lesson "Technique of autonomous survival in the wild" The purpose of the lesson: to develop students ' primary skills and survival skills in the wild. Tasks: 7 SHS Web of Conferences 87, 00056 (2020) SHS Web of Conferences 87, 00056 (2020) ICTP 2020 https://doi.org/10.1051/shsconf/20208700056 To introduce students to the basics of survival in the wild and the use of improvised means of protection from the adverse environmental factors. To give the basic skills of survival techniques in the forest and the construction of temporary shelters. p y Practice skills in building temporary shelters. p y Practice skills in building temporary shelters. Practice skills in using improvised means of protection against the impact of adverse environmental factors. Lesson №2 Lesson №2 Lesson №2 he Theme: "Safety instructions for tourist and local history events. Type of trips" The Theme: "Safety instructions for tourist and local history events. Type of trips" Fig. 1. security measures. Practical lesson: "Types of instructions", "Safety Instructions for tourists on a hike". The purpose of the lesson: to give primary knowledge about the types of instructions and learn the types of safety Instructions in the campaign. Tasks: Fig. 1. security measures. Practical lesson: "Types of instructions", "Safety Instructions for tourists on a hike". The purpose of the lesson: to give primary knowledge about the types of instructions and learn the types of safety Instructions in the campaign. Tasks: Practical lesson: "Types of instructions", "Safety Instructions for tourists on a hike". The purpose of the lesson: to give primary knowledge about the types of instructions and learn the types of safety Instructions in the campaign. T k Practical lesson: "Types of instructions", "Safety Instructions for tourists on a hike". The purpose of the lesson: to give primary knowledge about the types of instructions and learn the types of safety Instructions in the campaign. Tasks: Practical lesson: Types of instructions , Safety Instructions for tourists on a hike . The purpose of the lesson: to give primary knowledge about the types of instructions and learn the types of safety Instructions in the campaign. To introduce students to the types of training sessions. To learn different types of safety Instructions on a hike. Training methods: visual and demonstration. Venue: auditorium of KSU. Class type combined Form of classes: group, classroom. Equipment: multimedia equipment, presentation. Seminar-game for students in "Hooray, we are going on a hike", as part of the tourist and local history activities Seminar-game for students in "Hooray, we are going on a hike", as part of the tourist and local history activities Purpose of the workshop: Get organizational and practical skills on the topic: "Organization and conduct of a hiking trip" "Extreme situation in a hike". Objectives of the workshop: Objectives of the workshop: To prepare students to solve situational problems using the design method. To prepare students to solve situational problems using the design method. To give the concept of "what is an extreme situation" and to form the rules of personal behavior in the team together with students. To give the concept of "what is an extreme situation" and to form the rules of personal behavior in the team together with students. To promote awareness of self-organization, students ' understanding of collectivism, when solving the problem of survival in the natural environment. To promote awareness of self-organization, students ' understanding of collectivism, when solving the problem of survival in the natural environment. Correctional: to promote the formation of students ' communication skills. Fig. 1. security measures. 8 SHS Web of Conferences 87, 00056 (2020) ICTP 2020 SHS Web of Conferences 87, 00056 (2020) https://doi.org/10.1051/shsconf/20208700056 Problematic situation: the use of theoretical knowledge and skills "Organization and conduct of hiking" and "Behavior in an extreme situation" in practice. Problematic situation: the use of theoretical knowledge and skills Organization and conduct of hiking" and "Behavior in an extreme situation" in practice. Lesson №4 Practical lesson "setting up a Tent" Fig. 2. setting up the tent. The purpose of the lesson: to develop students ' skills to install various types of tents Lesson №4 Practical lesson "setting up a Tent" Lesson №4 Practical lesson "setting up a Tent" Lesson №4 Practical lesson "setting up a Tent" Fig. 2. setting up the tent. The purpose of the lesson: to develop students ' skills to install various types of tents The purpose of the lesson: to develop students ' skills to install various types of tents Tasks: To introduce students to the types of tents. To introduce students to the types of tents. To give primary skills in choosing a place to set up a tent. Demonstration of the installation of a classic tent. Formation of students ' skills to install various types of tents by practicing turnovers on the ground. Formation of students ' skills to install various types of tents by practicing turnovers on ground. 1. The skills of camping. 2. Demonstration of the installation of a classic tent. 3. Demonstration of the installation of a modern tent hemisphere. 3 Conclusions Makarenko considered the relationship of interdependence to be the most important lever of collective cohesion. "The members of the collective, "he said," are not free"," do not move in empty space", they are bound by their obligations or relations with the collective, their duty to the collective... the question of the relationship of a comrade to a comrade is a question of responsible dependence" [15]. knowledge necessary for survival in the modern world, and on the other hand, it will form a society in which people will not be strangers to each other, capable of empathy and mutual assistance. Participation in campaigns, expeditions, meetings, excursions and conferences will encourage the development of friendly relations. Now this part of the educational process is almost lost due to attempts to transfer tourist and local history activities to commercial tracks. Its development, the study of the ethnography, culture, and nature of our multi-ethnic state, promotes tolerance, understanding that we are united, that every shade of diversity of cultures and peoples inhabiting our common Homeland is a significant part of universal prosperity and success. In the consolidation of the tourist team, an important role is played by joint activities in the campaign, in the process of which there are relations of interdependence. A.S. Makarenko considered the relationship of interdependence to be the most important lever of collective cohesion. "The members of the collective, "he said," are not free"," do not move in empty space", they are bound by their obligations or relations with the collective, their duty to the collective... the question of the relationship of a comrade to a comrade is a question of responsible dependence" [15]. All subjects taught in higher education are designed to contain a moral foundation. Students, as future teachers, should be aware of their purpose and distinguish formal education from professionalism through life, excursions, trips to their native land. An important tool in teaching a modern teacher is tourism and local history. Proficient in this subject, the teacher will not only interest students, but also form their professionalism, active citizenship and life position. The introduction of tourism and local history activities in the interdisciplinary courses of higher education institutions will encourage the production of highly qualified professional teachers. 3 Conclusions The education of an active vital position and patriotism, civil responsibility should be specific examples: teaching tolerance - by studying Ethnography, education of patriotism – on the study of history, moral education and intelligence – the study of cultural heritage, etc. All this gives tourism and local history. During hikes and excursions, students not only gain knowledge of geography, history, and culture, but also improve themselves as individuals. Today, tourism and local history activities are a means of comprehensive development of the individual, a means of connecting higher education with society through the regional component. Students who are engaged in tourism and local history are more adaptable to life conditions, stress-resistant, are not afraid to take responsibility for themselves and will always find a way out of extraordinary situations. 3 Conclusions Regular and extracurricular tourist and local history activities will make it possible to produce not only specialists with professional knowledge, but also to get teachers of a new formation who can combine knowledge, an active life position, educational functions. It forms a new generation, figuratively speaking, combining "both lyricists and physicists", professional knowledge and universal values. Only a thinking, competent, adaptive specialist who is able to organize the conditions for achieving results in the labor code will be competitive and in demand in the labor market, will be able to provide knowledge and skills that are simply necessary in modern life. We can see that we are not protected from emergencies, when threats to the life of the population appear suddenly and unexpectedly. Knowledge of tourist and local history activities becomes especially relevant nowadays, and higher education should not be left out of this. Knowing the methods and methods of tourist and local history activities, self- survival techniques, we can successfully apply them. We can organize our safety, our survival not only in the wild, but also in megacities and in general at any point of residence and in any situation. Systematic introduction of the subject “tourist and local history activities" to the training of higher school teachers will make it possible to form a special system of interaction and mutual support among young people. On the one hand, this will provide the 9 SHS Web of Conferences 87, 00056 (2020) SHS Web of Conferences 87, 00056 (2020) ICTP 2020 https://doi.org/10.1051/shsconf/20208700056 knowledge necessary for survival in the modern world, and on the other hand, it will form a society in which people will not be strangers to each other, capable of empathy and mutual assistance. Participation in campaigns, expeditions, meetings, excursions and conferences will encourage the development of friendly relations. Now this part of the educational process is almost lost due to attempts to transfer tourist and local history activities to commercial tracks. Its development, the study of the ethnography, culture, and nature of our multi-ethnic state, promotes tolerance, understanding that we are united, that every shade of diversity of cultures and peoples inhabiting our common Homeland is a significant part of universal prosperity and success. In the consolidation of the tourist team, an important role is played by joint activities in the campaign, in the process of which there are relations of interdependence. A.S. References 1. V.A. Slastenin, Formation of the personality of a Soviet school Teacher in the process of professional training, 160 (Moscow: Prosveshchenie, 1976) 2. N.M. Izoria, Personal-oriented approach to training specialists of foreign language competence in the field of tourism, Innovative technologies for teaching cultural and leisure activities, 161-168 (Moscow, MGUKI, 2007) 3. Federal Law "On Education", 273 (dated December 29, 2012) 4. Federal State Educational Standard of Higher Education - Bachelor's degree in the field of training 03/44/01 Pedagogical education, 121 (dated 02.22.2018) 5. A.A. Ostapets, Pedagogy and psychology of tourist and local history activities of students: methodical edition, 87 (Moscow, 2001) 6. S.O. Schmidt, Local history and documentary monuments, 100 (Tver, 1992) 7. N.I. Reshetnikov, Local historian as a discoverer and Keeper of memory, Bulletin of children and youth tourism in Russia, 2 (26), 16-19 (1998) 10 SHS Web of Conferences 87, 00056 (2020) ICTP 2020 SHS Web of Conferences 87, 00056 (2020) https://doi.org/10.1051/shsconf/20208700056 ICTP 2020 8. I.V. Zorin, A.I. Zorin, Professional education and career in tourism: textbook, 528 (Moscow, 2005) 528 9. I.A. Drogov, Yu.S. Konstantinov, Problems and prospects of social and sports tourism: collection of scientific articles and materials of the International scientific and practical conference, 256 (Moscow: Mosgorsyutur, 2012) 10. G.V. Bukovskaya, Formation of ecological culture of Junior schoolchildren by means of local history and tourism activities, 19 (Moscow, 1997) 11. Z.I. Gordeeva, Goals, tasks and prospects of participation of pedagogical universities in training for tourist and local history activities of schoolchildren, Problems of youth tourism, 1, 42-46 (1998) 12. I.N. Pilat, Extracurricular tourist and local history work as a factor of moral education of teenagers, Autoref. dis. candidate of pedagogical Sciences, 19 (Moscow, 1970) 13. P.I. Istomin, Tourist activity of schoolchildren: Questions of theory and methodology, 94 (Moscow, 1987) 14. Fundamentals of the state youth policy of the Russian Federation for the period up to 2025 Order of November 29 (Moscow, 2014) 15. A.S. Makarenko, Essays. Text: in 7 vols. Akad. PED. Sciences of the RSFSR. Institute of theory and history of pedagogy (Moscow, 1957-1958) 11
https://openalex.org/W3011959663	https://europepmc.org/articles/pmc7146346?pdf=render	English	null	Grandmother’s Diet Matters: Early Life Programming with Sucrose Influences Metabolic and Lipid Parameters in Second Generation of Rats	Nutrients	2,020	cc-by	13,077	Received: 30 January 2020; Accepted: 19 March 2020; Published: 21 March 2020 Abstract: Early life exposure to certain environmental stimuli is related to the development of alternative phenotypes in mammals. A number of these phenotypes are related to an increased risk of disease later in life, creating a massive healthcare burden. With recent focus on the determination of underlying causes of common metabolic disorders, parental nutrition is of great interest, mainly due to a global shift towards a Western-type diet. Recent studies focusing on the increase of food or macronutrient intake don’t always consider the source of these nutrients as an important factor. In our study, we concentrate on the eﬀects of high-sucrose diet, which provides carbohydrates in form of sucrose as opposed to starch in standard diet, fed in pregnancy and lactation in two subsequent generations of spontaneously hypertensive rats (SHR) and congenic SHR-Zbtb16 rats. Maternal sucrose intake increased fasting glycaemia in SHR female oﬀspring in adulthood and increased their chow consumption in gravidity. High-sucrose diet fed to the maternal grandmother increased brown fat weight and HDL cholesterol levels in adult male oﬀspring of both strains, i.e., the grandsons. Fasting glycaemia was however decreased only in SHR oﬀspring. In conclusion, we show the second-generation eﬀects of maternal exposition to a high-sucrose diet, some modulated to a certain extent by variation in the Zbtb16 gene. Keywords: high sucrose diet; rat model; DOHAD; HDL cholesterol; brown fat Keywords: high sucrose diet; rat model; DOHAD; HDL cholesterol; brown fat Nutrients 2020, 12, 846; doi:10.3390/nu12030846 Grandmother’s Diet Matters: Early Life Progra with Sucrose Inﬂuences Metabolic and Lipid Parameters in Second Generation of Rats Elena Školníková , Lucie Šedová and Ondˇrej Šeda * Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and the General University Hospital, 128 00 Prague, Czech Republic; elena.skolnikova@gmail.com (E.Š.); lsedo@lf1.cuni.cz (L.Š.) * Correspondence: oseda@lf1.cuni.cz Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and the General University Hospital, 128 00 Prague, Czech Republic; elena.skolnikova@gmail.com (E.Š.); lsedo@lf1.cuni.cz (L.Š.) * Correspondence: oseda@lf1.cuni.cz Received: 30 January 2020; Accepted: 19 March 2020; Published: 21 March 2020 nutrients nutrients 1. Introduction The early development of a mammalian organism represents a critical time in the determination of health and disease in its adult life. During this period, a speciﬁc time windows occur, when the developing organism is particularly susceptible to environmental exposures capable of changing its phenotype outcome. Nutrition represents one of the most important factors that inﬂuence the highly sensitive developing fetus and does so through a complex of maternal physiology and the uteroplacental unit [1]. The availability and composition of nutrients aﬀect epigenetic marks as they are being established in the developing organism, aﬀect its development through changes in activity of its genes and by modulating metabolic pathways. Diet as well as other environmental factors inﬂuence the establishment or maintenance of speciﬁc epigenetic patterns through the activity of methyltransferases [2] or histone modiﬁcations [3,4]. When it comes to DNA methylation, not only do the activity of methyltransferases matter. The substrate–methyl donors also must be provided by the maternal diet [5]. It’s important to mention that epigenetic modiﬁcations can be maintained after mitotic cell divisions [6,7] and without regard to established pattern, the action of DNMT1 (DNA methyltransferase 1) is responsible for maintenance of these methylation patterns throughout life. That is why early life exposure to www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients Nutrients 2020, 12, 846 2 of 16 Nutrients 2020, 12, 846 environmental stimuli with the capacity to permanently alter epigenome can impact health/disease status later in life [6,7]. On the other hand, the importance of the genomic background of both mother and fetus for the risk of adult disease as well as for the sensitivity to environmental stimuli has also been repeatedly shown [8,9]. Truly transgenerational transfer can however only occur if none of the cell lines are directly inﬂuenced by the environmental stimulus. If F0 generation female is inﬂuenced by a stimulus during pregnancy, the F2 generation germline in developing F1 fetus is aﬀected, and therefore observed eﬀect is usually denoted as intergenerational [10,11]. Due to the raised interest in environmental factors able to alter the mammalian development towards disease in adult life (DOHAD, developmental origins of health and disease), many studies using animal models have focused on understanding the underlying mechanisms in past years. The eﬀects of micronutrient intake and various dietary regimens of parents on postnatal health of the oﬀspring have been extensively reported [12–17]. 1. Introduction Although many studies focus on the pressing problem of parental overnutrition and how it inﬂuences the progeny, the role of individual sources of macronutrients in these eﬀects is yet to be understood. Our study takes a closer look at how the source of carbohydrates (starch versus sucrose) in the diet matters, even though it provides a comparable amount of calories. In this paper, we present the eﬀects apparent in two subsequent generations of oﬀspring of two genetically distinct rat models, focusing on the second generation (F2). In addition to alternative carbohydrate source in maternal diet we introduced a small genetic diﬀerence in Zbtb16 (Zbtb16—Zinc Finger and BTB Domain Containing 16) gene between animals in the study design. Recent studies show that Zbtb16 acts as a pleiotropic factor involved in embryogenesis, cell diﬀerentiation and lipid and glucose metabolism [18]. We and others previously established the importance of Zbtb16 gene in metabolic syndrome and its nutrigenetic aspects [18–20]. In particular, Zbtb16 targeted models of mice [19] and rats [20] show signiﬁcantly reduced levels of triacylglycerols and cholesterol and exhibited lower levels of serum insulin and signiﬁcantly increased sensitivity of adipose and muscle tissue to insulin action, respectively. Therefore, we also tested the hypothesis that genetic variation in Zbtb16 will aﬀect the potential intergenerational eﬀects of sucrose feeding. Since the genetic diﬀerence between the two used strains is only subtle, we opted to enhance homogeneity of the experimental groups by using highly inbred strains of animals and limiting our focus only to male F2 oﬀspring of F1 programmed rat dams. 2. Materials and Methods chow consumed and again their glucose tolerance on the 10th day of pregnancy. Each group of F1 rat dams was fed standard diet whole 3 weeks of pregnancy and 4 weeks of lactation. The litter size was estricted to 8 pups both in SHR and SHR-Zbtb16 offspring which were weaned after 28 days and fed STD till adult age of 6 months creating the F2 generation. At that time, SHR and SHR-Zbtb16 male offspring of both control (n = 8/strain) and experimental (n = 8/strain) groups were subjected to OGTT, blood draw for metabolic and lipid profile assessment and sacrificed to determine the weights of heart, liver, kidneys, adrenals, interscapular brown fat, epididymal and retroperitoneal fat pads. The ipid profile was assessed using high performance liquid chromatography (HPLC) for determining riglyceride (TG) and cholesterol (C) concentrations in 20 lipoprotein fractions and the size of major classes of lipoprotein particles as described previously [25,26]. Figure 1. Experimental design. Schematic display of three generations of rats involved in the experiment valid for both SHR and SHR-Zbtb16 strain. Only F0 mothers of the experimental group Figure 1. Experimental design. Schematic display of three generations of rats involved in the experiment valid for both SHR and SHR-Zbtb16 strain. Only F0 mothers of the experimental group were fed high-sucrose diet. Current study focuses on F1 generation female groups mothering F2 generation males. STD: standard diet, HSD: high-sucrose diet. igure 1. Experimental design. Schematic display of three generations of rats involved in the xperiment valid for both SHR and SHR-Zbtb16 strain. Only F0 mothers of the experimental group Figure 1. Experimental design. Schematic display of three generations of rats involved in the experiment valid for both SHR and SHR-Zbtb16 strain. Only F0 mothers of the experimental group were fed high-sucrose diet. Current study focuses on F1 generation female groups mothering F2 generation males. STD: standard diet, HSD: high-sucrose diet. were fed high sucrose diet. Current study focuses on F1 generation female groups mothering F2 generation males. STD: standard diet, HSD: high-sucrose diet. F1 rat dams (n = 7/strain) were weighed regularly and subjected to OGTT at 16 weeks of age to determine the diﬀerences in glucose tolerance after being metabolically programmed by their maternal diet—either STD or HSD. Blood samples for metabolic and glycemic assessments were drawn after overnight fasting from the tail vein. 2. Materials and Methods Experiments were performed in agreement with the Animal Protection Law of the Czech Republic (311/1997) which is in compliance with the European Community Council recommendations for the use of laboratory animals 86/609/ECC and were approved by the Ethical Committee of the First Faculty of Medicine of the Charles University and by the Ministry of Education, Youth and Sports (protocol no. MSMT-14076/2015-14). We used the spontaneously hypertensive rat (SHR/OlaIpcv, RGD ID 631848) because of its inherent abnormal metabolic proﬁle [21] and its sensitivity manipulation by diet [22]. The SHR-Lx.PD5PD-Zbtb16 single congenic strain (SHR-Zbtb16 hereafter) was derived from polydactylous rat (PD/Cub, RGD ID 728161) [23] and carries the Zbtb16 gene of PD origin on the SHR genomic background [24,25]. Both strains are highly inbred and maintained by brother x sister mating at the Institute of Biology and Medical Genetics. All animals were held under controlled conditions (temperature, humidity) with free access to food and water. F0 generation of rat dams (grandmothers in this study) of SHR and SHR-Zbtb16 strains (n = 6/strain) came from standard breeding and were fed a standard diet till the age of 16 weeks when they entered the experimental protocol. To recapitulate, F0 rat dams were fed standard diet till breeding with corresponding (SHR × SHR, SHR-Zbtb16 × SHR-Zbtb16) males fed standard diet. After mating, rat dams were placed in the cages individually and fed either standard diet (STD, ssniﬀRat 3 of 16 med, nt of Nutrients 2020, 12, 846 with corresponding t id th i breeding V1324-000, ssniﬀSpezialdiäten GmbH, Soest, Germany) in control group or high-sucrose diet (HSD, proteins (19.6 cal%), fat (10.4 cal%), carbohydrates (sucrose, 70 cal%) [23] prepared by Institute for Clinical and Experimental Medicine, Prague, Czech Republic) in experimental group (Figure 1). The diets diﬀered in the carbohydrate fraction only, with starch in STD vs. sucrose in HSD as a source of carbohydrates; otherwise they contained equal amounts of macro- and micronutrients. Each group was fed either STD or HSD throughout pregnancy and lactation. The litter size was restricted to 8 pups both in SHR and SHR-Zbtb16 oﬀspring which were weaned after 28 days and fed standard diet till adulthood. Female oﬀspring of F0 generation were fed standard diet till the age of 4 months and used as F1 generation (mothers in this study) in the same breeding model as their mothers. 2. Materials and Methods The blood samples were obtained at intervals of 0, 30, 60, 120, and 180 min after intragastric glucose administration to conscious rats (3 g/kg body weight, 30% aqueous solution; Ascensia Elite Blood Glucose Meter; Bayer HealthCare, Mishawaka, IN, USA; validated by the Institute of Clinical Biochemistry and Laboratory Diagnostics of the First Faculty of Medicine). After the assessment of their adult glucose tolerance, the rat dams were mated with corresponding STD-fed males ((SHR x SHR, SHR-Zbtb16 x SHR-Zbtb16), not programmed, outside the experiment) and placed individually in cages. We assessed their weight gain, amount of chow consumed and again their glucose tolerance on the 10th day of pregnancy. Each group of F1 rat dams was fed standard diet whole 3 weeks of pregnancy and 4 weeks of lactation. The litter size was restricted to 8 pups both in SHR and SHR-Zbtb16 oﬀspring which were weaned after 28 days and fed STD till adult age of 6 months creating the F2 generation. At that time, SHR and SHR-Zbtb16 male oﬀspring of both control (n = 8/strain) and experimental (n = 8/strain) groups were subjected to OGTT, blood draw for metabolic and lipid proﬁle assessment and sacriﬁced to determine the weights of heart, liver, kidneys, adrenals, interscapular brown fat, epididymal and retroperitoneal fat pads. The lipid proﬁle was assessed using high performance liquid chromatography (HPLC) for determining triglyceride (TG) and cholesterol (C) concentrations in 20 lipoprotein fractions and the size of major classes of lipoprotein particles as described previously [25,26]. 4 of 16 4 of 16 Wh Nutrients 2020, 12, 846 u ie s 0 0, , O All statistical analyses were performed using STATISTICA 13.3 (TIBCO Software Inc.). When comparing morphometric and biochemical variables between groups, two-way ANOVA with STRAIN and DIET as major factors were used, followed by post-hoc Fisher’s test for comparison of the speciﬁc pairs of variables. Null hypothesis was rejected whenever p > 0.05. comparing morphometric and biochemical variables between groups, two-way ANOVA with STRAIN and DIET as major factors were used, followed by post-hoc Fisher’s test for comparison of the specific pairs of variables. Null hypothesis was rejected whenever p > 0.05. 3. Results 3.1. F1 Female Rats—Mothers There was a strain differ 3.1. F1 Female Rats—Mothers There was a strain differ There was a strain diﬀerence between control groups of female SHR F1 (Figure 2a) and SHR-Zbtb16 F1 rat dams (Figure 2c) in glucose tolerance prior to pregnancy (corresponding to week 4 in Figure 3). The diﬀerence in glucose tolerance between the strains diminished during pregnancy (Figure 2b,d). Zbtb16 F1 rat dams (Figure 2c) in glucose tolerance prior to pregnancy (corresponding to week 4 in Figure 3). The difference in glucose tolerance between the strains diminished during pregnancy (Figure 2b, d). (a) (b) Figure 2. Cont. (a) (b) 5 of 16 Nutrients 2020, 12, 846 Nutrients 2020, 12 (c) (d) Figure 2. The oral glucose tolerance test (OGTT). The course of glycaemic curves in rat dams in adulthood one week prior (a, c) and during pregnancy (b, d); SHR F1 controls—black boxes, F1 programmed SHR—blue boxes, SHR-Zbtb16 F1 controls—white circles, F1 programmed SHR- Zbtb16—green circles. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.05, p < 0.01, * p < 0.001. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. 3.1. F1 Female Rats—Mothers There was a strain differ (a) C represents STRAIN differences between SHR and SHR-Zbtb16 F1 controls in adulthood before the pregnancy, t = 30 min CCC p < 0.001 , t = 60 min C p < 0.05 , AUC180 CC p < 0.01, which were all higher in SHR; (b) # represents differences in non-pregnant and pregnant SHR F1 controls (effect of pregnancy), t = 0 min ## p < 0.01, higher in pregnant SHR F1 controls, t = 30 min ##, AUC180 ### p < 0.001 , higher in non-pregnant SHR F1 controls, + represents differences in non-pregnant and pregnant SHR F1- programmed dams (effect of pregnancy), t = 0 min ++ p < 0.01 , t = 90 min ++, which were all higher in non-pregnant SHR F1-programmed dams; (c) F represents strain differences between SHR and SHR- Zbtb16 F1-programmed dams in adulthood before the pregnancy, t = 0 min , t = 60 min , which were both higher in SHR F1-programmed dams; (d) C represents STRAIN differences between SHR and SHR-Zbtb16 F1 pregnant controls, AUC180 C p < 0.05, which was higher in SHR F1 controls, # represents differences in non-pregnant and pregnant SHR-Zbtb16 F1 controls (effect of pregnancy), t = 0 min ### p < 0.001, t = 180 min # p < 0.05, higher in pregnant SHR-Zbtb16 F1 controls, AUC180 # p < Figure 2. The oral glucose tolerance test (OGTT). The course of glycaemic curves in rat dams in adulthood one week prior (a,c) and during pregnancy (b,d); SHR F1 controls—black boxes, F1 programmed SHR—blue boxes, SHR-Zbtb16 F1 controls—white circles, F1 programmed SHR-Zbtb16—green circles. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 0.001. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. 3.1. F1 Female Rats—Mothers There was a strain differ (a) C represents STRAIN diﬀerences between SHR and SHR-Zbtb16 F1 controls in adulthood before the pregnancy, t = 30 min CCC p < 0.001 , t = 60 min C p < 0.05 , AUC180 CC p < 0.01, which were all higher in SHR; (b) # represents diﬀerences in non-pregnant and pregnant SHR F1 controls (eﬀect of pregnancy), t = 0 min ## p < 0.01, higher in pregnant SHR F1 controls, t = 30 min ##, AUC180 ### p < 0.001 , higher in non-pregnant SHR F1 controls, + represents diﬀerences in non-pregnant and pregnant SHR F1-programmed dams (eﬀect of pregnancy), t = 0 min ++ p < 0.01 , t = 90 min ++, which were all higher in non-pregnant SHR F1-programmed dams; (c) F represents strain diﬀerences between SHR and SHR-Zbtb16 F1-programmed dams in adulthood before the pregnancy, t = 0 min , t = 60 min , which were both higher in SHR F1-programmed dams; (d) C represents STRAIN diﬀerences between SHR and SHR-Zbtb16 F1 pregnant controls, AUC180 C p < 0.05, which was higher in SHR F1 controls, # represents diﬀerences in non-pregnant and pregnant SHR-Zbtb16 F1 controls (eﬀect of pregnancy), t = 0 min ### p < 0.001, t = 180 min # p < 0.05, higher in pregnant SHR-Zbtb16 F1 controls, AUC180 # p < 0.05, higher in non-pregnant SHR-Zbtb16 F1 controls, + represents diﬀerences in non-pregnant and pregnant SHR-Zbtb16 F1-programmed dams (eﬀect of pregnancy), t = 90 min ++ p < 0.01, t = 120 min + p < 0.05, which were all higher in non-pregnant SHR-Zbtb16 F1-programmed dams. (c) (d) (d) Figure 2. The oral glucose tolerance test (OGTT). The course of glycaemic curves in rat dams in adulthood one week prior (a, c) and during pregnancy (b, d); SHR F1 controls—black boxes, F1 programmed SHR—blue boxes, SHR-Zbtb16 F1 controls—white circles, F1 programmed SHR- Zbtb16—green circles. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 0.001. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. 3.1. F1 Female Rats—Mothers There was a strain differ (a) C represents STRAIN differences between SHR and SHR-Zbtb16 F1 controls in adulthood before the pregnancy, t = 30 min CCC p < 0.001 , t = 60 min C p < 0.05 , AUC180 CC p < 0.01, which were all higher in SHR; (b) # represents differences in non-pregnant and pregnant SHR F1 controls (effect of pregnancy), t = 0 min ## p < 0.01, higher in pregnant SHR F1 controls, t = 30 min ##, AUC180 ### p < 0.001 , higher in non-pregnant SHR F1 controls, + represents differences in non-pregnant and pregnant SHR F1- programmed dams (effect of pregnancy), t = 0 min ++ p < 0.01 , t = 90 min ++, which were all higher in non-pregnant SHR F1-programmed dams; (c) F represents strain differences between SHR and SHR- Zbtb16 F1-programmed dams in adulthood before the pregnancy, t = 0 min , t = 60 min , which were both higher in SHR F1-programmed dams; (d) C represents STRAIN differences between SHR and SHR-Zbtb16 F1 pregnant controls, AUC180 C p < 0.05, which was higher in SHR F1 controls, # represents differences in non-pregnant and pregnant SHR-Zbtb16 F1 controls (effect of pregnancy), t = 0 min ### p < 0.001, t = 180 min # p < 0.05, higher in pregnant SHR-Zbtb16 F1 controls, AUC180 # p < Figure 2. The oral glucose tolerance test (OGTT). The course of glycaemic curves in rat dams in adulthood one week prior (a,c) and during pregnancy (b,d); SHR F1 controls—black boxes, F1 programmed SHR—blue boxes, SHR-Zbtb16 F1 controls—white circles, F1 programmed SHR-Zbtb16—green circles. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 0.001. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. 3.1. F1 Female Rats—Mothers There was a strain differ (a) C represents STRAIN diﬀerences between SHR and SHR-Zbtb16 F1 controls in adulthood before the pregnancy, t = 30 min CCC p < 0.001 , t = 60 min C p < 0.05 , AUC180 CC p < 0.01, which were all higher in SHR; (b) # represents diﬀerences in non-pregnant and pregnant SHR F1 controls (eﬀect of pregnancy), t = 0 min ## p < 0.01, higher in pregnant SHR F1 controls, t = 30 min ##, AUC180 ### p < 0.001 , higher in non-pregnant SHR F1 controls, + represents diﬀerences in non-pregnant and pregnant SHR F1-programmed dams (eﬀect of pregnancy), t = 0 min ++ p < 0.01 , t = 90 min ++, which were all higher in non-pregnant SHR F1-programmed dams; (c) F represents strain diﬀerences between SHR and SHR-Zbtb16 F1-programmed dams in adulthood before the pregnancy, t = 0 min , t = 60 min , which were both higher in SHR F1-programmed dams; (d) C represents STRAIN diﬀerences between SHR and SHR-Zbtb16 F1 pregnant controls, AUC180 C p < 0.05, which was higher in SHR F1 controls, # represents diﬀerences in non-pregnant and pregnant SHR-Zbtb16 F1 controls (eﬀect of pregnancy), t = 0 min ### p < 0.001, t = 180 min # p < 0.05, higher in pregnant SHR-Zbtb16 F1 controls, AUC180 # p < 0.05, higher in non-pregnant SHR-Zbtb16 F1 controls, + represents diﬀerences in non-pregnant and pregnant SHR-Zbtb16 F1-programmed dams (eﬀect of pregnancy), t = 90 min ++ p < 0.01, t = 120 min + p < 0.05, which were all higher in non-pregnant SHR-Zbtb16 F1-programmed dams. Figure 2. The oral glucose tolerance test (OGTT). The course of glycaemic curves in rat dams in d lth d k i ( ) d d i (b d) SHR F1 t l bl k b F1 Figure 2. The oral glucose tolerance test (OGTT). The course of glycaemic curves in rat dams in adulthood 6 of 16 7 of 16 Nutrients 2020, 12, 846 Nutrients 2020, 12, x FO Figure 3. Body weight measurements of F1 SHR and SHR-Zbtb16 adult female rats during weeks prior to pregnancy (weeks 1–4), in pregnancy (weeks 5–7) and during lactation (weeks 8–11). Delivery occurred between weeks 7 and 8. SHR F1 controls—black boxes, F1-programmed SHR—blue boxes, SHR-Zbtb16 F1 controls—white circles, F1-programmed SHR-Zbtb16—green circles. Data are expressed as mean ± SEM. 3.1. F1 Female Rats—Mothers There was a strain differ The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, ** p < 0.01. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. F represents strain differences between F1-programmed Figure 3. Body weight measurements of F1 SHR and SHR-Zbtb16 adult female rats during weeks prior to pregnancy (weeks 1–4), in pregnancy (weeks 5–7) and during lactation (weeks 8–11). Delivery occurred between weeks 7 and 8. SHR F1 controls—black boxes, F1-programmed SHR—blue boxes, SHR-Zbtb16 F1 controls—white circles, F1-programmed SHR-Zbtb16—green circles. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, ** p < 0.01. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. F represents strain diﬀerences between F1-programmed SHR and SHR-Zbtb16 dams, week 2 , week 3 , week 4 , which were all higher in SHR F1-programmed dams. Figure 3. Body weight measurements of F1 SHR and SHR-Zbtb16 adult female rats during weeks prior to pregnancy (weeks 1–4), in pregnancy (weeks 5–7) and during lactation (weeks 8–11). Delivery occurred between weeks 7 and 8. SHR F1 controls—black boxes, F1-programmed SHR—blue boxes, SHR-Zbtb16 F1 controls—white circles, F1-programmed SHR-Zbtb16—green circles. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.05, ** p < 0.01. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. F represents strain differences between F1-programmed Figure 3. Body weight measurements of F1 SHR and SHR-Zbtb16 adult female rats during weeks prior to pregnancy (weeks 1–4), in pregnancy (weeks 5–7) and during lactation (weeks 8–11). Delivery occurred between weeks 7 and 8. SHR F1 controls—black boxes, F1-programmed SHR—blue boxes, SHR-Zbtb16 F1 controls—white circles, F1-programmed SHR-Zbtb16—green circles. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, ** p < 0.01. 3.1. F1 Female Rats—Mothers There was a strain differ Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. F represents strain diﬀerences between F1-programmed SHR and SHR-Zbtb16 dams, week 2 , week 3 , week 4 , which were all higher in SHR F1-programmed dams. SHR and SHR-Zbtb16 dams, week 2 , week 3 , week 4 , which were all higher in SHR F1- programmed dams. Both groups of rat dams programmed with maternal HSD exhibited signiﬁcantly higher levels of fasting blood glucose compared to the control groups. The eﬀect of pregnancy itself resulted in smaller area under the glycaemic curve in HSD-programmed groups (Table 1), as their response to glucose load had decreased. HSD-programmed females of both strains showed higher levels of fasting plasma insulin prior to pregnancy in comparison to control groups (Figure 4). Table 1. Areas under glycaemic curves (AUC) values calculated from course of oral glucose tolerance tests in SHR and SHR-Zbtb16 mothers and their male oﬀspring according to Heikkinen et al. [27]. Table 1. Areas under glycaemic curves (AUC) values calculated from course of oral glucose tolerance tests in SHR and SHR-Zbtb16 mothers and their male oﬀspring according to Heikkinen et al. [27]. Figure 4. Fasting insulin concentrations in SHR F1 control females (dark grey bars), SHR-Zbt control females (light grey bars), F1-programmed SHR females (blue bars), F1-programmed Zbtb16 females (green bars) in adulthood. The strain comparison using the post-hoc Fisher’ significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major fact indicated as follows: * p < 0.01, *** p < 0.001. Effect of programming in SHR and SHR-Zbtb16 is represented by blue and green asterisks (), respectively. tests in SHR and SHR-Zbtb16 mothers and their male oﬀspring according to Heikkinen et al. [2 AUC (mmol/L/180 min), mean ± SEM Adulthood Pregnancy F1 mothers SHR F1 control 587 ± 76 347 ± 44 SHR-Zbtb16 F1 control 383 ± 42 211 ± 43 SHR F1 programmed 385 ± 38 280 ± 32 SHR-Zbtb16 F1 programmed 345 ± 42 244 ± 36 F2 male oﬀspring SHR F2 control 372 ± 32 SHR-Zbtb16 F2 control 458 ± 19 SHR F2 programmed 379 ± 16 SHR-Zbtb16 F2 programmed 356 ± 21 7 of 16 1 Nutrients 2020, 12, 846 SHR and SHR Z programmed dam trients 2020, 12, 846 7 of g programmed dams. Figure 4. 3.1. F1 Female Rats—Mothers There was a strain differ Fasting insulin concentrations in SHR F1 control females (dark grey bars), SHR-Zbtb16 F1 control females (light grey bars), F1-programmed SHR females (blue bars), F1-programmed SHR- Zbtb16 females (green bars) in adulthood. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01, *** p < 0.001. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. Figure 4. Fasting insulin concentrations in SHR F1 control females (dark grey bars), SHR-Zbtb16 F1 control females (light grey bars), F1-programmed SHR females (blue bars), F1-programmed SHR-Zbtb16 females (green bars) in adulthood. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01, *** p < 0.001. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. Figure 4 Fa ti i uli o e t atio i SHR F1 o t ol fe ale (da k ey ba ) SHR Zbtb16 F1 Figure 4. Fasting insulin concentrations in SHR F1 control females (dark grey bars), SHR-Zbtb16 F1 Figure 4. Fasting insulin concentrations in SHR F1 control females (dark grey bars), SHR-Zbtb16 F1 control females (light grey bars), F1-programmed SHR females (blue bars), F1-programmed SHR- Zbtb16 females (green bars) in adulthood. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01, *** p < 0.001. Effect of programming in SHR and SHR-Zbtb16 strains i e e e ted by blue a d ee a te i k () e e ti ely Figure 4. Fasting insulin concentrations in SHR F1 control females (dark grey bars), SHR-Zbtb16 F1 control females (light grey bars), F1-programmed SHR females (blue bars), F1-programmed SHR-Zbtb16 females (green bars) in adulthood. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01, *** p < 0.001. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. Figure 4. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by After delivery we didn’t observe any significant differences in litter size or birth weight of the pups among the groups. Adult F2 male offspring of HSD—programmed mothers did not differ in body weight from control animals, however, SHR were heavier than SHR-Zbtb16, similar to their mothers (body weight, 312 ± 7 g in SHR F2 controls vs. 298 ± 3 g in SHR-Zbtb16 F2 controls, p = 0.01; 321 ± 3 g in SHR F2 programmed males vs. 301 ± 5 g in SHR-Zbtb16 F2 programmed males, p = 0.03). Morphometric analysis of the animals revealed significantly decreased liver mass/100g of body weight in F2 programmed animals with more prominent effect in SHR males (Figure 6b). Second- generation programming affected body fat distribution with increased weight of interscapular brown fat pad (Figure 6c) compared to respective control groups. While the F2 control SHR-Zbtb16 male offspring had lower weight of epididymal fat/100 g of body weight than F2 control SHR offspring, there was no difference between the animals from the F2 programmed groups (Figure 6d). After delivery we didn’t observe any signiﬁcant diﬀerences in litter size or birth weight of the pups among the groups. Adult F2 male oﬀspring of HSD—programmed mothers did not diﬀer in body weight from control animals, however, SHR were heavier than SHR-Zbtb16, similar to their mothers (body weight, 312 ± 7 g in SHR F2 controls vs. 298 ± 3 g in SHR-Zbtb16 F2 controls, p = 0.01; 321 ± 3 g in SHR F2 programmed males vs. 301 ± 5 g in SHR-Zbtb16 F2 programmed males, p = 0.03). Morphometric analysis of the animals revealed signiﬁcantly decreased liver mass/100g of body weight in F2 programmed animals with more prominent eﬀect in SHR males (Figure 6b). Second-generation programming aﬀected body fat distribution with increased weight of interscapular brown fat pad (Figure 6c) compared to respective control groups. While the F2 control SHR-Zbtb16 male oﬀspring had lower weight of epididymal fat/100 g of body weight than F2 control SHR oﬀspring, there was no diﬀerence between the animals from the F2 programmed groups (Figure 6d). 3.2. F2 Adult Male Offspring After delivery we didn’t observe any significant differences in litter size or birth weight of the pups among the groups. 3.1. F1 Female Rats—Mothers There was a strain differ Fasting insulin concentrations in SHR F1 control females (dark grey bars), SHR-Zbtb16 F1 control females (light grey bars), F1-programmed SHR females (blue bars), F1-programmed SHR- Zbtb16 females (green bars) in adulthood. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01, *** p < 0.001. Effect of programming in SHR and SHR-Zbtb16 strains i t d b bl d t i k () ti l Figure 4. Fasting insulin concentrations in SHR F1 control females (dark grey bars), SHR-Zbtb16 F1 control females (light grey bars), F1-programmed SHR females (blue bars), F1-programmed SHR-Zbtb16 females (green bars) in adulthood. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01, *** p < 0.001. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. As denoted in Figure 2, we observed several signiﬁcant diﬀerences in oral glucose tolerance test (OGTT) data between strains prior or during the pregnancy with glycaemia being higher in SHR compared to SHR-Zbtb16. Control groups of female rats of both strains showed similar weight during whole 11-week period of measurement, as opposed to HSD -programmed females showing slight strain diﬀerences prior to gravidity (Figure 3; body weight 3 weeks before pregnancy 160 ± 5 g in SHR vs. 154 ± 5 g in SHR-Zbtb16, p = 0.046; body weight 2 weeks before pregnancy, 165±5 g in SHR vs. 161±4 in SHR-Zbtb16, p = 0.033; body weight 1 week before pregnancy 171 ± 4 g in SHR vs. 166 ± 4 g in SHR-Zbtb16, p = 0.03) as SHR dams were heavier. The most evident trend was the body weight diﬀerence between adult control and HSD-programmed F1 SHR-Zbtb16 females at the same age, reaching as far as the second week of pregnancy, when the diﬀerence became nonsigniﬁcant and remained so until the period after delivery (Figure 3). Interestingly, HSD-programmed rat dams of both strains ingested signiﬁcantly larger amounts of chow during pregnancy and thus increased their caloric intake compared to control groups (Figure 5). utrients 2020, 12, 846 8 of Nutrients 2020, 12, x FOR PEER REVIEW 8 of 16 Figure 5. 3.1. F1 Female Rats—Mothers There was a strain differ Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. Figure 5. Diet consumption of F1 SHR and SHR-Zbtb16 adult female rats in gravidity. SHR F1 control females (grey bars), SHR-Zbtb16 F1 control females (grey patterned bars), F1-programmed SHR females (blue bars), F1-programmed SHR-Zbtb16 females (green patterned bars) daily consumption throughout 3 weeks of gravidity. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: ** p < 0.01. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. Figure 5. Diet consumption of F1 SHR and SHR-Zbtb16 adult female rats in gravidity. SHR F1 control females (grey bars), SHR-Zbtb16 F1 control females (grey patterned bars), F1-programmed SHR females (blue bars), F1-programmed SHR-Zbtb16 females (green patterned bars) daily consumption throughout 3 weeks of gravidity. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01. Effect of programming in SHR and SHR-Zbtb16 3.1. F1 Female Rats—Mothers There was a strain differ Diet consumption of F1 SHR and SHR-Zbtb16 adult female rats in gravidity. SHR F1 control females (grey bars), SHR-Zbtb16 F1 control females (grey patterned bars), F1-programmed SHR females (blue bars), F1-programmed SHR-Zbtb16 females (green patterned bars) daily consumption throughout 3 weeks of gravidity. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: ** p < 0.01. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. Figure 5. Diet consumption of F1 SHR and SHR-Zbtb16 adult female rats in gravidity. SHR F1 control females (grey bars), SHR-Zbtb16 F1 control females (grey patterned bars), F1-programmed SHR females (blue bars), F1-programmed SHR-Zbtb16 females (green patterned bars) daily consumption throughout 3 weeks of gravidity. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. Nutrients 2020, 12, x FOR PEER REVIEW 8 of 16 Figure 5. Diet consumption of F1 SHR and SHR-Zbtb16 adult female rats in gravidity. SHR F1 control females (grey bars), SHR-Zbtb16 F1 control females (grey patterned bars), F1-programmed SHR females (blue bars), F1-programmed SHR-Zbtb16 females (green patterned bars) daily consumption throughout 3 weeks of gravidity. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01. Effect of programming in SHR and SHR-Zbtb16 d b bl d k () l 8 of 16 16 Nutrients 2020, 12, 846 Nutrients 2020 12 x EVIEW Figure 5. Diet consumption of F1 SHR and SHR-Zbtb16 adult female rats in gravidity. SHR F1 control females (grey bars), SHR-Zbtb16 F1 control females (grey patterned bars), F1-programmed SHR females (blue bars), F1-programmed SHR-Zbtb16 females (green patterned bars) daily consumption throughout 3 weeks of gravidity. Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.01. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by Adult F2 male offspring of HSD—programmed mothers did not differ in body weight from control animals, however, SHR were heavier than SHR-Zbtb16, similar to their mothers (body weight, 312 ± 7 g in SHR F2 controls vs. 298 ± 3 g in SHR-Zbtb16 F2 controls, p = 0.01; 321 ± 3 g in SHR F2 programmed males vs. 301 ± 5 g in SHR-Zbtb16 F2 programmed males, p = 0.03). Morphometric analysis of the animals revealed significantly decreased liver mass/100g of body weight in F2 programmed animals with more prominent effect in SHR males (Figure 6b). Second- generation programming affected body fat distribution with increased weight of interscapular brown fat pad (Figure 6c) compared to respective control groups. While the F2 control SHR-Zbtb16 male offspring had lower weight of epididymal fat/100 g of body weight than F2 control SHR offspring, there was no difference between the animals from the F2 programmed groups (Figure 6d). (a) (b) (a) (b) Figure 6. Cont. (b) (a) (b) Figure 6. Cont. 9 of 16 9 of 16 Nutrients 2020, 12, 846 Nutrients 2020, 12, x FOR (c) (d) Figure 6. Morphometric assessment. Retroperitoneal fat pad weight (a), liver weight (b), interscapular brown fat weight (c), epidydimal fat pad weight (d) per 100 g of body weight in adult SHR F2 control male offspring (dark grey bars), SHR-Zbtb16 F2 control male offspring (light grey bars), F2- programmed SHR male offspring (blue patterned bars), F2-programmed SHR-Zbtb16 male offspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.05, ** p < 0.01. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. C represents STRAIN differences between F2 SHR and SHR-Zbtb16 control males, RPFP/100g BW CC p < 0.01, higher in SHR F2 control males; EFP/100 g BW C p < 0.05, higher in SHR F2 control males. F represents STRAIN differences between F2-programmed SHR and SHR-Zbtb16 males, RPFP/100 g BW , higher in SHR F2-programmed Figure 6. Morphometric assessment. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by F represents STRAIN differences between F2-programmed SHR and SHR-Zbtb16 males, RPFP/100 g BW , higher in SHR F2-programmed Figure 6. Morphometric assessment. Retroperitoneal fat pad weight (a), liver weight (b), interscapular brown fat weight (c), epidydimal fat pad weight (d) per 100 g of body weight in adult SHR F2 control male oﬀspring (dark grey bars), SHR-Zbtb16 F2 control male oﬀspring (light grey bars), F2-programmed SHR male oﬀspring (blue patterned bars), F2-programmed SHR-Zbtb16 male oﬀspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, ** p < 0.01. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. C represents STRAIN diﬀerences between F2 SHR and SHR-Zbtb16 control males, RPFP/100g BW CC p < 0.01, higher in SHR F2 control males; EFP/100 g BW C p < 0.05, higher in SHR F2 control males. F represents STRAIN diﬀerences between F2-programmed SHR and SHR-Zbtb16 males, RPFP/100 g BW , higher in SHR F2-programmed males. (c) (d) Figure 6. Morphometric assessment. Retroperitoneal fat pad weight (a), liver weight (b), interscapular brown fat weight (c), epidydimal fat pad weight (d) per 100 g of body weight in adult SHR F2 control male offspring (dark grey bars), SHR-Zbtb16 F2 control male offspring (light grey bars), F2- programmed SHR male offspring (blue patterned bars), F2-programmed SHR-Zbtb16 male offspring males. F2 programmed SHR males displayed lower fasting glycaemia compared to their control group (Figure 7a), with no corresponding difference present in SHR-Zbtb16. F2 programmed SHR-Zbtb16 males displayed a biphasic OGTT like their F1 programmed mothers (Figure 7b), although AUC was not significantly different between the groups (Figure 7c, Table 1). In both control and programmed groups, we observed higher glycemia in SHR compared to SHR-Zbtb16 three hours after the glucose bolus. Fasting plasma insulin concentrations of adult offspring showed no difference between the control groups and a strain-specific increase of insulin levels in F2 programmed group of SHR-Zbtb16 (Figure 8). (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.05, ** p < 0.01. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by Retroperitoneal fat pad weight (a), liver weight (b), interscapular brown fat weight (c), epidydimal fat pad weight (d) per 100 g of body weight in adult SHR F2 control male oﬀspring (dark grey bars), SHR-Zbtb16 F2 control male oﬀspring (light grey bars), F2-programmed SHR male oﬀspring (blue patterned bars), F2-programmed SHR-Zbtb16 male oﬀspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.05, ** p < 0.01. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. C represents STRAIN diﬀerences between F2 SHR and SHR-Zbtb16 control males, RPFP/100g BW CC p < 0.01, higher in SHR F2 control males; EFP/100 g BW C p < 0.05, higher in SHR F2 control males. F represents STRAIN diﬀerences between F2-programmed SHR and SHR-Zbtb16 males, RPFP/100 g BW , higher in SHR F2-programmed males. Nutrients 2020, 12, x FOR PEER REVIEW 9 of 16 (c) (d) Figure 6. Morphometric assessment. Retroperitoneal fat pad weight (a), liver weight (b), interscapular brown fat weight (c), epidydimal fat pad weight (d) per 100 g of body weight in adult SHR F2 control male offspring (dark grey bars), SHR-Zbtb16 F2 control male offspring (light grey bars), F2- programmed SHR male offspring (blue patterned bars) F2-programmed SHR-Zbtb16 male offspring (d) 9 of 16 (c) (d) (d) (c) Figure 6. Morphometric assessment. Retroperitoneal fat pad weight (a), liver weight (b), interscapular brown fat weight (c), epidydimal fat pad weight (d) per 100 g of body weight in adult SHR F2 control male offspring (dark grey bars), SHR-Zbtb16 F2 control male offspring (light grey bars), F2- programmed SHR male offspring (blue patterned bars), F2-programmed SHR-Zbtb16 male offspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.05, ** p < 0.01. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. C represents STRAIN differences between F2 SHR and SHR-Zbtb16 control males, RPFP/100g BW CC p < 0.01, higher in SHR F2 control males; EFP/100 g BW C p < 0.05, higher in SHR F2 control males. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. C represents STRAIN differences between F2 SHR and SHR-Zbtb16 control males, RPFP/100g BW CC p < 0.01, higher in SHR F2 control males; EFP/100 g BW C p < 0.05, higher in SHR F2 control males. F represents STRAIN differences between F2-programmed SHR and SHR-Zbtb16 males, RPFP/100 g BW , higher in SHR F2-programmed males. (a) (a) Figure 7. Cont. (a) Figure 7. Cont. 10 of 16 0 of 16 Nutrients 2020, 12, 846 Nutrients 2020, (b) (c) Figure 7. The oral glucose tolerance test (OGTT). The course of glycaemic curves in SHR F2 male offspring (a, control – black circles, programmed – blue circles), SHR-Zbtb16 F2 male offspring (b, control – black triangles, programmed – green triangles) during the oral glucose tolerance test and the corresponding areas under the glycaemic curves (c, AUC180). Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 0.001. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. (a) C represents STRAIN differences between F2 SHR and SHR-Zbtb16 control males, t = 0 min CCC p < 0.001, t = 30 min C p < 0.05, t = 180 min C, which were all higher in SHR F2 control males. (b) F represents STRAIN differences between F2-programmed SHR and SHR- Zbtb16 males, t = 60 min , t = 180 min , which were both higher in SHR F2-programmed males; (c) C represents STRAIN differences between F2 SHR and SHR-Zbtb16 control males, AUC180 C p < 0.05, higher in SHR-Zbtb16 F2 control males. F2 programmed SHR males displayed lower fasting glycaemia compared to their control group Figure 7. The oral glucose tolerance test (OGTT). The course of glycaemic curves in SHR F2 male oﬀspring (a, control – black circles, programmed – blue circles), SHR-Zbtb16 F2 male oﬀspring (b, control – black triangles, programmed – green triangles) during the oral glucose tolerance test and the corresponding areas under the glycaemic curves (c, AUC180). Data are expressed as mean ± SEM. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by The course of glycaemic curves in SHR F2 male ﬀspring (a, control – black circles, programmed – blue circles), SHR-Zbtb16 F2 male oﬀspring (b, ontrol – black triangles, programmed – green triangles) during the oral glucose tolerance test and the orresponding areas under the glycaemic curves (c, AUC180). Data are expressed as mean ± SEM. The rain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA r STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 001. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks ), respectively. (a) C represents STRAIN diﬀerences between F2 SHR and SHR-Zbtb16 control males, t 0 min CCC p < 0.001, t = 30 min C p < 0.05, t = 180 min C, which were all higher in SHR F2 control ales. (b) F represents STRAIN diﬀerences between F2-programmed SHR and SHR-Zbtb16 males, = 60 min , t = 180 min , which were both higher in SHR F2-programmed males; (c) C represents (b) (c) (b) (c) Figure 7 The oral glucose tolerance test (OGTT) The course of glycaemic curves in SHR F2 male Fi Th l l l (OGTT) Th f l i i SHR F2 (b) Figure 7. The oral glucose tolerance test (OGTT). The course of glycaemic curves in SHR F2 male offspring (a, control – black circles, programmed – blue circles), SHR-Zbtb16 F2 male offspring (b, control – black triangles, programmed – green triangles) during the oral glucose tolerance test and the corresponding areas under the glycaemic curves (c, AUC180). Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.05, p < 0.01, * p < 0.001. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. (a) C represents STRAIN differences between F2 SHR and SHR-Zbtb16 control males, t = 0 min CCC p < 0.001, t = 30 min C p < 0.05, t = 180 min C, which were all higher in SHR F2 control males. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 0.001. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. (a) C represents STRAIN diﬀerences between F2 SHR and SHR-Zbtb16 control males, t = 0 min CCC p < 0.001, t = 30 min C p < 0.05, t = 180 min C, which were all higher in SHR F2 control males. (b) F represents STRAIN diﬀerences between F2-programmed SHR and SHR-Zbtb16 males, t = 60 min , t = 180 min , which were both higher in SHR F2-programmed males; (c) C represents STRAIN diﬀerences between F2 SHR and SHR-Zbtb16 control males, AUC180 C p < 0.05, higher in SHR-Zbtb16 F2 control males. (b) (c) Figure 7. The oral glucose tolerance test (OGTT). The course of glycaemic curves in SHR F2 male offspring (a, control – black circles, programmed – blue circles), SHR-Zbtb16 F2 male offspring (b, control – black triangles, programmed – green triangles) during the oral glucose tolerance test and the corresponding areas under the glycaemic curves (c, AUC180). Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 0.001. Effect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. (a) C represents STRAIN differences between F2 SHR and SHR-Zbtb16 control males, t = 0 min CCC p < 0.001, t = 30 min C p < 0.05, t = 180 min C, which were all higher in SHR F2 control males. (b) F represents STRAIN differences between F2-programmed SHR and SHR- Zbtb16 males, t = 60 min , t = 180 min , which were both higher in SHR F2-programmed males; (c) C represents STRAIN differences between F2 SHR and SHR-Zbtb16 control males, AUC180 C p < 0.05, gure 7. The oral glucose tolerance test (OGTT). 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by (b) F represents STRAIN differences between F2-programmed SHR and SHR- Zbtb16 males, t = 60 min , t = 180 min , which were both higher in SHR F2-programmed males; (c) C represents STRAIN differences between F2 SHR and SHR-Zbtb16 control males, AUC180 C p < 0.05, higher in SHR-Zbtb16 F2 control males. F2 programmed SHR males displayed lower fasting glycaemia compared to their control group Figure 7. The oral glucose tolerance test (OGTT). The course of glycaemic curves in SHR F2 male oﬀspring (a, control – black circles, programmed – blue circles), SHR-Zbtb16 F2 male oﬀspring (b, control – black triangles, programmed – green triangles) during the oral glucose tolerance test and the corresponding areas under the glycaemic curves (c, AUC180). Data are expressed as mean ± SEM. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 0.001. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. (a) C represents STRAIN diﬀerences between F2 SHR and SHR-Zbtb16 control males, t = 0 min CCC p < 0.001, t = 30 min C p < 0.05, t = 180 min C, which were all higher in SHR F2 control males. (b) F represents STRAIN diﬀerences between F2-programmed SHR and SHR-Zbtb16 males, t = 60 min , t = 180 min , which were both higher in SHR F2-programmed males; (c) C represents STRAIN diﬀerences between F2 SHR and SHR-Zbtb16 control males, AUC180 C p < 0.05, higher in SHR-Zbtb16 F2 control males. 11 of 16 -Zbtb16 Nutrients 2020, 12, 846 control groups and (Figure 8) gure 8). Figure 8. Fasting insulin concentrations in adult SHR F2 control male offspring (dark grey bars), SHR- Zbtb16 F2 control male offspring (light grey bars), F2-programmed SHR male offspring (blue patterned bars), F2-programmed SHR-Zbtb16 male offspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05. Effect of programming in SHR Zbtb16 strain is represented by green asterisks () Figure 8. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by Fasting insulin concentrations in adult SHR F2 control male oﬀspring (dark grey bars), SHR-Zbtb16 F2 control male oﬀspring (light grey bars), F2-programmed SHR male oﬀspring (blue patterned bars), F2-programmed SHR-Zbtb16 male oﬀspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05. Eﬀect of programming in SHR-Zbtb16 strain is represented by green asterisks (). Nutrients 2020, 12, x FOR PEER REVIEW 11 of 16 bolus. Fasting plasma insulin concentrations of adult offspring showed no difference between the control groups and a strain-specific increase of insulin levels in F2 programmed group of SHR-Zbtb16 (Figure 8). PEER REVIEW ma insulin concentrations of adult offspring showed no strain-specific increase of insulin levels in F2 programme Figure 8. Fasting insulin concentrations in adult SHR F2 control male oﬀspring (dark grey bars), Figure 8. Fasting insulin concentrations in adult SHR F2 control male offspring (dark grey bars), SHR- Zbtb16 F2 control male offspring (light grey bars), F2-programmed SHR male offspring (blue patterned bars), F2-programmed SHR-Zbtb16 male offspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05. Effect of Figure 8. Fasting insulin concentrations in adult SHR F2 control male oﬀspring (dark grey bars), SHR-Zbtb16 F2 control male oﬀspring (light grey bars), F2-programmed SHR male oﬀspring (blue patterned bars), F2-programmed SHR-Zbtb16 male oﬀspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: * p < 0.05. Eﬀect of programming in SHR-Zbtb16 strain is represented by green asterisks (). Figure 8. Fasting insulin concentrations in adult SHR F2 control male offspring (dark grey bars), SHR- Zbtb16 F2 control male offspring (light grey bars), F2-programmed SHR male offspring (blue patterned bars), F2-programmed SHR-Zbtb16 male offspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05. Effect of Figure 8. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by The cholesterol (a,b) and triacylglycerol (c,d) conten t SHR F2 control male oﬀspring (dark grey bars), SHR-Zbtb bars), F2-programmed SHR male oﬀspring (blue patterned bar spring (green patterned bars) in 6 months of age. The strain co st signiﬁcant diﬀerence test of the two-way ANOVA for STRA indicated as follows: * p < 0.05, p < 0.01, * p < 0.001. Eﬀe tb16 strains is represented by blue and green asterisks (), r diﬀerences between F2 SHR and SHR-Zbtb16 control males, C h were all higher in SHR-Zbtb16 F2 control males ents 2020, 12, 846 Nutrients 2020, 12, x FOR PEER REVIEW 12 of 16 (b) (c) (d) Figure 9. Lipoprotein proﬁle. The cholesterol (a,b) and triacylglycerol (c,d) content in 20 lipoprote subfractions in adult SHR F2 control male oﬀspring (dark grey bars), SHR-Zbtb16 F2 control ma oﬀspring (light grey bars), F2-programmed SHR male oﬀspring (blue patterned bars), F2-programm SHR-Zbtb16 male oﬀspring (green patterned bars) in 6 months of age. The strain comparison using t post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRA as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 0.001. Eﬀect of programmi in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. (a,c) represents STRAIN diﬀerences between F2 SHR and SHR-Zbtb16 control males, C15 , C16 *, C17 TG10 , TG15 , which were all higher in SHR-Zbtb16 F2 control males. 12 of 16 Nutrients 2020, 12, 846 (b) (c) (b) (b) (c) (b) (b) (c) (c) (d) (d) Figure 9. Lipoprotein proﬁle. The cholesterol (a,b) and triacylglycerol (c,d) content in 20 lipoprotein subfractions in adult SHR F2 control male oﬀspring (dark grey bars), SHR-Zbtb16 F2 control male oﬀspring (light grey bars), F2-programmed SHR male oﬀspring (blue patterned bars), F2-programmed SHR-Zbtb16 male oﬀspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05, p < 0.01, * p < 0.001. Eﬀect of programming in SHR and SHR-Zbtb16 strains is represented by blue and green asterisks (), respectively. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by Fasting insulin concentrations in adult SHR F2 control male oﬀspring (dark grey bars), SHR-Zbtb16 F2 control male oﬀspring (light grey bars), F2-programmed SHR male oﬀspring (blue patterned bars), F2-programmed SHR-Zbtb16 male oﬀspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least signiﬁcant diﬀerence test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05. Eﬀect of programming in SHR-Zbtb16 strain is represented by green asterisks (). programming in SHR-Zbtb16 strain is represented by green asterisks ( ). We observed similar proﬁles of cholesterol and triacylglycerols distribution into lipoprotein fractions of both strains of oﬀspring. There were minor diﬀerences in very large to medium HDL cholesterol levels, which were higher in control SHR-Zbtb16 group, the same was true for small LDL and very large HDL triacylglycerols (Figure 9). Most prominent eﬀect of second-generation HSD - programming was a signiﬁcant increase in medium to very small HDL cholesterol in both strains (Figure 9a,b), with the additional increase of very large and large HDL particle levels in SHR-Zbtb16. Concurrently with the increase of HDL cholesterol, the size of HDL particles had decreased signiﬁcantly (HDL cholesterol particle size; 12.28 ± 0.06 nm (SHR F2 control males) vs. 11.93 ± 0.06 nm (SHR F2 programmed males), p = 0.002; 12.35 ± 0.05 nm (SHR-Zbtb16 F2 control males) vs. 11.94 ± 0.08 nm (SHR-Zbtb16 F2 programmed males), p = 0.0004). Triacylglycerol levels were lower in small to very small LDL and very large to medium HDL in F2 programmed oﬀspring compared to control groups, again with a more prominent eﬀect in SHR-Zbtb16 (Figure 9c,d). Figure 8. Fasting insulin concentrations in adult SHR F2 control male offspring (dark grey bars), SHR- Zbtb16 F2 control male offspring (light grey bars), F2-programmed SHR male offspring (blue patterned bars), F2-programmed SHR-Zbtb16 male offspring (green patterned bars) in 6 months of age. The strain comparison using the post-hoc Fisher’s least significant difference test of the two-way ANOVA for STRAIN and PROGRAM as major factors are indicated as follows: p < 0.05. Effect of programming in SHR-Zbtb16 strain is represented by green asterisks (). (a) (a) Figure 9. Cont. Figure 9. Cont. x FOR PEER REVIEW (b) (c) (d) n proﬁle. 4. Discussion We present data on how sucrose feeding in pregnancy and lactation of F0 generation rat dams aﬀect metabolic proﬁles of standard-diet-fed F1 and F2 oﬀspring generations. Particularly in F1, these eﬀects could be, in part, predictive adaptive responses (PARs), which are environmental responses without an immediate beneﬁt to the organism, but rather a distant beneﬁt, e.g., the ability to anticipate the future environment [28] and adapt. PARs presumably evolved to enable organisms to cope with transient changes in the environment and therefore “provide a process by which individuals adapt to their future postnatal environment by restricting their range of possible phenotypes to a narrower spectrum, without changing the genotype [28]”. In our study, we observed strain-speciﬁc body weight diﬀerences in the F1-programmed generation of females prior to pregnancy, during which the distinction disappeared. Control and F1-programmed females also diﬀered in their body weight, as the programming by maternal HSD contributed to lower body weight of F1 groups compared to controls and this diﬀerence persisted up to the ﬁrst week of pregnancy. Body weight of the lactating rat dams did not signiﬁcantly diﬀer, as opposed to the group of their F0 HSD-fed mothers, whose weight dropped signiﬁcantly in this period [8]. The fasting glycaemia and insulin levels were increased in F1-programmed females in adulthood, which suggests a possible inclination to impaired glucose tolerance. The eﬀect of pregnancy on glucose tolerance was comparable between both groups and strains, although SHR F1-programmed pregnant dams showed an additional decrease of blood glucose levels halfway through OGTT (t = 90 min). Interestingly, HSD programming seemed to alter the appetite of F1-programmed females, as they consumed more of the chow during pregnancy than their control group, which slightly increased their energy intake. This is consistent with previous studies which had shown that sucrose diet induced elevated food intake and appetite in oﬀspring exposed to maternal diet containing fructose, either bound (sucrose) or free fructose in form of high fructose corn syrup [29,30]. Eﬀects of maternal (F0) HSD feeding in F1 programmed males are a part of diﬀerent project and therefore not discussed here. Six-month-old SHR F2-programmed males showed lower fasting glycaemia compared to controls, as did SHR F1-programmed males [8]. 3.2. F2 Adult Male Offspring 3.2. F2 Adult Male Oﬀspring strains is represented by (a,c) C represents STRAIN diﬀerences between F2 SHR and SHR-Zbtb16 control males, C15 , C16 *, C17 , TG10 , TG15 , which were all higher in SHR-Zbtb16 F2 control males. Nutrients 2020, 12, 846 13 of 16 13 of 16 4. Discussion We observed that the eﬀect of HSD-programming of their mothers acted diﬀerently upon genomic background, as SHR-Zbtb16 F2-programmed males showed no diﬀerence in fasting glycaemia, but inclined to biphasic course of OGTT [31], with a signiﬁcant drop of blood glucose levels at t = 60 min with which they diﬀered not only from their controls but also SHR F2-programmed males. Fasting insulin levels of F2-programmed males were elevated compared to their controls in a similar fashion as in their F1-programmed mothers, although only SHR-Zbtb16 groups’ increase was statistically signiﬁcant. Decrease of small to very small LDL triacylglycerols in F2-programmed groups, with more prominent eﬀect in SHR-Zbtb16 was similar as it was in F1-programmed males [8], who directly interacted with HSD through the uteroplacental system and breast milk of F0 mothers. The persistence of this speciﬁc decrease seems to be determined by the HSD eﬀect even in second generation of oﬀspring. A similar pattern was observed in decrease of medium HDL triacylglycerols which was more prominent in SHR-Zbtb16 [8] F1-programmed males. In addition, we observed a decrease of very large HDL triacylglycerols in F2-programmed males. Interestingly, medium to very small HDL cholesterol levels have been increased signiﬁcantly only in F2-programmed males, together with a decrease of size of these particles. The maternal HSD-speciﬁc milieu possibly programmed the oﬀspring for the environment providing high amounts of sucrose, however in adult life their main source of carbohydrates from STD was starch. Predictive adaptive hypothesis postulates that environmental mismatch of early life versus adulthood can increase the risk of disease [32,33]. However, historically, it was poor maternal nutrition in early development versus overnutrition in adulthood due to westernized diet popularity that was the most studied model of disparity. Our study established the opposite conditions, whereby maternal diet of F0 generation abundant in sucrose, although the same in calorie content as STD, is followed by STD consumption after weaning of their F1 oﬀspring and whole pre- and postnatal life of their grandsons. If the strategies for maximizing the postnatal survival success are based on the anticipation Nutrients 2020, 12, 846 14 of 16 14 of 16 of a particular adult environment, it is possible that the metabolic systems of F1 and F2 oﬀspring were prepared to manage increased levels of sucrose and thus overproduction of triglycerides and responded with a higher baseline for HDL production in order to alleviate these eﬀects. 4. Discussion Triglycerides are being transferred from VLDL to HDL by the action of cholesterol ester transfer protein [34]. After hydrolysis by hepatic lipase they are cleared from plasma, which serves as a basis for protective eﬀect of HDL against dyslipidemia and coronary heart disease. However, a more comprehensive design, including all matched (including re-exposure to sucrose group) and mismatched pre- and postnatal environments, would be necessary to substantiate the above mechanism. Another eﬀect of HSD-programming in early life that persisted two generations of oﬀspring was a signiﬁcant increase of interscapular brown fat weight. We already noticed this eﬀect in F1-programmed adult males of both strains [8], although direct programming with HSD resulted in higher increase in F1-programmed males than the increase we observed in second generation. As shown lately, brown fat represents an essential regulator of the eﬀect of maternal nutritional programming [35]. Body fat distribution in terms of retroperitoneal fat and epididymal fat, partly correlating with human visceral fat [36], wasn’t aﬀected by the second-generation programming. Relative weights of retroperitoneal fat diﬀered among the strains in F2 generation of programmed males, with SHR having higher relative weights. Strains used in this study diﬀered in a single gene mutation—254 kb deletion in intronic region of Zbtb16 gene of the PD/Cub strain origin [37]. The genomic background of SHR was thought to exacerbate the eﬀects of Zbtb16 gene involved in pathogenesis of metabolic syndrome [18], but conversely was shown to improve the related parameters [20], which was to an extent apparent in our study as well. We are aware of several inherent limitations of this study. First, we studied only the male oﬀspring of two speciﬁc, inbred rat strains within a single protocol of exposition to HSD. This mostly followed our previous studies, maximizing the homogeneity of the control and experimental groups in order to observe the subtle eﬀects due to intergenerational transfer [8,38]. Also, the obtained results do not provide a direct mechanistic link between Zbtb16 and the observed metabolic changes. Thus, comprehensive genetic and epigenetic studies will be necessary to dissect these aspects in detail. In conclusion, we show the second-generation eﬀects of maternal exposition to a high-sucrose diet, some modulated to a certain extent by variation in the Zbtb16 gene. Author Contributions: Conceptualization, O.Š., L.Š. and E.Š.; Data curation, L.Š. and E.Š.; Resources, O.Š.; Supervision, O.Š.; Writing—original draft, E.Š.; Writing—review & editing, O.Š. and L.Š. 4. Discussion All authors have read and agreed to the published version of the manuscript. Funding: This research and APC was funded by Charles University, Prague, Czech Republic, grant number GAUK 132415, PROGRES Q25/LF1, SVV 260367 and Ministry of Health, Czech Republic–conceptual development of research organization 64165, General University Hospital in Prague, Czech Republic. Acknowledgments: We thank Michaela Jank˚u, Blanka Chylíková and Michaela Krupková for technical assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. . Delage, B.; Dashwood, R.H. Dietary manipulation of histone structure and function. Annu. Rev. Nutr. 2 28, 347–366. [CrossRef] References [CrossRef] [PubMed] 13. Muthayya, S.; Kurpad, A.V.; Duggan, C.P.; Bosch, R.J.; Dwarkanath, P.; Mhaskar, A.; Mhaskar, R.; Thomas, A.; Vaz, M.; Bhat, S.; et al. Low maternal vitamin B12 status is associated with intrauterine growth retardation in urban South Indians. Eur. J. Clin. Nutr. 2006, 60, 791–801. [CrossRef] [PubMed] 14. Yajnik, C.S.; Deshpande, S.S.; Jackson, A.A.; Refsum, H.; Rao, S.; Fisher, D.J.; Bhat, D.S.; Naik, S.S.; Coyaji, K.J.; Joglekar, C.V.; et al. Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the oﬀspring: The Pune Maternal Nutrition Study. Diabetologia 2008, 51, 29–38. [CrossRef] 15. Ainge, H.; Thompson, C.; Ozanne, S.E.; Rooney, K.B. A systematic review on animal models of maternal high fat feeding and oﬀspring glycaemic control. Int. J. Obes. 2011, 35, 325–335. [CrossRef] 16. Bocarsly, M.E.; Barson, J.R.; Hauca, J.M.; Hoebel, B.G.; Leibowitz, S.F.; Avena, N.M. Eﬀects of perinatal exposure to palatable diets on body weight and sensitivity to drugs of abuse in rats. Physiol. Behav. 2012, 107, 568–575. [CrossRef] 17. Ng, S.F.; Lin, R.C.; Laybutt, D.R.; Barres, R.; Owens, J.A.; Morris, M.J. Chronic high-Fat diet in fathers programs beta-Cell dysfunction in female rat oﬀspring. Nature 2010, 467, 963–966. [CrossRef] 18. Šeda, O.; Šedová, L.; Vˇcelák, J.; Vaˇnková, M.; Liška, F.; Bendlová, B. ZBTB16 and metabolic syndrome: A network perspective. Physiol. Res. 2017, 66, S357–S365. [CrossRef] 19. Chen, S.; Qian, J.; Shi, X.; Gao, T.; Liang, T.; Liu, C. Control of hepatic gluconeogenesis by the promyelocytic leukemia zinc ﬁnger protein. Mol. Endocrinol. 2014, 28, 1987–1998. [CrossRef] 20. Liška, F.; Landa, V.; Zídek, V.; Mlejnek, P.; Šilhavý, J.; Šimáková, M.; Strnad, H.; Trnovská, J.; Škop, V.; Kazdová, L. Downregulation of Plzf gene ameliorates metabolic and cardiac traits in the spontaneously hypertensive rat. Hypertension 2017, 69, 1084–1091. [CrossRef] 21. Aitman, T.J.; Gotoda, T.; Evans, A.L.; Imrie, H.; Heath, K.E.; Trembling, P.M.; Truman, H.; Wallace, C.A.; Rahman, A.; Doré, C.; et al. Quantitative trait loci for cellular defects in glucose and fatty acid metabolism in hypertensive rats. Nat. Genet. 1997, 16, 197–201. [CrossRef] [PubMed] 2. Pravenec, M.; Kˇren, V.; Landa, V.; Mlejnek, P.; Musilová, A.; Šilhavý, J.; Šimáková, M.; Zídek, V. Re progress in the genetics of spontaneously hypertensive rats. Physiol. Res. 2014, 63, S1–S8. 23. Sedova, L.; Kazdova, L.; Seda, O.; Krenova, D.; Kren, V. Rat inbred PD/Cub strain as a model of dyslipidemia and insulin resistance. Folia Biol. (Praha) 2000, 46, 99–106. [PubMed] 24. References 1. Bloomﬁeld, F.H.; Harding, J.E. Experimental aspects of nutrition and fetal growth. Fetal Matern. Med. Rev. 1998, 10, 91–107. [CrossRef] 1. Bloomﬁeld, F.H.; Harding, J.E. Experimental aspects of nutrition and fetal growth. Fetal Matern. Med. Rev. 1998, 10, 91–107. [CrossRef] 2. Singal, R.; Ginder, G.D. DNA methylation. Blood 1999, 93, 4059–4070. [CrossRef] [PubMed] 3. Delage, B.; Dashwood, R.H. Dietary manipulation of histone structure and function. Annu. Rev. Nutr. 2008, 28, 347–366. [CrossRef] 3. Delage, B.; Dashwood, R.H. Dietary manipulation of histone structure and function. Annu. Rev. Nutr. 2008, 28, 347–366. [CrossRef] 4. Fernandez-Twinn, D.S.; Hjort, L.; Novakovic, B.; Ozanne, S.E.; Saﬀery, R. Intrauterine programming of obesity and type 2 diabetes. Diabetologia 2019, 62, 1789–1801. [CrossRef] 4. Fernandez-Twinn, D.S.; Hjort, L.; Novakovic, B.; Ozanne, S.E.; Saﬀery, R. Intrauterine programming of obesity and type 2 diabetes. Diabetologia 2019, 62, 1789–1801. [CrossRef] y yp g 5. Waterland, R.A.; Jirtle, R.L. Transposable elements: Targets for early nutritional eﬀects on epigenetic gene regulation. Mol. Cell Biol. 2003, 23, 5293–5300. [CrossRef] 5. Waterland, R.A.; Jirtle, R.L. Transposable elements: Targets regulation. Mol. Cell Biol. 2003, 23, 5293–5300. [CrossRef] 5. Waterland, R.A.; Jirtle, R.L. Transposable elements: Targets for early nutritional eﬀects on epigenetic gene regulation. Mol. Cell Biol. 2003, 23, 5293–5300. [CrossRef] Nutrients 2020, 12, 846 15 of 16 15 of 16 6. Skinner, M.K. Role of epigenetics in developmental biology and transgenerational inheritance. Birth Defects Res. C Embryo Today 2011, 93, 51–55. [CrossRef] 7. Skinner, M.K. Environmental epigenetic transgenerational inheritance and somatic epigenetic mitotic stability. Epigenetics 2011, 6, 838–842. 8. Skolnikova, E.; Sedova, L.; Krenova, D.; Kren, V.; Seda, O. Mutation in Zbtb16 gene plays a role in lipid proﬁles of pregnant rats and their oﬀspring after high-Sucrose diet feeding. Atherosclerosis 2017, 263, E36–E37. [CrossRef] 9. Warrington, N.M.; Beaumont, R.N.; Horikoshi, M.; Day, F.R.; Helgeland, Ø.; Laurin, C.; Bacelis, J.; Peng, S.; Hao, K.; Feenstra, B.; et al. Maternal and fetal genetic eﬀects on birth weight and their relevance to cardio-Metabolic risk factors. Nat. Genet. 2019, 51, 804–814. [CrossRef] 10. Heard, E.; Martienssen, R.A. Transgenerational epigenetic inheritance: Myths and mechanisms. Cell 2014, 2014 157, 95–109. [CrossRef] 11. Skinner, M.K. What is an epigenetic transgenerational phenotype? F3 or F2. Reprod. Toxicol. 2008, 25, 2–6. [CrossRef] [PubMed] 12. Jou, M.Y.; Lönnerdal, B.; Philipps, A.F. Maternal zinc restriction aﬀects postnatal growth and glucose homeostasis in rat oﬀspring diﬀerently depending upon adequacy of their nutrient intake. Pediatr. Res. 2012, 71, 228e34. References Seda, O.; Liska, F.; Sedova, L.; Kazdova, L.; Krenova, D.; Kren, V. A 14-Gene region of rat chromosome 8 in SHR-Derived polydactylous congenic substrain aﬀects muscle-Speciﬁc insulin resistance, dyslipidaemia and visceral adiposity. Folia Biol.-Prague 2005, 51, 53–61. 25. Krupková, M.; Liška, F.; Kazdová, L.; Šedová, L.; Kábelová, A.; Kˇrenová, D.; Kˇren, V.; Šeda, O. Single-Gene Congenic Strain Reveals the Eﬀect of Zbtb16 on Dexamethasone-Induced Insulin Resistance. Front. Endocrinol. (Lausanne) 2018, 9, 185. [CrossRef] 26. Usui, S.; Hara, Y.; Hosaki, S.; Okazaki, M. A new on-Line dual enzymatic method for simultaneous quantiﬁcation of cholesterol and triglycerides in lipoproteins by HPLC. J. Lipid Res. 2002, 43, 805–814. Nutrients 2020, 12, 846 16 of 16 16 of 16 27. Heikkinen, S.; Argmann, C.A.; Champy, M.F.; Auwerx, J. Evaluation of glucose homeostasis. Curr. Protoc. Mol. Biol. 2007. [CrossRef] 28. Gluckman, P.D.; Hanson, M.A. Developmental origins of disease paradigm: A mechanistic and evolutionary perspective. Pediatr. Res. 2004, 56, 311–317. [CrossRef] 29. Kisioglu, B.; Nergiz-Unal, R. Potential eﬀect of maternal dietary sucrose or fructose syrup on CD36, leptin, and ghrelin-Mediated fetal programming of obesity. Nutr. Neurosci. 2018. [CrossRef] 30. Lowette, K.; Roosen, L.; Tack, J.; Vanden Berghe, P. Eﬀects of high-fructose diets on central appetite signaling and cognitive function. Front. Nutr. 2015, 2, 5. [CrossRef] 31. Tschritter, O.; Fritsche, A.; Shirkavand, F.; Machicao, F.; Häring, H.; Stumvoll, M. Assessing the shape of the glucose curve during an oral glucose tolerance test. Diabetes Care 2003, 26, 1026–1033. [CrossRef] [PubMed] 31. Tschritter, O.; Fritsche, A.; Shirkavand, F.; Machicao, F.; Häring, H.; Stumvoll, M. Assessing the shape of the glucose curve during an oral glucose tolerance test. Diabetes Care 2003, 26, 1026–1033. [CrossRef] [PubMed] 32. Gluckman, P.D.; Hanson, M.A. The developmental origins of the metabolic syndrome. Trends Endocrinol. 32. Gluckman, P.D.; Hanson, M.A. The developmental origins of the metabolic syndrome. Trends Endocrinol. Metab. 2004, 15, 183–187. [CrossRef] [PubMed] 33. Gluckman, P.D.; Hanson, M.A.; Spencer, H.G. Predictive adaptive responses and human evolution. Trends Ecol. Evol. 2005, 20, 527–533. [CrossRef] [PubMed] 34. Hopkins, G.J.; Barter, P.J. Role of triglyceride-Rich lipoproteins and hepatic lipase in determining the particle size and composition of high density lipoproteins. J. Lipid Res. 1986, 27, 1265–1277. 35. Dumortier, O.; Roger, E.; Pisani, D.F.; Casamento, V.; Gautier, N.; Lebrun, P.; Johnston, H.; Lopez, P.; Amri, E.Z.; Jousse, C. Age-dependent control of energy homeostasis by brown adipose tissue in progeny subjected to maternal diet-Induced fetal programming. References Diabetes 2017, 66, 627–639. [CrossRef] 36. Chusyd, D.E.; Wang, D.; Huﬀman, D.M.; Nagy, T.R. Relationships between rodent white adipose fat pads and human white adipose fat depots. Front. Nutr. 2016, 3, 10. [CrossRef] 37. Liska, F.; Snajdr, P.; Sedova, L.; Seda, O.; Chylikova, B.; Slamova, P.; Krejci, E.; Sedmera, D.; Grim, M.; Krenova, D.; et al. Deletion of a conserved noncoding sequence in Plzf intron leads to Plzf down-Regulation in limb bud and polydactyly in the rat. Dev. Dyn. 2009, 238, 673–684. [CrossRef] 38. Sedova, L.; Seda, O.; Kazdová, L.; Chylíková, B.; Hamet, P.; Tremblay, J.; Kren, V.; Krenová, D. Sucrose feeding during pregnancy and lactation elicits distinct metabolic response in oﬀspring of an inbred genetic model of metabolic syndrome. Am. J. Physiol. Endocrinol. Metab. 2017, 292, E1318–E1324. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2098273728	https://escholarship.org/content/qt3tf344wz/qt3tf344wz.pdf?t=mscgmy	English	null	Circadian rhythms constrain leaf and canopy gas exchange in an Amazonian forest	Geophysical research letters	2,006	cc-by	4,899	UC Irvine Faculty Publications Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/3.0/ Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/3.0/ Peer reviewed UC Irvine Faculty Publications Title Circadian rhythms constrain leaf and canopy gas exchange in an Amazonian forest Permalink https://escholarship.org/uc/item/3tf344wz Journal Geophysical Research Letters, 33(15) ISSN 0094-8276 Authors Doughty, Christopher E. Goulden, Michael L. Miller, Scott D. et al. Publication Date 2006-08-08 DOI 10.1029/2006GL026750 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/3.0/ Peer reviewed UC Irvine Faculty Publications Title Circadian rhythms constrain leaf and canopy gas exchange in an Amazonian forest Permalink https://escholarship.org/uc/item/3tf344wz Journal Geophysical Research Letters, 33(15) ISSN 0094-8276 Authors Doughty, Christopher E. Goulden, Michael L. Miller, Scott D. et al. Publication Date 2006-08-08 DOI 10.1029/2006GL026750 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/3.0/ Peer reviewed UC Irvine Faculty Publications Title Circadian rhythms constrain leaf and canopy gas exchange in an Amazonian forest Permalink https://escholarship.org/uc/item/3tf344wz Journal Geophysical Research Letters, 33(15) ISSN 0094-8276 Authors Doughty, Christopher E. Goulden, Michael L. Miller, Scott D. et al. Publication Date 2006-08-08 DOI 10.1029/2006GL026750 Copyright Information This work is made available under the terms of a Creative Commons Attribution Lice availalbe at https://creativecommons.org/licenses/by/3.0/ Peer reviewed Circadian rhythms constrain leaf and canopy gas exchange in an Amazonian forest Christopher E. Doughty,1 Michael L. Goulden,1 Scott D. Miller,1 and Humberto R. da Rocha2 Received 28 April 2006; revised 19 June 2006; accepted 28 June 2006; published 8 August 2006. we are unaware of surveys that document the occurrence of photosynthetic circadian rhythms in other plant functional types such as large trees, or of efforts to establish the role of circadian rhythms in controlling whole-ecosystem gas ex- change over a day. [1] We used a controlled-environment leaf gas-exchange system and the micrometeorological technique eddy covariance to determine whether circadian rhythms constrain the rates of leaf and canopy gas exchange in an Amazonian forest over a day. When exposed to continuous and constant light for 20 to 48 hours leaves of eleven of seventeen species reduced their photosynthetic rates and closed their stomata during the normally dark period and resumed active gas exchange during the normally light period. Similarly, the rate of whole-forest CO2 uptake at a predetermined irradiance declined during the late afternoon and early morning and increased during the middle of the day. We attribute these cycles to circadian rhythms that are analogous to ones that have been reported for herbaceous plants in the laboratory. The importance of endogenous gas exchange rhythms presents a previously unrecognized challenge for efforts to both interpret and model land- atmosphere energy and mass exchange. Citation: Doughty, C. E., M. L. Goulden, S. D. Miller, and H. R. da Rocha (2006), Circadian rhythms constrain leaf and canopy gas exchange in an Amazonian forest, Geophys. Res. Lett., 33, L15404, doi:10.1029/ 2006GL026750. g y [3] We used a controlled-environment leaf gas-exchange system and the micrometeorological technique eddy covari- ance to determine whether circadian rhythms constrain the rates of leaf and canopy gas exchange in an Amazonian forest over a day. We focused on Amazonian forest because tropical forests play key roles in the global hydrological, energy, and carbon cycles. Understanding of the controls on tropical forest gas exchange lags that for temperate ecosys- tems, even though tropical forest accounts for 45% of global terrestrial evapotranspiration [Hetherington and Woodward, 2003] and at least 30% of global terrestrial primary production [Field et al., 1998]. Powered by the California Digital Library University of California eScholarship.org eScholarship.org GEOPHYSICAL RESEARCH LETTERS, VOL. 33, L15404, doi:10.1029/2006GL026750, 2006 1Department of Earth System Science, University of California, Irvine, California, USA. 2Department of Atmospheric Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil. 2. Methods [4] Measurements were made between June 2000 and August 2004 at the LBA (Large-Scale Biosphere- Atmosphere Experiment in Amazonia) km 83 and 67 sites in the Tapajo´s National Forest, Para´, Brazil [Goulden et al., 2004; Saleska et al., 2003]. The vegetation was semideci- dous closed tropical forest with canopy emergents on flat upland terrain. 1. Introduction [2] Research on the controls of ecosystem gas exchange has focused on the direct effects of light, moisture avail- ability and other aspects of the physical environment on plant physiology [Jones, 1992; Lambers et al., 1998; Sellers et al., 1997; Bonan, 2002]. The possibility that plant circadian rhythms [McClung, 2001; Johnson et al., 1998; Webb, 2003], associated with the up and down regulation of activity by endogenous biological clocks, constrain the rates of land-atmosphere exchange over a day has received scant attention. This lack of attention is surprising since there is strong evidence that circadian rhythms control many pro- cesses in C3 plants [Harmer et al., 2002] including stomatal conductance [Mansfield and Heath, 1963; Stalfelt, 1963] and leaf photosynthesis [Hennessey and Field, 1991; Williams and Gorton, 1998; Dodd et al., 2005]. Nonethe- less, previous work on photosynthetic circadian rhythms has focused on short-lived herbaceous plants growing in pots in the laboratory, and we are unaware of observations of photosynthetic circadian rhythms in the field. Moreover, p [5] The gas exchange by 56 leaves (Table 1) exposed to constant conditions for 20 to 48 hours in light and 6 leaves in darkness was measured with a LiCor 6400 gas exchange system. Plants were identified following Ribero et al. [1999]. We were unable to identify some plants to species after consulting experts on the local flora, and some of these species may be unknown to science. Most of the illuminated measurements were made at a Photon Flux Density (PPFD) of 100 mmol m2 s1, a leaf temperature of 30C, a chamber CO2 concentration of 370 mmol mol1, and either a constant chamber vapor pressure or a constant flow through the chamber. Some runs were made at a PPFD of 1000 mmol m2 s1 or without temperature control or without CO2 control. Repeated measurements on Micropholis sp. indi- cated the occurrence of an endogenous gas exchange rhythm was independent of the configuration of the gas exchange system and the environmental conditions within the chamber. The neighboring leaves on the branch being tested were kept in darkness, and a larger section of the branch was shaded under a tarp, throughout each run. Leaves were accessed on the ground or from scaffolding. Copyright 2006 by the American Geophysical Union. 0094-8276/06/2006GL026750 Table 1. Occurrence of Endogenous Gas Exchange Rhythms Table 1. Occurrence of Endogenous Gas Exchange Rhythms Serjania sp. Lecythis sp. Minquartia quianensis Aubl. Sclerolobium paraense Faramea platyneura Arrabidaea sp. aPhotosynthesis, stomatal conductance, and Ci remained constant or declined gradually over time. bPhotosynthesis, stomatal conductance, and Ci declined during the normally dark period and recovered during the next normally light period. cPhotosynthesis and stomatal conductance declined and Ci increased during the normally dark period and recovered during the next normally light i d y g y bPhotosynthesis, stomatal conductance, and Ci declined during the normally dark period and recovered during the next normally light period. cPhotosynthesis and stomatal conductance declined and Ci increased during the normally dark period and recovered during the next normally light period. bPhotosynthesis, stomatal conductance, and Ci declined during the normally dark period and recovered during the ne cPhotosynthesis and stomatal conductance declined and Ci increased during the normally dark period and recovere period. p dPhotosynthesis and stomatal conductance declined with no subsequent recovery during the next normally light period. tosynthesis and stomatal conductance declined with no subseq draulic communication imposed a spurious 24-hour gas exchange cycle on the investigated leaf. However, hydraulic coupling cannot explain the observed gas exchange trend since the associated changes in leaf water potential would be expected to decrease leaf photosynthesis during daytime, whereas the opposite trend was observed. Moreover, photosynthetic recovery typically started before dawn (Figures 1 and 2), indicating that it was not initiated by exposure of the remainder of the plant to light. a micrometeorological technique that measures the net turbulent exchange of CO2 by a patch of forest upwind of a meteorological tower. The area sampled varies with the meteorological conditions, decreasing to 10 ha during daytime and increasing to as much as 1000 ha on atmospherically-stable nights. The fluxes were averaged for 30-minute periods and corrected for changes in CO2 storage between the measurement height and the ground. DOUGHTY ET AL.: CIRCADIAN RHYTHMS CONSTRAIN GAS EXCHANGE L15404 L15404 Table 1. Occurrence of Endogenous Gas Exchange Rhythms Family Leaves Sampled Functional Group Plants with no apparent endogenous rhythma Chimarrhis turbinate Rubiaceae 6 Subcanopy Protium punticulatum Macbr. Burseraceae 3 Canopy Plants with an endogenous rhythm in stomatal conductance but not photosynthetic capacityb Distachya huber Cecropiaceae 4 Pioneer Serjania sp. Sapindaceae 1 Liana Lecythis sp. Lecythidaceae 4 Subcanopy Prionostemma aspera Hippocrateaceae 1 Liana Plants with an endogenous photosynthetic capacity rhythmc Derris amazonica Leguminosae-Papilionoideae 2 Liana Duguetia flagellaris Annonaceae 2 Understory Eschweilera amazonica Lecythidaceae 3 Canopy Micropholis sp. Sapotaceae 16 Canopy Lecythis lurida Lecythidaceae 3 Canopy Copaifera duckei Leguminosae-Caesalpinioideae 3 Canopy Manilkara huberi Sapotaceae 6 Canopy Plants with a sharp photosynthetic decline and no subsequent recoveryd Minquartia quianensis Aubl. Olacaceae 2 Canopy Sclerolobium paraense Leguminosae-Caesalpinioideae 4 Subcanopy Faramea platyneura Rubiaceae 1 Subcanopy Arrabidaea sp. Bignoniaceae 1 Liana aPhotosynthesis, stomatal conductance, and Ci remained constant or declined gradually over time. bPhotosynthesis, stomatal conductance, and Ci declined during the normally dark period and recovered during the next normally light period. cPhotosynthesis and stomatal conductance declined and Ci increased during the normally dark period and recovered during the next normally light period. 3. Results and Discussion [7] We exposed 62 leaves on 17 plants representing 17 species to constant irradiance and temperature for 20 to 48 hours while continuously measuring the rate of gas exchange. Seven of the species exhibited an endogenous rhythm in photosynthetic capacity, and four additional species exhibited an endogenous rhythm in stomatal con- ductance but not photosynthetic capacity (Table 1). For example, the net CO2 Assimilation (An) by Micropholis sp., a canopy tree, decreased after 1300 Local Time (LT), remained near zero during the evening, and recovered beginning before dawn (Figure 1). The An decline during the normally-dark period was not a result of stomatal closure and a lower intercellular CO2 concentration (Ci). Micropholis’s photosynthesis increased during daytime, when Ci was low, and declined at night, when Ci was high. The concentration of CO2 within the chamber was held constant during these measurements, and the change in Ci resulted from, rather than caused, the photosynthetic trend. [8] A limitation to our approach is that a single leaf was held in constant conditions while most of the rest of the plant was exposed to ambient conditions. The study leaf’s water potential presumably declined during daytime and recovered at night in concert with the water potential of nearby leaves (Figure 2a), raising the possibility that hy- Figure 1. Leaf net CO2 exchange (An, filled circles) and calculated intercellular CO2 concentration (Ci, open squares) by a Micropholis sp. leaf exposed to continuous light for 24 hours (PPFD 100 mmole m2 s1, leaf T 26 C, relative humidity 70%, chamber CO2 370 mmol mol1). The gas exchange measurements were made on a canopy leaf growing 30-m above ground level in an Amazonian tropical forest. The dashed line shows the average incident PPFD at the site; the shaded area shows the normally-dark period. Figure 1. Leaf net CO2 exchange (An, filled circles) and calculated intercellular CO2 concentration (Ci, open squares) by a Micropholis sp. leaf exposed to continuous light for 24 hours (PPFD 100 mmole m2 s1, leaf T 26 C, relative humidity 70%, chamber CO2 370 mmol mol1). The gas exchange measurements were made on a canopy leaf growing 30-m above ground level in an Amazonian tropical forest. The dashed line shows the average incident PPFD at the site; the shaded area shows the normally-dark period. 1. Introduction p [5] The gas exchange by 56 leaves (Table 1) exposed to constant conditions for 20 to 48 hours in light and 6 leaves in darkness was measured with a LiCor 6400 gas exchange system. Plants were identified following Ribero et al. [1999]. We were unable to identify some plants to species after consulting experts on the local flora, and some of these species may be unknown to science. Most of the illuminated measurements were made at a Photon Flux Density (PPFD) of 100 mmol m2 s1, a leaf temperature of 30C, a chamber CO2 concentration of 370 mmol mol1, and either a constant chamber vapor pressure or a constant flow through the chamber. Some runs were made at a PPFD of 1000 mmol m2 s1 or without temperature control or without CO2 control. Repeated measurements on Micropholis sp. indi- cated the occurrence of an endogenous gas exchange rhythm was independent of the configuration of the gas exchange system and the environmental conditions within the chamber. The neighboring leaves on the branch being tested were kept in darkness, and a larger section of the branch was shaded under a tarp, throughout each run. Leaves were accessed on the ground or from scaffolding. [6] We used the eddy covariance technique to measure the rates of whole ecosystem CO2 exchange from June 2000 to August 2004 [Goulden et al., 2004]. Eddy covariance is Copyright 2006 by the American Geophysical Union. 0094-8276/06/2006GL026750 L15404 1 of 5 DOUGHTY ET AL.: CIRCADIAN RHYTHMS CONSTRAIN GAS EXCHANGE 3. Results and Discussion y [8] A limitation to our approach is that a single leaf was held in constant conditions while most of the rest of the plant was exposed to ambient conditions. The study leaf’s water potential presumably declined during daytime and recovered at night in concert with the water potential of nearby leaves (Figure 2a), raising the possibility that hy- 2 of 5 DOUGHTY ET AL.: CIRCADIAN RHYTHMS CONSTRAIN GAS EXCHANGE L15404 L15404 Figure 2. (a) Leaf water potential for Micropholis sp. leaves exposed to ambient conditions. (b) Leaf CO2 exchange (An) by Micropholis sp. leaves in continuous light for 24 hours (filled circles PPFD 100 mmole m2 s1, leaf T 26 C, relative humidity 70%, chamber CO2 370 mmol mol1; open squares PPFD 1000 mmole m2 s1, leaf T 26 C, relative humidity 75%, chamber CO2 370 mmol mol1). (c) Leaf respiration by a Micropholis sp. leaf in constant darkness for 24 hours (filled triangles PPFD 0 mmole m2 s1, leaf T 30 C, relative humidity 65%, chamber CO2 370 mmol mol1). Shaded area shows the normally-dark period. which, in turn, caused photosynthesis to decline during the normally dark period even though the biochemical capacity to fix carbon apparently remained constant. The final seven species, including Micropholis sp., exhibited endogenous rhythms in the biochemical capacity to fix carbon. The patterns of gas exchange were generally consistent between different leaves on a plant and between different times of the year for a plant. The occurrence of an endogenous rhythm was not confined to a single plant functional group, but was observed in an understory plant, three lianas, a pioneer, and six canopy or subcanopy trees. The rhythms we observed for Micropholis and other plants appear similar to, and in some cases more extreme than, the gas exchange rhythms that have been attributed to circadian oscillators in other species [Hennessey and Field, 1991; Williams and Gorton, 1998; Dodd et al., 2005]. ] [11] We analyzed 16,138 half-hour measurements of whole-forest CO2 exchange (Net Ecosystem Exchange; NEE) made at the LBA km-83 site from 2000 to 2004 to determine whether ecosystem gas exchange also exhibits an endogenous rhythm. NEE at a PPFD of 500 to 600 mmol m2s1 increased from an uptake rate of 3.6 mmol m2s1 at 645LT to a maximum of 16.1 mmol m2s1 at 945LT before declining to 0.4 mmol m2s1 at 1615LT (Figure 3b). 3. Results and Discussion The changing relationship between irradiance and NEE (Figure 3a) paralleled that observed at the leaf-level (Figure 2b), and the diurnal NEE changes might have resulted from an endogenous gas exchange rhythm similar to that observed for individual leaves. However, several possible alternative explanations for the NEE observations merit consideration as well. Figure 2. (a) Leaf water potential for Micropholis sp. leaves exposed to ambient conditions. (b) Leaf CO2 exchange (An) by Micropholis sp. leaves in continuous light for 24 hours (filled circles PPFD 100 mmole m2 s1, leaf T 26 C, relative humidity 70%, chamber CO2 370 mmol mol1; open squares PPFD 1000 mmole m2 s1, leaf T 26 C, relative humidity 75%, chamber CO2 370 mmol mol1). (c) Leaf respiration by a Micropholis sp. leaf in constant darkness for 24 hours (filled triangles PPFD 0 mmole m2 s1, leaf T 30 C, relative humidity 65%, chamber CO2 370 mmol mol1). Shaded area shows the normally-dark period. Figure 3. (a) Net Ecosystem CO2 exchange (NEE; uptake shown as a positive flux) measured by eddy covariance as a function of incident PPFD. Light curves are averages (n > 60) within 100 mmol m2 s1 wide PPFD bins for one-hour intervals centered on the Local Time (LT) shown. Ninety five percent confidence intervals are shown for the 730– 830 curve; confidence intervals for the other times are similar. NEE was screened to exclude periods with a friction velocity (U) < 0.2 m s1. (b) Average NEE (n > 20) and 95% confidence intervals for half-hour intervals with a PPFD of 500 to 600 mmol m2s1 as a function of Local Time. [9] Micropholis’s photosynthesis was always strongly controlled by light, though the amplitude of the light response changed with time of day (Figure 2). The photo- synthetic uptake at PPFDs of 100 mmol m2 s1 and 1000 mmol m2 s1 declined after 1300LT and recovered beginning before 0600LT (Figure 2b). Dark respiration also declined (became less negative) beginning shortly after 1300LT and recovered beginning around 0600LT (Figure 2c). There was no obvious difference in period or phase between light intensities. The similar trend at all light levels suggests a general up-regulation of metabolism during the normally light period and down-regulation during the nor- mally dark period. Figure 3. (a) Net Ecosystem CO2 exchange (NEE; uptake shown as a positive flux) measured by eddy covariance as a function of incident PPFD. 3. Results and Discussion Moreover, most studies indicate that the ratio of diffuse to direct light peaks early and late in the day [Campbell and Norman, 1998], and diurnal changes in light quality should have maximized rather than suppressed NEE at a given irradiance during these periods [Gu et al., 2002]. possibility is that the nocturnal decline in photosynthesis reflects a general down regulation of metabolism that conserves energy. Micropholis’s rates of photosynthesis and respiration change in concert (Figures 2b and 2c), raising the possibility that leaves adjust their metabolism in anticipation of the average light conditions. Hence, a leaf’s light response might change from one that is typical for a sun leaf during the middle of the day to one that is typical for a shade leaf during the early morning, late afternoon, and night. g [15] The local climate at the site may have promoted the evolutionary development of circadian rhythms. The site is 3 south of the equator, resulting in a nearly constant 12-hour photoperiod. The daily maximum air temperature was always within a few degrees of 29C from 2000 to 2004, and most plants were sufficiently deeply rooted to avoid water stress, resulting in nearly constant rates of midday canopy gas exchange year-round [Goulden et al., 2004]. This predictable environment, especially with respect to the aspects of the climate that directly control photosyn- thesis, may have selected for the anticipatory, biological clock-driven regulation of photosynthetic capacity. The size of tropical trees may also have led to adaptations that increase coordination among leaves and allow large plants to act in a physiologically integrated manner. p y g y g [16] The endogenous photosynthesis patterns coincide with the natural photoperiod, and the diel pattern of gas exchange predicted assuming constant physiology under current conditions may not differ markedly from that predicted after accounting for the endogenous cycle [Williams and Gorton, 1998]. Some researchers may emphasize this issue and conclude that endogenous rhythms are unimportant; a perspective that we believe misses the point. Both modeling and observational research on eco- system gas exchange have focused almost entirely on the direct effects of the physical environment on plant physiol- ogy. We have shown that the exclusive focus on the direct physical controls on physiology is unwarranted, and that tropical plants possess endogenous rhythms that are poorly understood with respect to both mechanism and adaptive significance. 3. Results and Discussion Light curves are averages (n > 60) within 100 mmol m2 s1 wide PPFD bins for one-hour intervals centered on the Local Time (LT) shown. Ninety five percent confidence intervals are shown for the 730– 830 curve; confidence intervals for the other times are similar. NEE was screened to exclude periods with a friction velocity (U) < 0.2 m s1. (b) Average NEE (n > 20) and 95% confidence intervals for half-hour intervals with a PPFD of 500 to 600 mmol m2s1 as a function of Local Time. [10] Co-occurring species at the Tapajo´s National Forest differ in the degree to which endogenous factors control leaf gas exchange (Table 1). Two species exhibited no obvious endogenous gas exchange trend. Four other species exhibited a marked decline in gas exchange during the first afternoon without subsequent recovery. Four other species exhibited an endogenous trend in stomatal conductance, 3 of 5 DOUGHTY ET AL.: CIRCADIAN RHYTHMS CONSTRAIN GAS EXCHANGE L15404 L15404 [12] Increased soil respiration due to afternoon warming might have caused part of the afternoon NEE decline. However, automated chamber observations at the site showed that soil respiration increased by less than 1 mmol m2s1 from morning to afternoon [Goulden et al., 2004]. Alternatively, decreased afternoon photosynthesis due to increased stress might have caused part of the afternoon NEE decline. However, it is unlikely that increased stress alone caused the dramatic NEE reduction from 1300 to 1700LT since air temperature and vapor saturation deficit were nearly constant, evapotranspiration was decreasing, and leaf water potential was recovering during this period (Figure 2a) [da Rocha et al., 2004]. Moreover, the diurnal change in NEE for the subset of observations with a PPFD of 400 to 700 mmol m2s1 and measurable rainfall within the preceding 2 hours was similar to that for observations with a PPFD of 400 to 700 mmol m2s1 and no rain within 24 hours (plots not shown). Recent rain and the resulting wet canopy would be expected to decrease stress, whereas NEE declined by 11 mmol m2s1 from 1230 to 1630LT on both wet and dry afternoons. Finally, a midday increase in the ratio of diffuse to direct beam radiation might have increased photosynthesis. However, this effect was probably small since the midday light environment was dominated by patchy sunlight, which has only a minor effect on NEE [Rocha et al., 2004]. 3. Results and Discussion [14] Our observations extend previous laboratory studies to show that endogenous gas exchange rhythms are com- mon in canopy trees and understory plants growing natu- rally in a tropical forest. The adaptive significance of circadian gas exchange rhythms is poorly understood. One 3. Results and Discussion We conclude that the gas exchange at our study site is strongly co-limited over the day by the plants’ physiological capacity as regulated by circadian rhythms and by the direct effect of the physical environment on physiology. Both factors are important in controlling the diel patterns of gas exchange, and the goal for plant physiologists, micrometeorologists and modelers is to build a broad understanding of tropical forest physiology that accounts for both the endogenous and exogenous controls on land-atmosphere exchange. [13] We believe the diurnal NEE shifts are far larger than can be accounted for by the combined effects of soil respiration, stress, and light quality. We acknowledge that whole ecosystem exchange observations of the type ana- lyzed may be confounded by multicollinearity and are unlikely to provide the definitive proof of an endogenous circadian rhythm that is possible under tightly controlled laboratory conditions. Similarly, we note that the logistical difficulty of fieldwork in a tropical forest canopy prevented us from fully documenting that the rhythms we observed at the leaf level have all the hallmarks of circadian oscillators [McClung, 2001; Johnson et al., 1998; Webb, 2003]. None- theless, we emphasize that the diurnal changes in CO2 exchange we observed for the whole forest (Figure 3) appear very similar to those that we observed for individual leaves (Figures 1 and 2), which, in turn, appear very similar to the circadian rhythms that have been intensively inves- tigated in the laboratory [Hennessey and Field, 1991; Williams and Gorton, 1998; Dodd et al., 2005]. The predawn increase in leaf-level CO2 uptake (Figures 1 and 2), and the morning increase in whole-forest CO2 uptake (Figure 3), are particularly difficult to explain if gas ex- change is driven solely by the direct effect of the physical environment on physiology. In fact, a biological-clock- driven circadian rhythm is the only established mechanism we know of that can account for all of our observations. [17] Acknowledgments. We thank Helber Freitas, Michela Figueira, Augusto Maia, and Albert de Sousa for field help, Clara Tinoco, Jim Randerson, Michael Keller, Jeff Chambers, Adrian Rocha, Diane Pataki, and the late Tim Hennessey for comments, suggestions, and ideas; Tomas Domingues and Jim Ehlerhinger for advice and equipment loan; UFPA in Santare´m and Bele´m for help identifying species; and the LBA-ECO Santare´m field office for support. This work was funded by NASA as a component of LBA-ECO. Bonan, G. B. (2002), Ecological Climatology: Concepts and Applications, Cambridge Univ. Press, New York. Campbell, G. S., and J. M. Norman (1998), An Introduction to Environ- mental Biophysics, Springer, New York. DOUGHTY ET AL.: CIRCADIAN RHYTHMS CONSTRAIN GAS EXCHANGE L15404 DOUGHTY ET AL.: CIRCADIAN RHYTHMS CONSTRAIN GAS EXCHANGE L15404 da Rocha, H. R., et al. (2004), Seasonality of water and heat fluxes over a tropical forest in eastern Amazonia, Ecol. Appl., 14, S22–S32. Mansfield, T. A., and O. V. S. Heath (1963), Studies in stomatal behaviour. 9. Photoperiodic effects on rhythmic phenomena in xanthium pennsylva- nicum, J. Exp. Bot., 14, 334–352. p pp Dodd, A. N., et al. (2005), Plant circadian clocks increase photosynth- esis, growth, survival, and competitive advantage, Science, 309, 630– 633. , p , , McClung, C. R. (2001), Circadian rhythms in plants, Annu. Rev. Plant Physiol. Plant Mol. Biol., 52, 139–162. Field, C. B., M. J. Behrenfeld, J. T. Randerson, and P. Falkowski (1998), Primary production of the biosphere: Integrating terrestrial and oceanic components, Science, 281, 237–240. Ribero, J. E. et al. (1999), Flora da Reserva Ducke (in Portuguese), 799 pp., Dep. for Int. Dev., Manaus, Brazil. pp p Rocha, A. V., et al. (2004), Photosynthetic and water use efficiency re- sponses to diffuse radiation by an aspen-dominated northern hardwood forest, For. Sci., 50, 793–801. p Goulden, M. L., et al. (2004), Diel and seasonal patterns of tropical forest CO2 exchange, Ecol. Appl., 14, S24–S54. , , , Saleska, S. R., et al. (2003), Carbon in Amazon forests: Unexpected sea- sonal fluxes and disturbance-induced losses, Science, 302, 1554–1557. Gu, L., D. Baldocchi, S. B. Verma, T. A. Black, T. Vesala, E. M. Falge, and P. R. Dowty (2002), Advantages of diffuse radiation for terrestrial eco- system productivity, J. Geophys. Res., 107(D6), 4050, doi:10.1029/ 2001JD001242. Sellers, P. J., et al. (1997), Modeling the exchanges of energy, water, and carbon between continents and the atmosphere, Science, 275, 502–509. Harmer, S. L., et al. (2002), Orchestrated transcription of key pathways in Arabidopsis by the circadian clock, Science, 290, 2110–2113. Stalfelt, M. G. (1963), Diurnal dark reactions in stomatal movements, Phy- siol. Plantarum, 16, 756–766. Webb, A. A. R. (2003), The physiology of circadian rhythms in plants, New Phytol., 160, 281–303. Hennessey, T. L., and C. B. Field (1991), Circadian-rhythms in photosynth- esis-oscillations in carbon assimilation and stomatal conductance under constant conditions, Plant Physiol., 96, 831–836. y Williams, W. E., and H. L. Gorton (1998), Circadian rhythms have insig- nificant effects on plant gas exchange under field conditions, Physiol Plantarum, 103, 247–256. , y , , Hetherington, M., and F. I. C. E. Doughty, M. L. Goulden, and S. D. Miller, Department of Earth System Science, University of California, Croul Hall, Irvine, CA 92697– 3100, USA. (mgoulden@uci.edu) References Bonan, G. B. (2002), Ecological Climatology: Concepts and Applications, Cambridge Univ. Press, New York. Campbell, G. S., and J. M. Norman (1998), An Introduction to Environ- mental Biophysics, Springer, New York. 4 of 5 DOUGHTY ET AL.: CIRCADIAN RHYTHMS CONSTRAIN GAS EXCHANGE Woodward (2003), The role of stomata in sensing and driving environmental change, Nature, 424, 901–908. Johnson, C. H., M. Knight, A. Trewavas, and T. Kondo (1998), Biological Rhythms and Photoperiodism in Plants, edited by P. Lumsden and A. Millar, pp. 1–34, Oxford Univ. Press, New York. A. Millar, pp. 1–34, Oxford Univ. Press, New York H. R. da Rocha, Department of Atmospheric Sciences, University of Sa˜o Paulo, Sa˜o Paulo, SP Brazil. Jones, H. G. (1992), Plants and Microclimate, Cambridge Univ. Press, New York. C. E. Doughty, M. L. Goulden, and S. D. Miller, Department of Earth System Science, University of California, Croul Hall, Irvine, CA 92697– 3100, USA. (mgoulden@uci.edu) Lambers, H., F. S. Chapin III, and T. L. Pons (1998), Plant Physiological Ecology, Springer, New York. 5 of 5
https://openalex.org/W3167154406	https://malariajournal.biomedcentral.com/track/pdf/10.1186/s12936-021-03936-3	English	null	Evidence supporting deployment of next generation insecticide treated nets in Burkina Faso: bioassays with either chlorfenapyr or piperonyl butoxide increase mortality of pyrethroid-resistant Anopheles gambiae	Malaria journal	2,021	cc-by	8,938	Hien et al. Malar J (2021) 20:406 https://doi.org/10.1186/s12936-021-03936-3 Hien et al. Malar J (2021) 20:406 https://doi.org/10.1186/s12936-021-03936-3 Malaria Journal Open Access Evidence supporting deployment of next generation insecticide treated nets in Burkina Faso: bioassays with either chlorfenapyr or piperonyl butoxide increase mortality of pyrethroid‑resistant Anopheles gambiae Aristide S. Hien1 , Dieudonné D. Soma1, Samina Maiga1, Dramane Coulibaly1, Abdoulaye Diabaté1, Allison Belemvire2, Mame B. Diouf3, Djenam Jacob4, Adama Koné5, Ellen Dotson6, Taiwo S. Awolola6,7, Richard M. Oxborough4 and Roch K. Dabiré1* Abstract Background: Pyrethroid resistance poses a major threat to the efficacy of insecticide-treated nets (ITNs) in Burkina Faso and throughout sub-Saharan Africa, particularly where resistance is present at high intensity. For such areas, there are alternative ITNs available, including the synergist piperonyl butoxide (PBO)-based ITNs and dual active ingre- dient ITNs such as Interceptor G2 (treated with chlorfenapyr and alpha-cypermethrin). Before deploying alternative ITNs on a large scale it is crucial to characterize the resistance profiles of primary malaria vector species for evidence- based decision making. Methods: Larvae from the predominant vector, Anopheles gambiae sensu lato (s.l.) were collected from 15 sites located throughout Burkina Faso and reared to adults for bioassays to assess insecticide resistance status. Resistance intensity assays were conducted using WHO tube tests to determine the level of resistance to pyrethroids commonly used on ITNs at 1×, 5 × and 10 × times the diagnostic dose. WHO tube tests were also used for PBO synergist bioas- says with deltamethrin and permethrin. Bottle bioassays were conducted to determine susceptibility to chlorfenapyr at a dose of 100 µg/bottle. Results: WHO tube tests revealed high intensity resistance in An. gambiae s.l. to deltamethrin and alpha-cyperme- thrin in all sites tested. Resistance intensity to permethrin was either moderate or high in 13 sites. PBO pre-exposure followed by deltamethrin restored full susceptibility in one site and partially restored susceptibility in all but one of the remaining sites (often reaching mortality greater than 80%). PBO pre-exposure followed by permethrin partially restored susceptibility in 12 sites. There was no significant increase in permethrin mortality after PBO pre-exposure in ( ), Burkina Faso Full list of author information is available at the end of the article Burkina Faso Full list of author information is available at the end of the article Background mutations and metabolic resistance patterns [13, 14], there is limited bioassay information regarding the rela- tive improvement in mortality provided by piperonyl butoxide (PBO) synergists within wild An. gambiae s.l. populations. Reduced mosquito mortality by pyrethroid ITNs was reported in experimental hut trials in Benin in 2007 [15] and more recently in Burkina Faso [16] with free-flying An. gambiae s.l. In order to preserve the effi- cacy of ITNs and other insecticide-based control meth- ods such as IRS, the World Health Organization (WHO) has developed a global plan for insecticide resistance management (GPIRM) [17]. Among the key elements are: (i) rotation of insecticides; (ii) mixtures of at least two different insecticides; (iii) alternating use of at least two insecticides from different classes; and, (iv) mosaic use of insecticides. g Burkina Faso relies on mass distribution of insecticide- treated nets (ITNs) every three years as the primary method of vector control, while indoor residual spray- ing (IRS) has been implemented in two or three districts per year since 2018 [1]. Millions of pyrethroid ITNs have been distributed in Burkina Faso during the past 20 years which, along with agricultural use of pyrethroids, has increased the selection pressure on malaria vector popu- lations. Pyrethroid resistance was observed in Anopheles gambiae sensu lato (s.l.) populations for the first time in Côte d’Ivoire in 1993 [2], and within a few years was detected throughout all countries in West Africa, includ- ing Burkina Faso, and is now found across all of sub-Saha- ran Africa [3, 4]. The voltage-gated sodium channel gene (Vgsc)-L1014F mutation (also known as kdr-west) was assumed to be the main mechanism conferring resistance to pyrethroids in West Africa [5, 6], while in East Africa, a different amino acid substitution at the same locus known as Vgsc-L1014S (also known as kdr-east) had been detected [7]. Voltage-gated sodium channel gene muta- tions were first identified within An. gambiae populations [5, 6] across West Africa before it was detected within Anopheles coluzzii [8], most likely as a result of introgres- sion, while its occurrence in wild Anopheles arabiensis populations was identified as an independent mutation [9]. While Vgsc mutations are widespread and contribute to phenotypic pyrethroid resistance, metabolic resistance mechanisms are generally more important drivers of high intensity pyrethroid resistance [10].fi A limiting factor preventing implementation of these strategies has been the lack of alternative classes of insec- ticide for ITNs. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hien et al. Malar J (2021) 20:406 Hien et al. Malar J (2021) 20:406 Page 2 of 12 Kampti, Karangasso-Vigué or Mangodara; while in Seguenega, Orodara and Bobo-Dioulasso there was a significant increase in mortality, but rates remained below 50%. Susceptibility to chlorfenapyr was confirmed in 14 sites. Conclusion: High pyrethroid resistance intensity in An. gambiae s.l. is widespread across Burkina Faso and may be a predictor of reduced pyrethroid ITN effectiveness. PBO + deltamethrin ITNs would likely provide greater control than pyrethroid nets. However, since susceptibility in bioassays was not restored in most sites following pre-exposure to PBO, Interceptor G2 may be a better long-term solution as susceptibility was recorded to chlorfenapyr in nearly all sites. This study provides evidence supporting the introduction of both Interceptor G2 nets and PBO nets, which were distributed in Burkina Faso in 2019 as part of a mass campaign. Keywords: Pyrethroid, Insecticide resistance, Piperonyl butoxide, Chlorfenapyr, Anopheles gambiae, Insecticide- treated net, Malaria, Burkina Faso Background However, in recent years several PBO synergist nets have received prequalification (PQ) listing by the WHO [18]. PBO is a synergist that acts by inhibit- ing metabolic enzymes within the mosquito, particularly cytochrome P450s that detoxify or sequester pyrethroids [19]. Although synergists do not typically act as insecti- cides, they can increase or restore the potency of insecti- cides by overcoming existing resistance mechanisms [19, 20]. As a result, PBO nets should produce an increased killing effect of malaria vectors where the major pyre- throid resistance mechanisms are due to increased oxi- dase activity. Two randomized control trials in Tanzania and Uganda have demonstrated a clinical benefit of PBO nets in areas of moderate pyrethroid resistance, with a reduction in malaria prevalence detected for at least six months after net distribution compared to conventional pyrethroid nets [21, 22]. Pyrethroid resistance poses a threat to the efficacy of ITNs, particularly when resistance is present at high intensity [11, 12]. In Burkina Faso, pyrethroid resist- ance has been observed nationwide for several years and there are fears that pyrethroid ITNs may no longer provide the desired levels of individual and community protection [6]. While there are several localized studies in Burkina Faso that have described the genetic muta- tions involved in pyrethroid resistance, including Vgsc- 1014F and 1014S mutations in association with N1575Y A new ‘dual active ingredient’ net with two different active ingredients (AIs) is Interceptor® G2 (combining chlorfenapyr and alpha-cypermethrin), which received WHO PQ listing in 2018 [18]. Chlorfenapyr is a pyr- role compound with a non-neurotoxic mode of action Hien et al. Malar J (2021) 20:406 Page 3 of 12 Page 3 of 12 Pyrethroid insecticide susceptibility and resistance intensity tests using WHO tube testsh Three pyrethroid insecticides were used for susceptibil- ity tests at their diagnostic concentrations: permethrin 0.75%, deltamethrin 0.05% and alpha-cypermethrin 0.05%. Pyrethroid resistance intensity was monitored in 14 locations (resistance intensity testing was not con- ducted in Bobo-Dioulasso) with alpha-cypermethrin, deltamethrin and permethrin papers treated at 1, 5 and 10 times the diagnostic concentration using WHO tubes. Prior to the field susceptibility assays, two impregnated papers with each insecticide were randomly selected from each box and tested against a laboratory-main- tained susceptible strain of An. gambiae Kisumu to verify the quality of the paper. On average, batches of 20–25 sugar-fed female An. gambiae s.l. aged 3–5 days old were introduced into each WHO tube and exposed to the insecticide-treated paper for one hour [28] at 27 ± 2 °C temperature and 70 ± 5% relative humidity (Additional file 2). Four replicates were conducted, making a total of approximately 100 mosquitoes tested for each insecti- cide per site. Negative control bioassays were conducted for each test by exposing a total of 50 An. gambiae s.l. (two replicates of 25) using tubes lined with filter papers treated with silicone oil. Positive control bioassays were also conducted for each insecticide with an insectary colony of susceptible An. gambiae Kisumu (4 replicates per insecticide). The knock-down rates were recorded at 5, 10, 15, 20, 30, 40, 50 and 60 min after the start of expo- sure with mortality recorded 24 h after insecticide expo- sure [28]. All mosquitoes were held for 24 h in laboratory conditions at 27 ± 2 °C temperature and 70 ± 5% relative Chlorfenapyr susceptibility tests using CDC bottle bioassays During the time of data collection there was no pub- lished guidance from WHO regarding chlorfenapyr sus- ceptibility test procedures or diagnostic concentrations. Preliminary bottle bioassay testing by Center for Disease Control and Prevention (CDC) protocol established a tentative diagnostic dose of 100 µg/bottle. Therefore, 250-ml Wheaton bottles were treated in the IRSS labo- ratory using technical grade chlorfenapyr dissolved in acetone at a dosage of 100 µg/bottle. Mortality rates were assessed at 24, 48 and 72 h after exposure. Four replicates of 25 An. gambiae s.l. were conducted, making a total of approximately 100 mosquitoes tested per site. Tests were conducted during the daytime with effort made to keep testing and holding conditions within WHO guidelines of 27 ± 2 °C and relative humidity of 75 ± 10% [28]. PBO synergist assays using WHO tube tests y g y g Synergist bioassays were also conducted by exposing batches from 20 to 25 sugar-fed female An. gambiae s.l. in WHO tubes lined with PBO (4%)-impregnated papers for one hour before being transferred to a second tube with a pyrethroid insecticide (either permethrin (0.75%), deltamethrin (0.05%) or alpha-cypermethrin (0.05%)) impregnated paper for one hour (Additional file 3). The number of dead mosquitoes was recorded 24 h after the end of the exposure period at 27 ± 2 °C temperature and 70 ± 5% relative humidity. Negative controls consisted of two batches of around 20–25 mosquitoes exposed to PBO without subsequent exposure to pyrethroid insecti- cide and also silicone oil-treated papers. Each test con- sisted of four replicates. Synergist tests were conducted in all 15 sites with deltamethrin and permethrin but only five sites with alpha-cypermethrin due to insufficient mosquitoes in 10 sites during larval collections. Mosquito collections Mosquito larval collections were carried out between August and October 2019 during the period of high malaria vector density. Anopheles gambiae s.l. larvae were collected from temporary water pools from all 15 sites before transport to the Research Institute of Health Sciences (IRSS) insectary and were reared to the adult stage prior to use in bioassays. Female An. gambiae s.l. were then used for insecticide susceptibility assays according to WHO procedures [28]. Species composi- tion among the An. gambiae species complex was deter- mined through PCR after completion of bioassay and is described below. Methods Study sitesh In the central Sudano-Sahelian zone (Solenzo, Nouna, Boromo, Ouagadougou, and Kaya), the average yearly rainfall is 600–900 mm per year, with a shorter rainy sea- son than in the southwest, extending from June to Sep- tember 2019. humidity to evaluate the mortality. Insecticide-treated papers were supplied by the WHO Collaborating Centre of Universiti Sains Malaysia (USM). Methods Study sitesh which shows no cross resistance to existing pyrethroid resistance mechanisms [23, 24]. Experimental hut studies of Interceptor G2 have shown high efficacy and wash durability against pyrethroid resistant malaria vectors in Benin [25], Burkina Faso [26] and Côte d’Ivoire [27]. The NMCP of Burkina Faso selected 21 sentinel sites for malaria vector insecticide resistance monitoring studies (Fig. 1). The sites were dispersed nationwide through the three eco-climatic zones of the country (Sahelian, Sudan- Sahelian, Sudan). For security reasons, it was not possible to monitor all 21 sites in 2019, especially sites located in the Sahelian zone and eastern part of country, therefore, the study covered 15 sites located in the three climatic areas, including IRS sites and neighbouring unsprayed control sites (Additional file 1). The Sudanian climatic zone covers the southwestern area (including Orodara, Bobo-Dioulasso, Soumousso, Karangasso-Vigué, Dié- bougou, Gaoua, Kampti, and Mangodara sites) and is the wettest in the country, with the mean annual rainfall averaging 1,000–1200 mm with most occurring in the rainy season extending from May to November, whereas the northern Sahelian zone (Kongoussi and Seguenega) is relatively dry, with less than 600 mm annual rainfall. To guide National Malaria Control Programme (NMCP) decision-making regarding choice of ITN type, it is crucial to regularly gather insecticide sus- ceptibility data for those insecticides used on ITNs, namely pyrethroids with and without PBO syner- gist, and chlorfenapyr. This study evaluated the level of pyrethroid resistance intensity in An. gambiae s.l. against the main pyrethroids used on ITNs followed by synergist assays to determine whether the syn- ergist PBO restores susceptibility. In addition, the study determined susceptibility of An. gambiae s.l. to chlorfenapyr. Fig. 1 Location of 15 NMCP selected insecticide resistance monitoring sites where testing was conducted in 2019 (including 3 sites where IRS was conducted and 3 neighbouring unsprayed site) Fig. 1 Location of 15 NMCP selected insecticide resistance monitoring sites where testing was conducted in 2019 (including 3 sites where IRS was conducted and 3 neighbouring unsprayed site) Hien et al. Malar J (2021) 20:406 Page 4 of 12 In the central Sudano-Sahelian zone (Solenzo, Nouna, Boromo, Ouagadougou, and Kaya), the average yearly rainfall is 600–900 mm per year, with a shorter rainy sea- son than in the southwest, extending from June to Sep- tember 2019. Molecular assays to determine Anopheles gambiae s.l. species composition and characterization of resistance mutations After the bioassay tests, all mosquitoes were placed individually in 1.5-ml tubes with silica gel before being stored in a − 20 °C freezer at IRSS until further labora- tory analysis. A sub-set of approximately 50 female An. gambiae s.l. per site were tested by PCR to determine species composition. Genomic DNA of mosquitoes was extracted with 2% cetyl trimethyl ammonium bromide (2% CTAB). Species of the An. gambiae complex were identified by PCR as either An. gambiae sensu stricto (s.s.), An. coluzzii or An. arabiensis using the Sine 200X Hien et al. Malar J (2021) 20:406 Page 5 of 12 protocol of Santolamazza et al. [29]. Mutations involved in insecticide resistance were identified by PCR using the protocols of Martinez-Torres et al. [30] and Ranson et al. [7] for the voltage-gated sodium channel (Vgsc) L1014F and L1014S mutations. were estimated for sites located in Sudan areas where the mass coverage of PBO-pyrethroid ITNs were targeted. The genotypic frequencies of Vgsc1014F and 1014S in mosquito populations were compared to Hardy–Wein- berg expectations using GenePOP software [32]. Pyrethroid susceptibility • Complete restoration of susceptibility following pre- exposure to PBO (i.e. ≥ 98% mean mortality) implies that a monooxygenase-based resistance mechanism fully accounts for expression of the resistant pheno- type in the test population. WHO tube tests revealed that An. gambiae s.l. were resistant to the three pyrethroid insecticides tested, with mortality rates less than 70% at all sites for deltamethrin, permethrin and alpha-cypermethrin diagnostic doses (Fig. 3). Mean mortality rates across the 14 sites were 33.2% for deltamethrin, 24.5% for permethrin and 19.0% for alpha-cypermethrin (Fig. 3). There was particularly low mortality in Kaya for all three pyrethroids: 11.8% (95% CI:0.1–28.6) mortality for deltamethrin, 13.6% (95% CI:0.1–27.2) for permethrin and 2.0% (95% CI:0.1–7.8) for alpha-cypermethrin. There were many examples of sites having very low mortality rates for individual pyre- throid insecticides. The susceptible An. gambiae Kisumu strain exhibited 100% mortality to all three pyrethroids, therefore confirming that insecticide-treated papers were dosed correctly. Control mortality was less than 5% in all bioassays. • Partial restoration of susceptibility following pre- exposure to PBO (i.e. mean mortality in the PBO followed by insecticide samples is greater than mean mortality in the insecticide only samples but less than 98%) implies that a monooxygenase-based resistance mechanism only partially accounts for expression of the resistant phenotype and that other resistance mechanisms are likely to be present in the test popu- lation. • No restoration of susceptibility following pre-expo- sure to PBO (mean mortality in the PBO followed by insecticide samples is equal to or lower than mean mortality in the insecticide only samples) implies that the resistance phenotype detected is not based on mono-oxgenase-mediated detoxification. Data analysis Distribution of Anopheles gambiae species Of 700 An. gambiae s.l. analysed by PCR, 51.8% were An. gambiae s.s., 30.6% An. coluzzii and 17.6% An. arabien- sis. Anopheles gambiae s.s. was the predominant species in the Sudan area (61.6%), except in Kampti and Bobo- Dioulasso where more than 50% were An. arabiensis (Fig. 2). In the Sudano-Sahelian zone An. coluzzii was more common in Nouna and Solenzo, where it was the dominant species (> 50%); however, in Boromo and Oua- gadougou, more than 80% were An. gambiae s.s. Anoph- eles coluzzii was the predominant species in Sahelian area (67.3%). In all three eco-climatic zones, An. gambiae s.s. was found in sympatry with An. coluzzii. Anopheles arabiensis was found in relatively high proportions in the Sudanian zone at 27.4% (95%CI: 0.6–51.3). Data were entered into Microsoft Excel and analysed with STATA version 13.0 (College Station, TX 77,845, USA). WHO criteria were used to classify wild An. gambiae s.l. as ‘resistant’ if less than 90% mortality was observed, resistance needing confirmation if mortality was between 90–97% and susceptible if between 98–100% [28]. Resist- ance intensity was defined as being high resistance intensity if mortality at the 10 × dose was less than 98%, moderate intensity if less than 98% at the 5 × dose but greater than 98% at 10x, and low intensity resistance if mortality was greater than 98% at the 5 × dose [28].h The results of synergist assays were analysed by insecti- cide by comparing results of PBO plus pyrethroid versus pyrethroid only. The data were then interpreted following WHO criteria [28]: Pyrethroid resistance intensityh The results showed high resistance intensity in all 14 sites to deltamethrin and alpha-cypermethrin (mortal- ity less than 98% at the 10 × dose) with An. gambiae s.l. (Fig. 3A, B). Mortality rates varied between 45.6% in Kaya to 96.0% in Karangasso-Vigue at the 10 × dose of del- tamethrin (Fig. 3A). The results with alpha-cypermethrin at the 10 × dose showed mortality rates between 45.8% in Nouna and 96.3% in Kampti (Fig. 3B). The resistance intensity of An. gambiae s.l. to permethrin was more varied with eight sites classified as high intensity, five as Descriptive statistics were used to calculate the mean mortality rates and differences between the average mor- tality rate. Mortality rates from bioassays were calculated with their 95% confidence interval (CI) and compared by ecological zone using Chi-squared test. The knockdown times for 50 and 95% of tested mosquitoes (KdT50 and KdT95) were calculated using a log time probit model [31] for mosquito exposure to PBO followed by deltame- thrin and permethrin exposure. The KdT50 and KdT95 Hien et al. Malar J (2021) 20:406 Page 6 of 12 Fig. 2 Members of the Anopheles gambiae s.l. present at each study sites (n≈50 females mosquitoes analysed per site) Fig. 3 Percentage mortality (24 h) of Anopheles gambiae s.l. in WHO tube tests at 1, 5, and 10 times the diagnostic concentration of A deltamethrin (0.05%, 0.25%, 0.50%), B alpha-cypermethrin (0.05, 0.25, 0.50%) and C permethrin (0.75, 3.75, 7.50%) (n≈100 female mosquitoes per dose/ insecticide) Fig. 2 Members of the Anopheles gambiae s.l. present at each study sites (n≈50 females mosquitoes analysed per site) Fig. 2 Members of the Anopheles gambiae s.l. present at each study sites (n≈50 females mosquitoes analysed per site) Fig. 2 Members of the Anopheles gambiae s.l. present at each study sites (n≈50 females mosquitoes analysed per site) Fig. 3 Percentage mortality (24 h) of Anopheles gambiae s.l. in WHO tube tests at 1, 5, and 10 times the diagnostic concentration of A deltamethrin (0.05%, 0.25%, 0.50%), B alpha-cypermethrin (0.05, 0.25, 0.50%) and C permethrin (0.75, 3.75, 7.50%) (n≈100 female mosquitoes per dose/ insecticide) Fig. 3 Percentage mortality (24 h) of Anopheles gambiae s.l. in WHO tube tests at 1, 5, and 10 times the diagnostic concentration of A deltamethrin (0.05%, 0.25%, 0.50%), B alpha-cypermethrin (0.05, 0.25, 0.50%) and C permethrin (0.75, 3.75, 7.50%) (n≈100 female mosquitoes per dose/ insecticide) Fig. Pyrethroid resistance intensityh 3 Percentage mortality (24 h) of Anopheles gambiae s.l. in WHO tube tests at 1, 5, and 10 times the diagnostic concentration of A deltamethrin (0.05%, 0.25%, 0.50%), B alpha-cypermethrin (0.05, 0.25, 0.50%) and C permethrin (0.75, 3.75, 7.50%) (n≈100 female mosquitoes per dose/ insecticide) Fig. 3 Percentage mortality (24 h) of Anopheles gambiae s.l. in WHO tube tests at 1, 5, and 10 times the diagnostic concentration of A deltamethrin (0.05%, 0.25%, 0.50%), B alpha-cypermethrin (0.05, 0.25, 0.50%) and C permethrin (0.75, 3.75, 7.50%) (n≈100 female mosquitoes per dose/ insecticide) Page 7 of 12 Hien et al. Malar J (2021) 20:406 Hien et al. Malar J (2021) 20:406 moderate and one with low resistance intensity (Diebou- gou) (Fig. 3C). to PBO followed by alpha-cypermethrin in three of five sites (Ouagadougou, Karangasso-Vigué and Soumousso) (Fig. 4C). In the remaining two sites of Kongoussi and Seguenega there was partial restoration of susceptibility, but mortality rates did not reach 50%. PBO plus pyrethroid synergist assays Pre-exposure to PBO (4%) followed by a pyrethroid insecticide significantly improved mortality rates in all sites for deltamethrin (P < 0.05) although there was great variation between sites (Fig. 4A). PBO pre-exposure fol- lowed by deltamethrin restored full susceptibility only in Gaoua but partially restored susceptibility in the remain- ing sites (often reaching mortality greater than 80%) except Solenzo where the mortality remained below 50%. Pre-exposure to PBO (4%) followed by a pyrethroid insecticide significantly improved mortality rates in all sites for deltamethrin (P < 0.05) although there was great variation between sites (Fig. 4A). PBO pre-exposure fol- lowed by deltamethrin restored full susceptibility only in Gaoua but partially restored susceptibility in the remain- ing sites (often reaching mortality greater than 80%) except Solenzo where the mortality remained below 50%. PBO pre-exposure followed by permethrin did not restore full susceptibility in any site, but did partially restore susceptibility in 12 sites from the 14 sites tested. However, there was no significant increase in mortal- ity in Kampti, Karangasso-Vigué or Mangodara. While in Seguenega, Orodara and Bobo-Dioulasso there was a significant increase in mortality, but rates remained below 50% (Fig. 4B). Synergist tests with alpha-cyperme- thrin were only conducted in five sites. Mortality of An. gambiae s.l. reached more than 90% after pre-exposure The analysis of knockdown times (KDT) showed that the pre-exposure of An. gambiae s.l. to PBO reduced both the KDT50 and KDT95 (time needed to achieve 50 and 95% mosquito knockdown) compared to those mos- quitoes only exposed to deltamethrin or permethrin. The KDT50 and KDT95 times with deltamethrin after exposure to PBO were 2 to 3 times faster compared to those obtained with deltamethrin only (Table 1). KDT50 and KDT95 values for PBO after exposure to permethrin showed similar trends with two-fold faster knockdown than permethrin only (Table 1). PBO pre-exposure followed by permethrin did not restore full susceptibility in any site, but did partially restore susceptibility in 12 sites from the 14 sites tested. However, there was no significant increase in mortal- ity in Kampti, Karangasso-Vigué or Mangodara. While in Seguenega, Orodara and Bobo-Dioulasso there was a significant increase in mortality, but rates remained below 50% (Fig. 4B). Synergist tests with alpha-cyperme- thrin were only conducted in five sites. Mortality of An. gambiae s.l. reached more than 90% after pre-exposure Chlorfenapyr susceptibilityh The results of susceptibility tests performed with An. gambiae s.l. against chlorfenapyr at 100 µg/bottle revealed high mortality (98–100%) 24 h after exposure in most sites. Susceptibility to chlorfenapyr was confirmed Fig. 4 Percentage mortality (24 h) of Anopheles gambiae s.l. in WHO tube tests with A deltamethrin (0.05%) with/without PBO (4%) and B permethrin (0.75%) with/without PBO (4%) C alpha-cypermethrin (0.05%) with/without PBO (4%) tage mortality (24 h) of Anopheles gambiae s.l. in WHO tube tests with A deltamethrin (0.05%) with/without PBO (4%) and B .75%) with/without PBO (4%) C alpha-cypermethrin (0.05%) with/without PBO (4%) Hien et al. Malar J (2021) 20:406 Page 8 of 12 in 14 sites using 72 h mortality data, with Boromo th only site where mortality was less than 98% (Fig. 5; Addi tional file 4). Frequency of the Vgsc‑L1014F and 1014S mutations The Vgsc-L1014F mutation was observed at varying fre quencies between sites and by mosquito species (Table 2) This mutation was found at a higher frequency within An. gambiae s.s. populations with allele frequencies vary ing from 0.53 in Boromo to 0.98 in Soumousso (for site where more than 20 samples were tested). The mean fre quency within An. gambiae s.s. (n = 310) across all site was 0.71 for Vgsc-L1014F and 0.13 for Vgsc-L1014S, with 5% of this species having both alleles present. In An. coluzzii (n = 200), the mean Vgsc-L1014F fre quency was lower than that in An. gambiae s.s. at 0.52 The mean Vgsc-L1014S frequency was 0.19 in An Table 1 Time to knockdown (in minutes) for 50% and 95% ( following exposure to pyrethroids only and PBO plus pyrethroid Climatical area Insecticides KDT50 (min) Sudan Deltamethrin 48.61 Sudan PBO + deltamethrin 22.97 Sudan Permethrin 154.21 Sudan PBO + permethrin 78.29 Fig. 5 Percentage mortality (24 and 72 h) of Anopheles gambiae s.l. in s AI/bottle Table 1 Time to knockdown (in minutes) for 50% and 95% (KDT50 and KDT95) of Anopheles gambiae s.l. from the Sudanian area following exposure to pyrethroids only and PBO plus pyrethroid Climatical area Insecticides KDT50 (min) [95% IC] KDT50 KDT95 (min) [95% IC] KDT95 Sudan Deltamethrin 48.61 [45.43–52.59] 146.48 [123.23–182.75] Sudan PBO + deltamethrin 22.97 [22.06–23.89] 48.46 [45.31–52.41] Sudan Permethrin 154.21 [111.65–262.32] 1051.22 [520.39–3427.81] Sudan PBO + permethrin 78.29 [66.69–97.07] 530.84 [348.65–955.87] in 14 sites using 72 h mortality data, with Boromo the only site where mortality was less than 98% (Fig. Chlorfenapyr susceptibilityh 5; Addi- tional file 4). coluzzii. More surprisingly, both allele mutations were also present in An. arabiensis populations at a similar frequency to An. coluzzii, with a mean of 0.52 for Vgsc- L1014F and 0.19 for Vgsc-L1014S. Comparing by climatic zones, the Vgsc-L1014F mutation was higher in Suda- nian and Sudano-Sahelian areas (χ2 = 12.91, df = 1.311, P < 0.0001) than that in Sahelian sites. Frequency of the Vgsc‑L1014F and 1014S mutationsh The Vgsc-L1014F mutation was observed at varying fre- quencies between sites and by mosquito species (Table 2). This mutation was found at a higher frequency within An. gambiae s.s. populations with allele frequencies vary- ing from 0.53 in Boromo to 0.98 in Soumousso (for sites where more than 20 samples were tested). The mean fre- quency within An. gambiae s.s. (n = 310) across all sites was 0.71 for Vgsc-L1014F and 0.13 for Vgsc-L1014S, with 5% of this species having both alleles present. The L1014S mutation is now widespread throughout the country within An. gambiae s.s. and An. coluzzii and was found in six sites within An. arabiensis populations, although the frequency was relatively low in all sites with 0.29 for An. arabiensis in Kampti being the highest fre- quency for any site (where > 10 samples were tested). Significant deviations from Hardy–Weinberg expec- tations were observed in An. gambiae populations from Kongoussi, Seguenega, Ouagadougou, Boromo, and Kaya, and An. coluzzii populations from Kampti, Gaoua, In An. coluzzii (n = 200), the mean Vgsc-L1014F fre- quency was lower than that in An. gambiae s.s. at 0.52. The mean Vgsc-L1014S frequency was 0.19 in An. Fig. 5 Percentage mortality (24 and 72 h) of Anopheles gambiae s.l. in susceptibility tests with chlorfenapyr in bottle bioassays at a dose of 100 µg AI/bottle Fig. 5 Percentage mortality (24 and 72 h) of Anopheles gambiae s.l. in susceptibility tests with chlorfenapyr in bottle bioassays at a dose of 100 µg Fig. 5 Percentage mortality (24 and 72 h) of Anopheles gambiae s.l. in susceptibility tests with chlorfenapyr in bottle bioassays at a dose of 100 µg AI/bottle Hien et al. Malar J (2021) 20:406 Page 9 of 12 Solenzo, Boromo, and Seguenega. This was due to strong heterozygote deficits. Discussion This study have demonstrated for the first time, using standardized WHO protocols [28], that high intensity pyrethroid resistance is not geographically confined to areas of intense agriculture but is widespread throughout Burkina Faso. Given that the primary malaria vector control strategy in Burkina Faso is nationwide univer- sal coverage with ITNs, of particular concern is the high intensity of resistance to deltamethrin and alpha- cypermethrin documented at every site tested. Pyre- throid resistance intensity is an important measure that may indicate the potential for reduced effectiveness of pyrethroid ITNs. Frequency of the Vgsc‑L1014F and 1014S mutationsh WHO suggest that “confirmed levels Table 2 Allele frequency of the Vgsc-L1014F and -L1014S mutations in Anopheles gambiae s.l. populations in 2019 Species Sites N L1014L L1014F L1014S L1014F L1014L L1014L Allele frequency L1014L L1014F L1014S L1014S L1014F L1014S L1014F L1014S An. gambiae s.s Kampti 13 0 11 2 0 0 0 0.85 0.15 Gaoua 27 0 21 3 1 1 1 0.81 0.15 Solenzo 9 1 5 2 0 1 0 0.61 0.22 Nouna 2 0 1 1 0 0 0 0.50 0.50 Kongoussi 2 0 0 0 1 0 1 0.25 0.50 Seguenega 46 2 9 3 11 14 7 0.34 0.14 Orodara 44 2 38 0 0 4 0 0.91 0 Soumousso 42 1 41 0 0 0 0 0.98 0 Ouagadougou 40 1 37 0 0 0 2 0.93 0.03 Boromo 34 7 14 1 3 5 4 0.53 0.13 Mangodara 43 7 27 3 0 6 0 0.70 0.07 Kaya 8 2 0 0 1 4 1 0.25 0.06 Total 310 23 204 15 17 35 16 0.71 0.13 An. coluzzii Kampti 9 1 2 0 1 4 1 0.50 0.11 Gaoua 3 0 2 0 0 1 0.67 0.17 Solenzo 26 3 10 1 7 4 1 0.60 0.19 Nouna 43 7 16 6 4 7 3 0.50 0.22 Kongoussi 47 2 16 2 8 19 0 0.63 0.13 Seguenega 10 0 4 0 2 3 1 0.65 0.15 Orodara 2 0 0 2 0 0 0 0 1.00 Soumousso 1 0 1 0 0 0 0 1.00 0 Ouagadougou 0 0 0 0 0 0 0 NA NA Boromo 10 1 3 0 0 3 3 0.45 0.15 Mangodara 7 0 2 5 0 0 0 0.29 0.71 Kaya 42 10 8 3 1 16 5 0.39 0.14 Total 200 24 64 19 23 56 15 0.52 0.19 An. Frequency of the Vgsc‑L1014F and 1014S mutationsh arabiensis Kampti 28 0 11 0 11 1 5 0.60 0.29 Gaoua 19 0 9 1 5 1 3 0.63 0.26 Solenzo 9 6 2 0 1 0 0 0.28 0.06 Nouna 5 2 0 0 0 2 1 0.20 0.10 Kongoussi 0 0 0 0 0 0 0 NA NA Seguenega 0 0 0 0 0 0 0 NA NA Orodara 4 0 4 0 0 0 0 1.00 0 Soumousso 6 4 0 2 0 0 0 0 0.33 Ouagadougou 10 0 10 0 0 0 0 1.00 0 Boromo 3 0 3 0 0 0 0 1.00 0 Mangodara 0 0 0 0 0 0 0 NA NA Kaya 0 0 0 0 0 0 0 NA NA Total 84 12 39 3 17 4 9 0.59 0.19 Solenzo, Boromo, and Seguenega. This was due to strong heterozygote deficits. Burkina Faso. Given that the primary malaria vector control strategy in Burkina Faso is nationwide univer- sal coverage with ITNs, of particular concern is the high intensity of resistance to deltamethrin and alpha- cypermethrin documented at every site tested. Pyre- throid resistance intensity is an important measure that may indicate the potential for reduced effectiveness of pyrethroid ITNs. WHO suggest that “confirmed levels Discussionh [34] showed in Vallée du Kou and Tengrela (southwestern Burkina Faso) evidence of syner- gism but pre-exposure to PBO did not fully restore sus- ceptibility to deltamethrin or permethrin in bioassays or in experimental hut studies. Bayili et al. [35] also showed in experimental hut studies in Vallée du Kou that del- tamethrin plus PBO nets provided additional benefit over the equivalent pyrethroid-only LLINs, but mortality and blood-feeding inhibition rates were relatively low. While these studies provided important information, they were limited in geographical scope. Results from study showed that mosquito mortality rates nationwide significantly increased when An. gambiae s.l. were pre-exposed to PBO in association with permethrin, deltamethrin and alpha-cypermethrin even in areas of high resistance intensity in western part of country (Mangodara, Gaoua, Kampti). In all sites, pre-exposure to PBO also reduced the mean KDT compared to exposure to only pyre- throids. While randomized controlled trials have shown the benefit of PBO nets in Tanzania [21] and Uganda [22] in areas of moderate pyrethroid resistance, it is not clear what level of increased mortality in susceptibility bioassays is required to correlate with epidemiological impact. WHO provide limited guidance, stating that the added benefit of pyrethroid PBO nets compared to pyre- throid-only LLINs is expected to be the greatest where y Laboratory analysis showed that the frequency of the Vgsc-L101F mutation is moderate or high in An. gambiae s.s., An. coluzzii and An. arabiensis. The frequency of the Vgsc-L1014S mutation is also increasing in frequency compared to prior years, particularly in An. gambiae s.s. [38]. Surprising results found also were the similar allele mutations frequencies in both species An. coluzzi and An. arabiensis. Indeed, this finding could be explained by the colonization of An. coluzzi breeding sites by An. arabiensis species particularly in urban sites exposed to insecticides pressure. Previous microarray analysis of An. coluzzii from Vallée du Kou has implicated several P450 genes including CYP6P3 and CYP6Z2 [39]. Similarly, Toé et al. [34] found CYP6Z2 and CYP6Z3 the most highly overexpressed genes in Vallée du Kou and Tengrela. This suggests that there are complex underlying resist- ance mechanisms involved, including target site resist- ance, metabolic resistance mechanisms and there may be other mechanisms not yet detected, such as increased cuticle thickness. Discussionh This study have demonstrated for the first time, using standardized WHO protocols [28], that high intensity pyrethroid resistance is not geographically confined to areas of intense agriculture but is widespread throughout Hien et al. Malar J (2021) 20:406 Page 10 of 12 pyrethroid resistance is at “intermediate levels”, where mosquito mortality after exposure to a pyrethroid insec- ticide in WHO test kits ranges from 10 to 80%, but do not state what level of mortality increase is biologically significant [36]. The most recent PMI Malaria Opera- tional Plan Guidance recommends that PBO nets only be considered in areas where PBO increases pyrethroid sus- ceptibility by at least 10% (absolute terms) [37]. Based on results from study, PBO plus deltamethrin ITNs are the most promising option in Burkina Faso as greater than 70% mortality was reached from bioassays in 12 of 15 sites, with susceptibility restored in one site and a greater than 10% absolute increase in mortality in all sites. of resistance, especially at ten times the discriminating concentration may indicate or predict operational con- trol failure and highlight a particularly urgent need to develop an appropriate resistance management strategy” [28]. High intensity resistance was previously only docu- mented in intensive agricultural areas, such as Vallée du Kou, an area of concentrated rice cultivation 25 km from Bobo-Dioulasso, where deltamethrin resistance up to 1,000 fold was reported in An. coluzzii in 2013 compared to the susceptible Kisumu strain of An. gambiae [16]. To supplement universal coverage with pyrethroid ITNs, which are deployed via mass campaigns every three years, US President’s Malaria Initiative (PMI)- funded IRS with non-pyrethroid insecticides (organo- phosphates and neonicotinoids) has also been conducted annually since 2018 in three high burden districts (Kampti, Solenzo, Kongoussi) in accordance with the WHO GPIRM recommendations. However, the rela- tively high cost of IRS makes the prospect of national use of this strategy low and PBO or dual active ingredient nets are likely to be the primary response for control of pyrethroid-resistant malaria vectors. A barrier limiting the purchase of PBO nets by NMCPs and donors is that PBO ITNs cost approximately 40% more than equivalent pyrethroid ITNs [33]. Therefore, it is important to tar- get distribution to locations where PBO synergist bioas- says indicate full or partial restoration of susceptibility to pyrethroids. Toé et al. Discussionh It is important for further studies to be conducted over a wider geographical distribution to do molecular and genetic investigations to have a better understanding of the specific cytochrome P450 enzymes and mono-oxygenases. While there is a good prospect that PBO plus deltame- thrin ITNs will provide greater control than pyrethroid ITNs in most locations of Burkina Faso in the short term, there are already examples in parts of Côte d’Ivoire [40] and Mozambique [41] where PBO nets no longer pro- vide additional benefit due to the high intensity of pyre- throid resistance and the resistance mechanisms present. Interceptor G2 may be a longer term alternative for improved control of pyrethroid-resistant mosquitoes as chlorfenapyr has a completely different mode of action and works through oxidative phosphorylation of the cell mitochondria [42]. Large-scale testing of susceptibility to chlorfenapyr at the interim diagnostic dose of 100 µg/bot- tle was conducted for the first time in Burkina Faso and showed susceptibility in nearly all sites. Experimental hut trials in Burkina Faso [26], Benin [25] and Côte d’Ivoire Hien et al. Malar J (2021) 20:406 Page 11 of 12 Page 11 of 12 [27] have shown higher vector mortality with Interceptor G2 even when washed 20 times to simulate field use. The results of randomized controlled trials being conducted in Benin and Tanzania to determine the epidemiological impact of Interceptor G2 nets compared to pyrethroid nets are eagerly awaited before widespread distribution can be recommended by WHO. In the interim, as part of the ‘New Nets Project’, pilot distribution of PBO and Interceptor G2 nets was conducted in parts of Burkina Faso in 2019 [43]. Districts were selected to receive PBO and Interceptor G2 nets based on the results of insecti- cide resistance monitoring, along with other operational criteria. Given the increased costs of PBO and Intercep- tor G2 nets it is important to continue monitoring their lifetime durability including physical integrity, residual insecticidal efficacy and chemical retention over three years. Funding This study was supported by the United States President’s Malaria Initiative through the United States Agency for International Development funded VectorLink Project. Acknowledgements h k Our sincere thanks go to residents of the different regional heath directors and collaborators for their support during the sample collections. Our gratitude also goes to the fieldworkers for their hard work. Availability of data and materials All data generated or analysed during this study are included in this published article and additional files. Consent for publication Not applicable. Consent for publication Not applicable. References 1. President’s Malaria Initiative Burkina Faso Malaria Operational Plan FY 2019. https://www.pmi.gov/docs/default-source/default-document-libra ry/malaria-operational-plans/fy19/fy-2019-burkina-faso-malaria-opera tional-plan.pdf?sfvrsn=3#:~:text=ITNs%3A%20The%20national%20str ategy%20for,routine%20antenatal%20care%20and%20expanded. Accessed 25 May 2021. 1. President’s Malaria Initiative Burkina Faso Malaria Operational Plan FY 2019. https://www.pmi.gov/docs/default-source/default-document-libra ry/malaria-operational-plans/fy19/fy-2019-burkina-faso-malaria-opera tional-plan.pdf?sfvrsn=3#:~:text=ITNs%3A%20The%20national%20str ategy%20for,routine%20antenatal%20care%20and%20expanded. Accessed 25 May 2021. Disclaimeri The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of U.S. Centers for Disease Con- trol and Prevention, USAID or PMI. Authors’ contributions RMO, ED, TSA, AB, AK, DJ and RKD, conceived and designed the experiments. ASH, SDD, SM and DC participated to the experiments and analysed the data. ASH, SDD, RMO and DKR wrote the paper. AD, ED, TSA, AB and MBD contrib- uted to the paper writing. All authors read and approved the final manuscript. Abbreviations CDC C f D CDC: Center for Disease Control and Prevention; CI: Confidence interval; GPIRM: Global plan for insecticide resistance management; IRSS: Institut de Recherche en Sciences de la Santé; IRS: Indoor residual spraying; ITNs: Insecticide-treated nets; Kdr: Knockdown resistance; KdT50: Knockdown times for 50%; KdT95: Knockdown times for 95%; LLINs: Long-lasting insecticidal nets; NMCP: National Malaria Control Programme; PBO: Piperonyl butoxide; PMI: US President’s Malaria Initiative; s.l.: Sensu lato; s.s.: Sensu stricto; USM: Uni- versiti Sains Malaysia; Vgsc: Voltage-gated sodium channel gene; Vgsc-L1014F: kdr-West; Vgsc-L1014S: kdr-East; WHO: World Health Organization. Received: 9 June 2021 Accepted: 30 September 2021 Author details 1 Institut de Recherche en Sciences de La Santé (IRSS), Bobo‑Dioulasso, Burkina Faso. 2 U.S. President’s Malaria Initiative, U.S. Agency for International Develop- ment, Washington, DC, USA. 3 U.S. President’s Malaria Initiative, US Embassy Ouagadougou, Ouagadougou, Burkina Faso. 4 PMI VectorLink Project, Abt Associates Inc, 6130 Executive Blvd, Rockville, MD 20852, USA. 5 PMI VectorLink Burkina Faso, Abt Associates Inc, Ouagadougou, Burkina Faso. 6 Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. 7 Centers for Disease Control and Prevention, U.S. President’s Malaria Initiative, Atlanta, GA, USA. 2. Elissa N, Mouchet J, Riviere F, Meunier JY, Yao K. Resistance of Anopheles gambiae s.s. to pyrethroids in Côte d’Ivoire. Ann Soc Belg Med Trop. 1993;73:291–4. References 1. President’s Malaria Initiative Burkina Faso Malaria Operational Plan FY 2019. https://www.pmi.gov/docs/default-source/default-document-libra ry/malaria-operational-plans/fy19/fy-2019-burkina-faso-malaria-opera tional-plan.pdf?sfvrsn=3#:~:text=ITNs%3A%20The%20national%20str ategy%20for,routine%20antenatal%20care%20and%20expanded. Accessed 25 May 2021. 2. Elissa N, Mouchet J, Riviere F, Meunier JY, Yao K. Resistance of Anopheles gambiae s.s. to pyrethroids in Côte d’Ivoire. Ann Soc Belg Med Trop. 1993;73:291–4. 3. Dabiré RK, Namountougou M, Sawadogo SP, Yaro LB, Toé HK, Ouari A, et al. Population dynamics of Anopheles gambiae s.l. in Bobo-Dioulasso city: bionomics, infection rate and susceptibility to insecticides. Parasit Vectors. 2012;5:127. 4. Moyes CL, Wiebe A, Gleave K, Trett A, Hancock PA, Padonou GG, et al. Analysis-ready datasets for insecticide resistance phenotype and genotype frequency in African malaria vectors. Sci Data. 2019;6:121. Competing interests Competing interests The authors declare that they have no competing interests. 4. Moyes CL, Wiebe A, Gleave K, Trett A, Hancock PA, Padonou GG, et al. Analysis-ready datasets for insecticide resistance phenotype and genotype frequency in African malaria vectors. Sci Data. 2019;6:121. Declarations High pyrethroid resistance intensity in malaria vector species is widespread across Burkina Faso and may be a predictor of reduced pyrethroid ITN effectiveness. Pre- exposure to the synergist PBO significantly increased vector mortality, resulting in partial or full restoration of susceptibility in most sites. PBO plus deltamethrin ITNs should be prioritized for distribution in Burkina Faso as this combination provided the highest levels of mortal- ity increase. However, susceptibility was not restored in most sites and dual active ingredient nets, such as Inter- ceptor G,2 may be a better long-term solution. Ethics approval and consent to participate Not applicable. Hien et al. Malar J (2021) 20:406 Hien et al. Malar J (2021) 20:406 Page 12 of 12 Page 12 of 12 5. Chandre F, Darrier F, Manga L, Akogbeto M, Faye O, Mouchet J, et al. Status of pyrethroid resistance in Anopheles gambiae sensu lato. Bull World Health Organ. 1999;77:230–4. 25. N’Guessan R, Odjo A, Ngufor C, Malone D, Rowland M. A chlorfenapyr mix- ture net Interceptor® G2 shows high efficacy and wash durability against resistant mosquitoes in West Africa. PLoS ONE. 2016;11:e0165925. 6. Dabiré KR, Diabaté A, Namountougou M, Djogbenou L, Wondji C, Chandre F, Simard F, Ouédraogo J-B, Martin T, Weill M, Baldet T. Trends in insecticide resistance in natural populations of malaria vectors in Burkina Faso, West Africa: 10 years’ surveys. In: Perveen F, editor. Insecticides – pest engineering Croatia: InTech; 2012. p. 479–502. 26. Bayili K, N’do S, Namountougou M, Sanou R, Ouattara A, Dabiré RK, et al. Eval- uation of efficacy of Interceptor® G2, a long-lasting insecticide net coated with a mixture of chlorfenapyr and alpha-cypermethrin, against pyrethroid resistant Anopheles gambiae s.l. in Burkina Faso. Malar J. 2017;16:190. resistant Anopheles gambiae s.l. in Burkina Faso. Malar J. 2017;16:190. 27. Camara S, Ahoua Alou LP, Koffi AA, Clegban YCM, Kabran JP, Koffi FM, et al. Efficacy of Interceptor® G2, a new long-lasting insecticidal net against wild pyrethroid-resistant Anopheles gambiae s.s. from Côte d’Ivoire: a semi-field trial. Parasite. 2018;25:42. 7. Ranson H, Jensen B, Vulule JM, Wang X, Hemingway J, Collins FH. Identifica- tion of a point mutation in the voltage-gated sodium channel gene of Ken- yan Anopheles gambiae associated with resistance to DDT and pyrethroids. Insect Mol Biol. 2000;9:491–7. 7. Ranson H, Jensen B, Vulule JM, Wang X, Hemingway J, Collins FH. Identifica- tion of a point mutation in the voltage-gated sodium channel gene of Ken- yan Anopheles gambiae associated with resistance to DDT and pyrethroids. Insect Mol Biol. 2000;9:491–7. 28. WHO. Test procedures for insecticide resistance monitoring in malaria vec- tor mosquitoes. 2nd ed. Geneva: World Health Organization; 2016. 8. Diabate A, Baldet T, Chandre C, Dabire KR, Kengne P, Guiguemde TR, et al. kdr mutation, a genetic marker to assess events of introgression between the molecular M and S forms of Anopheles gambiae (Diptera: Culicidae) in the tropical savannah area of West Africa. J Med Entomol. 2003;40:195–8. 29. Santolamazza F, Mancini E, Simard F, Qi Y, Tu Z, della Torre A. Hien et al. Malar J (2021) 20:406 Insertion polymorphisms of SINE200 retrotransposons within speciation islands of Anopheles gambiae molecular forms. Malar J. 2008;7:163. e tropical savannah area of West Africa. J Med Entomol. 2003;40:195– 9. Diabaté A, Baldet T, Chandre F, Dabire KR, Simard F, Ouedraogo JB, et al. First report of a kdr mutation in Anopheles arabiensis from Burkina Faso, West Africa. J Am Mosq Control Assoc. 2004;20:195–6. 30. Martinez-Torres D, Chandre F, Williamson MS, Darriet F, Bergé JB, Devonshire AL, et al. Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s. Insect Mol Biol. 1998;7:179–84. 10. Ranson H, Lissenden N. Insecticide resistance in African Anopheles mosqui- toes: a worsening situation that needs urgent action to maintain malaria control. Trends Parasitol. 2016;32:187–96. 31. Finney DJ. Probit analysis. 3rd ed. Cambridge: Cambridge University Press; 1971. 11. Churcher TS, Lissenden N, Griffin JT, Worrall E, Ranson H. The impact of pyre- throid resistance on the efficacy and effectiveness of bednets for malaria control in Africa. Elife. 2016;5:16090. 32. Rousset F, Raymond M. Testing heterozygote excess and deficiency. Genet- ics. 1995;140:1413–9. 33. Long-lasting insecticidal net (LLIN) price data. https://www.unicef.org/ supply/reports/long-lasting-insecticidal-net-llin-price-data. Accessed 2 May 2021. 12. Hemingway J, Ranson H, Magill A, Kolaczinski J, Fornadel C, Gimnig J, et al. Averting a malaria disaster: will insecticide resistance derail malaria control? Lancet. 2016;387:1785–8. 34 Toe KH, Müller P, Badolo A, Traore A, Sagnon N, Dabiré RK, et al. Do bednets including piperonyl butoxide offer additional protection against popula- tions of Anopheles gambiae s.l. that are highly resistant to pyrethroids? An experimental hut evaluation in Burkina Fasov. Med Vet Entomol. 2018;32:407–16. 13. Jones CM, Liyanapathirana M, Agossa FR, Weetman D, Ranson H, Don- nelly MJ, et al. Footprints of positive selection associated with a mutation (N1575Y) in the voltage-gated sodium channel of Anopheles gambiae. Proc Natl Acad Sci USA. 2012;109:6614–9. 14 Namountougou M, Simard F, Baldet T, Diabaté A, Ouédraogo JB, Martin T, et al. Multiple insecticide resistance in Anopheles gambiae s.l. populations from Burkina Faso. West Africa PLoS One. 2012;7:e48412. 35 Bayili K, N’Do S, Yadav RS, Namountougou M, Ouattara A, Dabiré RK, et al. Experimental hut evaluation of DawaPlus 3.0 LN and DawaPlus 4.0 LN treated with deltamethrin and PBO against free-flying popula- tions of Anopheles gambiae s.l. in Vallée du Kou. Burkina Faso PLoS One. 2019;14:e0226191. 15. N’Guessan R, Corbel V, Akogbéto M, Rowland M. Hien et al. Malar J (2021) 20:406 Riveron JM, Huijben S, Tchapga W, Tchouakui M, Wondji MJ, Tchoupo M, et al. Escalation of pyrethroid resistance in the malaria vector Anopheles funestus induces a loss of efficacy of piperonyl butoxide-based insecticide- treated nets in Mozambique. J Infect Dis. 2019;220:467–75. 22. Staedke SG, Gonahasa S, Dorsey G, Kamya MR, Maiteki-Sebuguzi C, Lynd A, et al. Effect of long-lasting insecticidal nets with and without piperonyl butoxide on malaria indicators in Uganda (LLINEUP): a pragmatic, cluster- randomised trial embedded in a national LLIN distribution campaign. Lancet. 2020;395:1292–303. 42. Black BC, Hollingworth RM, Ahmmadsahib KI, Kukel CD, Donovan S. Insec- ticidal action and mitochondrial uncoupling activity of AC-303,630 and related halogenated pyrroles. Pest Biochem Physiol. 1994;50:115–28. 42. Black BC, Hollingworth RM, Ahmmadsahib KI, Kukel CD, Donovan S. Insec- ticidal action and mitochondrial uncoupling activity of AC-303,630 and related halogenated pyrroles. Pest Biochem Physiol. 1994;50:115–28. 43. Building a stronger line of defense against malaria. https://agriculture.basf. com/en/Pest-Control/Commitment-to-Public-Health/The-New-Nets-Proje ct.html. Accessed 25 May 2021. 43. Building a stronger line of defense against malaria. https://agriculture.basf. com/en/Pest-Control/Commitment-to-Public-Health/The-New-Nets-Proje ct.html. Accessed 25 May 2021. 23. Oxborough RM, N’Guessan R, Jones R, Kitau J, Ngufor C, Malone D, et al. The activity of the pyrrole insecticide chlorfenapyr in mosquito bioassay: towards a more rational testing and screening of non-neurotoxic insecti- cides for malaria vector control. Malar J. 2015;14:124. Hien et al. Malar J (2021) 20:406 Reduced efficacy of insecticide-treated nets and indoor residual spraying for malaria control in pyrethroid resistance area. Benin Emerg Infect Dis. 2007;13:199–206. 36. Conditions for deployment of mosquito nets treated with a pyrethroid and piperonyl butoxide. http://apps.who.int/iris/bitstream/handle/10665/ 258939/WHO-HTM-GMP-2017.17-eng.pdf?sequence=5. Accessed 18 Mar 2018. 16. Toé KH, Jones CM, N’Fale S, Ismail HM, Dabiré RK, Ranson H. Increased pyre- throid resistance in malaria vectors and decreased bed net effectiveness. Burkina Faso Emerg Infect Dis. 2014;20:1691–6. 17. The Global Plan for Insecticide Resistance Management in Malaria Vectors (GPIRM). http://www.who.int/malaria/publications/atoz/gpirm/en/. 37. U.S. President’s malaria initiative FY 2020 guidance. https://www.pmi.gov/ pmi-technical-guidance-5/. Accessed 3 Sept 2020. 38. Namountougou M, Diabaté A, Etang J, Bass C, Sawadogo SP, Gnankinié O, et al. First report of the L1014S kdr mutation in wild populations of Anoph- eles gambiae M and S molecular forms in Burkina Faso (West Africa). Acta Trop. 2013;125:123–7. 18. List of WHO prequalified vector control products. https://www.who.int/ pq-vector-control/prequalified-lists/VCP_PQ-List_26August2020.pdf?ua=1. Accessed 26 Aug 2020. 19. Moores GD, Philippou D, Borzatta V, Trincia P, Jewess P, Gunning R, et al. An analogue of piperonyl butoxide facilitates the characterisation of metabolic resistance. Pest Manag Sci. 2009;65:150–4. 39 Kwiatkowska RM, Platt N, Poupardin R, Irving H, Dabire RK, Mitchell S, et al. Dissecting the mechanisms responsible for the multiple insecticide resistance phenotype in Anopheles gambiae s.s., M form, from Vallée du Kou Burkina Faso. Gene. 2013;519:98–106. 20. Tungu P, Magesa S, Maxwell C, Malima R, Masue D, Sudi W, et al. Evaluation of PermaNet 3.0 a deltamethrin-PBO combination net against Anopheles gambiae and pyrethroid resistant Culex quinquefasciatus mosquitoes: an experimental hut trial in Tanzania. Malar J. 2010;9:21. 40. Kouassi BL, Edi C, Tia E, Konan LY, Akré MA, Koffi AA, et al. Susceptibility of Anopheles gambiae from Côte d’Ivoire to insecticides used on insecticide- treated nets: evaluating the additional entomological impact of piperonyl butoxide and chlorfenapyr. Malar J. 2020;19:454. 40. Kouassi BL, Edi C, Tia E, Konan LY, Akré MA, Koffi AA, et al. Susceptibility of Anopheles gambiae from Côte d’Ivoire to insecticides used on insecticide- treated nets: evaluating the additional entomological impact of piperonyl butoxide and chlorfenapyr. Malar J. 2020;19:454. 21. Protopopoff N, Mosha JF, Lukole E, Charlwood JD, Wright A, Mwalimu CD, et al. Effectiveness of a long-lasting piperonyl butoxide-treated insecticidal net and indoor residual spray interventions, separately and together, against malaria transmitted by pyrethroid-resistant mosquitoes: a cluster, randomised controlled, two-by-two factorial design trial. Lancet. 2018;391:1577–88. 41. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12936-021-03936-3. Additional file 1. Study sites distributed in three eco-climatical areas with different agricultural practices. Additional file 2. Temperature and humidity conditions of WHO suscepti- bility and CDC bottle bioassays. Additional file 3. Synergist assays using pyrethroid insecticides and piperonyl butoxide data. Additional file 4. CDC bottle assays using 100ug chlorfenapyr data. 3. Dabiré RK, Namountougou M, Sawadogo SP, Yaro LB, Toé HK, Ouari A, et al. Population dynamics of Anopheles gambiae s.l. in Bobo-Dioulasso city: bionomics, infection rate and susceptibility to insecticides. Parasit Vectors. 2012;5:127. 4. Moyes CL, Wiebe A, Gleave K, Trett A, Hancock PA, Padonou GG, et al. Analysis-ready datasets for insecticide resistance phenotype and genotype frequency in African malaria vectors. Sci Data. 2019;6:121. Hien et al. Malar J (2021) 20:406 Publisher’s Note S i N i 24. N’Guessan R, Boko P, Odjo A, Akogbéto M, Yates A, Rowland M. Chlorfenapyr a pyrrole insecticide for the control of pyrethroid or DDT resistant Anopheles gambiae (Diptera: Culicidae) mosquitoes. Acta Trop. 2007;102:69–78. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W2899302240	http://ejournal.uika-bogor.ac.id/index.php/TADIBUNA/article/download/1361/1057	Indonesian	null	Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said	Ta'dibuna	2,018	cc-by-sa	7,506	PENDIDIKAN ISLAM DALAM PERSPEKTIF ULAMA BUGIS K.H. LANRE SAID Muhammad Zaitun Rasmin Wahdah Islamiyah Foundation zaitunrasmin@gmail.com Muhammad Zaitun Rasmin Wahdah Islamiyah Foundation zaitunrasmin@gmail.com ABSTRAK Sebagai sebuah usaha untuk melestarikan ajaran pemahaman dan ideologi, pendidikan Islam akan terus eksis selama umat Islam masih menjalankan kewajiban beragama. Dengan pendidikan ajaran agama tetap terjaga, para ulama sebagai agen pendidikan terus muncul tiap saat. Di Indonesia, perkembangan pendidikan Islam cukup pesat, baik sebelum maupun setelah kemerdekaan. Ulama Indonesia yang telah menimba ilmu di luar maupun dalam negeri berlomba mendirikan lembaga pendidikan dengan konsep yang mereka rumuskan. Hasilnya, lahirlah berbagai lembaga pendidikan Islam yang kelak melahirkan tokoh-tokoh bangsa. Tren perkembangan pendidikan Islam yang bermula dari Sumatera, lalu ke Jawa, akhirnya ke Sulawesi Selatan. Di kepulauan ini, berdiri beberapa lembaga pendidikan kaderisasi ulama, khususnya ulama Bugis. Ulama Bugis telah membuktikan bahwa mereka tetap eksis dengan perubahan zaman yang begitu cepat. Merespons dengan baik setiap pergantian zaman, tetap menjadi bagian penting dalam membangun bangsa lewat dunia pendidikan dengan mencerdaskan kehidupan bangsa, membangun masyarakat beradab. Mereka pada akhirnya menjadi agen islamisasi di wilayah Indonesia Timur pada paruh kedua abad ke-XX. Di antara ulama terkemuka Bugis adalah Lanre Said. Beliau telah meletakkan dasar-dasar pemikiran pendidikan Islam di nusantara terkhusus masyarakat Bugis, terutama pendidikan Tahfidzul Qur’an. Dengan hidupnya budaya hafal Al-Qur’an maka umat Islam tidak akan susah diajak berlayar ke pulau Al-Qur’an. Kata kunci: Pendidikan Islam; Tahfidz Qur’an; Ulama Bugis; Lanre Said I. PENDAHULUAN Indonesia sebagai negara yang pemeluk Islamnya terbanyak di dunia sudah barang tentu mempunyai sejarah tentang keislaman. Termasuk perkembangan islamisasi, terutama di bidang ilmu pengetahuan. Penyebaran pengaruh Islam yang berasal dari Jazirah Arab ke Asia dan benua lainnya, menimbulkan munculnya pusat-pusat agama Islam di kawasan tersebut yang berguna sebagai pusat pemerintahan dan peradaban, sekaligus sebagai pusat pendidikan sebagai sarana utama dalam berdakwah dan melakukan regenerasi dai, pendidikan, hingga ulama penuntun umat. Para dai yang berprofesi ganda sebagai pedagang yang menjalin hubungan dengan penguasa dan pengusaha di Indonesia yang berasal dari berbagai latar belakang mulai dari Cina India, Persia, Arab, Mesir dan Turki. Adanya interaksi sosial antara pedagang muslim yang dai itu dengan masyarakat setempat inilah yang akhirnya memberi pengaruh masuknya nilai-nilai dan ajaran Islam sehingga semakin banyak yang memeluk agama Islam. 61 Rasmin, Hafidhuddin, Husaini, Mujahidin Rasmin, Hafidhuddin, Husaini, Mujahidin Teori dakwah secara sistematis yang dilakukan para dai pedagang dalam penyampaian dakwahnya adalah sebagai berikut: Mula-mula para dai pedagang berdatangan ke pusat perdagangan, lalu kemudian mulai ada yang bertempat tinggal, baik sementara maupun menetap. Lambat laun tempat tinggal mereka berkembang menjadi perkampungan muslim dari negeri asing yang disebut pekojan. Status sosial yang tinggi, memudahkan mereka mengawini pribumi baik rakyat biasa maupun anak bangsawan. Sebelum pernikahan, calon istrinya diislamkan dahulu dengan mengucapkan dua kalimat syahadat. Lalu berkembang menjadi perkampungan, masyarakat dan kerajaan Islam. Sehingga dengan demikian, para pedagang mempunyai andil besar dalam penyebaran Islam melalui pendidikan sosial kemasyarakatan, seperti cara berdagang Islam, cara bermasyarakat, upacara pernikahan sampai pada cara bersosialisasi sehari-hari yang telah mereka praktikkan dalam kehidupan kesehariannya. Namun, ada pula yang memang sengaja datang berdakwah tanpa bermaksud berdagang, dan sasaran utama mereka adalah para penguasa setempat. Dai semacam ini adalah biasanya utusan dari kerajaan Islam di tempat lain. Untuk kasus semacam ini dapat dilihat yang terjadi di kerajaan Gowa-Tallo atau Makassar. Setelah Islam diterima oleh masyarakat Indonesia tanpa melalui konfrontasi fisik secara berarti, maka masalah yang muncul belakangan adalah mengajarkan para mualaf tersebut agama Islam dengan baik dan benar. Karena terbatasnya jumlah personil guru- guru yang dapat mendidik umat di sebuah tempat dengan durasi waktu tertentu, menjadikan mayoritas bangsa Indonesia yang Islamnya belum maksimal. Ada yang telah turun-temurun memeluk Islam namun belum juga dapat membaca Al-Qur’an dengan betul, belum menunaikan salat wajib lima waktu dengan sempurna, bahkan rukun-rukun Islam lainnya, kecuali syahadat belum dipahami dan dilaksanakan secara benar. Para ulama Bugis sejak dulu dikenal sebagai peletak dasar-dasar pendidikan Islam. I. PENDAHULUAN Mereka merespons dengan baik setiap pergantian zaman, serta tetap menjadi bagian penting dalam membangun bangsa lewat dunia pendidikan dengan mencerdaskan kehidupan bangsa dan membangun masyarakat beradab. II. METODE PENELITIAN Pendekatan yang digunakan dalam penelitian ini adalah pendekatan kualitatif, yaitu prosedur penelitian yang mendeskripsikan perilaku orang, peristiwa atau tempat tertentu secara rinci dan mendalam (Umar, 2000). Pendekatan ini bertujuan untuk menggambarkan sesuatu yang tengah berlangsung pada saat riset dilakukan dan memeriksa sebab-sebab dari sesuatu gejala tertentu (Ahmad Kadir, 2003). Dalam hubungan ini, peneliti akan mendeskripsikan tentang pemikiran pendidikan KH. Lanre Said. Adapun sumber data ada dua yaitu, data primer dan data sekunder. Data primer Ta’dibuna, Vol. 7, No. 1, April 2018 62 Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said pada penelitian pemikiran pendidikan KH. Lanre Said adalah data karya-karya orisinal KH. Lanre Said yang merupakan materi pendidikan yang ia ajarkan kepada para santrinya, juga observasi langsung ke lembaga pendidikan yang didirikan oleh KH. Lanre Said, termasuk melakukan wawancara kepada narasumber yang dipandang memiliki otoritas terkait pengetahuan tentang KH. Lanre Said berasal dari mantan santri- santrinya. Metodologi yang digunakan dalam penelitian ini adalah deskriptif kualitatif yang di dalamnya meliputi: metode kajian pustaka; metode pengamatan; metode pengamatan berpartisipasi (participant observation); metode wawancara sambil lalu; metode wawancara mendalam; dan metode mendengarkan. Dalam proses analisis data, ada tiga komponen utama yang harus dilakukan yaitu, reduksi data, sajian data, dan penarikan kesimpulan ataupun verifikasi. Tiga komponen ini terlibat dalam proses yang saling berkaitan serta menentukan hasil akhir analisis (Ahmad Kadir, 2003). Sajian data adalah suatu rakitan organisasi informasi yang memungkinkan kesimpulan penelitian dapat dilakukan. Sajian data meliputi rangkaian deskriptif yang dijalin sedemikian rupa sehingga memperlihatkan satu kesatuan yang utuh. Kesemuanya itu dirancang guna merakit informasi secara teratur supaya mudah dilihat dan dimengerti dalam bentuk yang kompak. Dari awal pengumpulan data, peneliti sudah harus memahami apa arti dari berbagai hal yang ditemukan dengan mulai melakukan pencatatan pola-pola, pernyataan-pernyataan, konfigurasi-konfigurasi yang mungkin, sebab-akibat dan berbagai preposisi. Kesimpulan-kesimpulan tersebut pada waktu awal kurang jelas, kemudian semakin meningkat secara eksplisit dan memiliki landasan yang kuat. Kesimpulan akhir tidak akan terjadi sampai proses pengumpulan data berakhir. Kesimpulan yang perlu diverifikasi dilakukan pada waktu menulis dengan melihat kembali fieldnotes yang tersedia (Abd Kadir, 2008) III. HASIL DAN PEMBAHASAN A. Pemikiran Pendidikan Islam di Indonesia Diskursus tentang pemikiran pendidikan Islam di Indonesia dalam telaah historis sosiologis serta wacana dan dinamika yang berkembang di dalamnya akan kita belah menjadi dua bagian, yaitu periode sebelum Indonesia merdeka 1900-1945 dan periode setelah Indonesia merdeka, tahun 1945-sekarang. Pada pembahasan ini akan dikaji masing-masing kedua periode tersebut, termasuk dinamika yang muncul dalam hal-hal yang terkait dengan pengembangan pendidikan Islam, terutama di kalangan para pemikir, pengembang, dan pengelola pendidikan Islam dari satu periode ke periode selanjutnya di negeri ini (Hasbullah, 1999). Setidaknya ada dua tujuan pendidikan dengan tingkat keragamannya masing-masing jika ditinjau dari secara teoritis, pertama adalah pendidikan berorientasi Ta’dibuna, Vol. 7, No. 1, April 2018 63 Rasmin, Hafidhuddin, Husaini, Mujahidin kemasyarakatan, yaitu pandangan yang menganggap pendidikan sebagai sarana utama dalam menciptakan rakyat yang baik, baik untuk sistem pemerintahan demokratis, oligarkis, maupun monarkis. Pandangan teoritis yang kedua lebih pada orientasi individu, yang lebih memfokuskan diri pada kebutuhan, daya tampung, dan minat pelajar. Asumsi bahwa manusia adalah hewan yang bermasyarakat (social animal) dan ilmu pengetahuan pada dasarnya dibina di atas dasar-dasar kehidupan bermasyarakat, mereka yang berpandangan kemasyarakatan berpendapat bahwa pendidikan bertujuan mempersiapkan manusia yang bisa berperan dan menyesuaikan diri dalam masyarakatnya masing-masing. Berdasarkan hal ini, tujuan dan target pendidikan dengan sendirinya diambil dari dan diupayakan untuk memperkuat kepercayaan, sikap, ilmu pengetahuan, dan sejumlah keahlian yang sudah diterima dan sangat berguna bagi masyarakat. Konsekuensinya, karena kepercayaan, sikap, ilmu pengetahuan, dan keahlian yang bermanfaat dan diterima oleh sebuah masyarakat itu senantiasa berubah, mereka lalu berpendapat bahwa pendidikan dalam masyarakat tersebut harus bisa mempersiapkan peserta didiknya untuk menghadapi segala bentuk perubahan yang ada. Pendidikan Indonesia tidak akan pernah lebih baik jika generalisasi sains masih ada, apalagi jika fardu kifayah sains lebih disukai dan mengabaikan fardu 'ain sains. Kurikulum pendidikan yang dilakukan secara hierarkis dengan klasifikasi fardu 'ain dan fardu kifayah sains adalah cara untuk menjadikan Indonesia sebagai muslim yang baik dan ulama yang baik (Ardiansyah, Hafidhuddin, Mujahidin, & Syafrin, 2017). Mereka yang meyakini bahwa pendidikan sebagai sesuatu yang memainkan peranan penting dalam membentuk masyarakat beranggapan bahwa masyarakat jauh lebih penting dari pada individu. Ciri khas pendidikan yang berorientasi masyarakat adalah mengutamakan kebutuhan masyarakat dan menjadikan minat peserta didik sebagai prioritas kedua. Pada era modern ini, tokoh-tokoh filsafat pendidikan yang berhaluan sosialis dan komunis dengan terang-terang menekankan dimensi sosial daripada dimensi demokrasi liberal yang lebih mengutamakan individu daripada masyarakat (Husaini, 2013). 1. Corak Pendidikan di Indonesia Warna pendidikan pada periode sebelum Indonesia merdeka setidaknya ada dua corak, yang pertama jenis pendidikan yang berpusat pada pondok pesantren yang didirikan dan dikelola oleh para kiai dan santri (Damanhuri, Mujahidin, & Hafidhuddin, 2013). Kedua, corak baru yang dibawa dari kolonial Barat seperti sekolah-sekolah yang didirikan oleh pemerintah Belanda (Muljana, 2008). Ciri-ciri dari corak lama adalah (1) adanya kiai atau ulama yang hanya menguasai ilmu-ilmu agama; (2) kurang diberikan pengetahuan umum kepada peserta didik atau bahkan tidak sama sekali; (3) sikap eksklusif disebabkan sikap nonkooperasi secara total dari pihak pesantren terhadap apa saja yang beraroma Barat, dan aliran kebangunan Islam tidak leluasa untuk bisa masuk karena dihalang-halangi oleh pemerintah Belanda. Di lain pihak Belanda memiliki sistem Ta’dibuna, Vol. 7, No. 1, April 2018 64 Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said pendidikan yang bertolak belakang dengan kaum pribumi seperti, hanya menonjolkan sisi intelek dan sekaligus hendak melahirkan kaum intelek semata dan umumnya bersikap negatif terhadap agama Islam (Hasbullah, 1999). Pada corak pendidikan pesantren, tujuan utamanya adalah mengeluarkan calon lulusan yang difokuskan untuk menguasai ilmu-ilmu agama. Tradisi pondok pesantren setidaknya memiliki lima elemen dasar, yakni, pondok, masjid, pengajaran kitab-kitab Islam klasik (kitab kuning), dan kiai (Mujahidin, 2005). Menurut Martin van Bruinessen, salah satu tradisi agung di Indonesia adalah tradisi pengajaran agama Islam, misalnya di pondok pesantren, yang bertujuan untuk mentransmisikan Islam tradisional sebagaimana yang terdapat dalam kitab-kitab klasik yang ditulis berabad-abad yang lalu. Pondok pesantren tradisional sistem pengajarannya diselenggarakan di bawah kepemimpinan kiai yang dibantu oleh beberapa orang ulama atau para ustaz yang hidup bersama di tengah-tengah para santri dengan masjid atau surau sebagai pusat kegiatan keagamaan, gedung sekolah atau ruang-ruang belajar mengajar, serta pondok sebagai tempat tinggal para santri. Proses belajar mengajar dilakukan melalui struktur, metode, dan literatur tradisional, baik berupa pendidikan formal di sekolah atau madrasah dengan jenjang yang bertingkat, atau pun pemberian pengajaran dengan sistem halaqah. Ciri utama dari pengajaran tradisional adalah pemberian ajarannya yang ditekankan pada penghafalan, pemahaman, dan pengamalan atas suatu kitab tertentu (Bruinessen, 1999). Pendidikan dalam dunia pesantren, sebagaimana yang penulis rasakan, hakikat pendidikan adalah yang mampu memahami kitab-kitab keagamaan yang sulit sekalipun, serta mampu mengajarkan ilmunya pada orang lain. Untuk itulah jenjang pendidikan dipondok pesantren dibutuhkan, termasuk pada awal-awal belajar seorang siswa harus menguasai ilmu-ilmu alat, terutama bahasa Arab dan segala instrumennya. Hakikat peserta didik atau santri adalah seorang yang sedang belajar memahami agama dan mengembangkan perasaan beragama dengan mendalam. 1. Corak Pendidikan di Indonesia Kurikulum adalah rencana pelajaran sebagaimana tertuang dalam kitab-kitab keagamaan produk ulama terdahulu. Evaluasi adalah penilaian kemampuan santri dalam menguasai kitab-kitab yang dipelajari untuk selanjutnya meningkat dalam mempelajari kitab yang kitab-kitab lanjutan. Pada tahun 1909, madrasah-madrasah dengan sistem berkelas (klasikal) mulai muncul, menurut penelitian Mahmud Yunus, pendidikan Islam yang kali pertama memiliki kelas dan memakai bangku, meja, dan papan tulis adalah Madrasah Adabiyah (Adabiyah School) di Padang. Madrasah Adabiyah adalah madrasah pertama di Minangkabau, bahkan di seluruh Indonesia, yang didirikan oleh Syaikh Abdullah Ahmad pada tahun 1909. Madrasah ini hidup sampai tahun 1914, kemudian diubah menjadi HIS Ta’dibuna, Vol. 7, No. 1, April 2018 65 Rasmin, Hafidhuddin, Husaini, Mujahidin Adabiyah pada tahun 1915, yang merupakan HIS pertama di Minangkabau yang memasukkan pelajaran agama Islam dalam pengajarannya (Manti, Husaini, Mujahidin, & Hafidhuddin, 2016; Yunus, 1996). Munculnya sekolah-sekolah Islam yang bersepadu dengan sistem pendidikan modern juga tak terlepas dari banyaknya alumni Al-Azhar Mesir yang telah menyelesaikan pendidikannya di sana (Yunus, 1996). Mereka adalah hasil dari sistem pendidikan yang telah direformasi oleh Muhammad Abduh. Setibanya di Indonesia, mereka mengelola dan mengajar di sekolah-sekolah agama serta memasukkan mata pelajaran umum. Lembaga pendidikan yang demikian dinamai Madrasah Guru Islam atau Sekolah Menengah Islam (SMI). Di antara madrasah yang juga termasuk awal adalah Al-Jami’ah Islamiyah, di Sungayang Batusangkar, didirikan oleh Mahmud Yunus pada 20 Maret 1931; Normal Islam (Kuliah Mu’allim Islamiah), didirikan oleh Persatuan Guru-guru Agama Islam (PGAI) di Padang pada tanggal 1 April 931 dan dipimpin oleh Mahmud Yunus, dengan demikian Mahmud Yunus memimpin dua madrasah tingkat menengah dan tinggi di atas; Islamic College, didirikan oleh Persatuan Muslim Indonesia (Permi) di Padang pada tanggal 1 Mei 1931 dan dipimpin oleh Mr. Abdul Hakim. Kemudian digantikan oleh Mukhtar Yahya tahun 1935 (Yunus, 1996). Selanjutnya berdirilah beberapa madrasah yang memasukkan pengetahuan umum dalam rencana pendidikannya, di antaranya, Training College didirikan oleh Nasruddin Thaha di Payakumbuh tahun 1934; Kulliah Muballghin/Muballighat, didirikan oleh Muhammadiyah di Padang Panjang; Kulliah Muallimat Islamiah, didirikan oleh Rgk. Rahmah Al-Yunusiah di Padang Pandang tanggal 1 Februari 1937; Kulliah Dianah, didirikan oleh Syaikh Ibrahim Musa di Parabek pada tahun 1940 dan dipimpin oleh H. Bustami A. Gani; Kulliatul Ulum, didirikan oleh Thawalib Padang Panjang dan dipimpin oleh Engku Mudo Abdul hakim; Kulliah Syariah, didirikan oleh Tarbiyah Islamiah di Padang Panjang; Nasional Islamic College, didirikan oleh alumni Islamic College di Padang; Modern Islamic College didirikan oleh St. 1. Corak Pendidikan di Indonesia Sulaiman dan kawan-kawan di Bukittinggi (Yunus, 1996), Secara garis besar, dapat ditarik benang merah bahwa sebelum Indonesia merdeka terdapat berbagai corak pengembangan pendidikan Islam, yaitu, pertama, isolatif- tradisional, dalam arti tidak menerima apa saja yang berbau Barat (kolonial) dan terhambatnya pengaruh pemikiran-pemikiran modern dalam Islam untuk masuk ke dalamnya, sebagaimana tampak pada pendidikan pondok pesantren tradisional yang hanya menonjolkan ilmu-ilmu agama Islam, sedangkan pengetahuan umum tidak diberikan, kecuali beberapa keterampilan seperti bertani, bertukang, beternak dan berdagang demi untuk bekal hidup seadanya, karena memang kesederhanaan menjadi ciri khas seorang penganjur dan pendidik agama (kiai). Hakikat pendidikan Islam, sebagaimana dalam tradisi pondok pesantren adalah sebagai suatu upaya untuk Ta’dibuna, Vol. 7, No. 1, April 2018 66 Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said melestarikan dan mempertahankan khazanah pemikiran ulama terdahulu seperti yang telah tertuang dalam kitab-kitab muktabar. Tujuan pendidikan tak lain dan bukan kecuali untuk melahirkan para ulama sebagai pewaris para nabi, dan umara yang berjiwa ulama. Kedua, sintesis, yakni kolaborasi antara corak lama (pondok pesantren tradisional) dan corak baru yaitu nuansa modern sebagaimana model kolonial Belanda yang berwujud sekolah formal sebagaimana madrasah. Dalam realitasnya, corak pemikiran sintesis ini mengandung beberapa variasi pola pendidikan Islam, seperti pola pendidikan madrasah mengikuti format pendidikan Barat terutama sistem pengajarannya secara klasikal, tetapi sistem pendidikan tetap lebih menonjolkan ilmu-ilmu agama Islam, sebagaimana dikembangkan pada Madrasah Sumatera Thawalib, Madrasah Tebuireng asuhan KH. Hasyim Asy’ari, atau Madrasah Arabiyah Islamiyah (MAI) Sengkang dibawa Anregurutta Haji Muhammad As’ad; pola pendidikan madrasah yang mengutamakan pelajaran agama, tetapi mata pelajaran umum secara terbatas juga diberikan seperti yang dikembangkan oleh Madrasah Diniyah Zaenuddin Labay el-Yunusi dan Madrasah Salafiyah Tebuireng pimpinan KH. Ilyas; pola pendidikan madrasah yang menggabungkan secara lebih seimbang antara muatan keagamaan dan non-keagamaan, seperti yang dikembangkan oleh Muhammadiyah, dan Pondok Modern Gontor Ponorogo, dan pola pendidikan sekolah yang mengikuti pola pemerintah Hindia Belanda dengan ditambah beberapa mata pelajaran agama, sebagaimana yang dikembangkan oleh Madrasah Adabiyah dan sekolah-sekolah yang dikelola oleh Muhammadiyah (Mahlani, 2013). 2. Pendidikan Islam di Sulawesi Selatan Di Sulawesi Selatan, secara umum para raja-raja memberi keleluasaan kepada para dai dan ulama sekaligus pendidik untuk mengembangkan syiar agama Islam dan pendidikan. Raja Gowa yang bergelar Imangimangi Daeng Matuju Karaeng Bontonompo Sultan Muhammad Tahir Muhibuddin (1936 – 1946) menggagas pembukaan Madrasah Islamiyah, bertempat di Jongaya, Gowa (Palimai, 2010). Pengajaran agama Islam yang diberikan berdasarkan mazhab Syafii. Pimpinan Madrasah dipegang oleh Asy Syekh Abdullah bin Shadaqah Dahlan, penganjur mazhab Imam Syafii yang taat. Madrasah ini dibuka, setelah beberapa bulan Sultan Muhammad Tahir naik takhta di Gowa pada tahun 1936. Para murid-murid madrasah ini berasal dari daerah Takalar, Jeneponto, dan dari daerah Gowa sendiri. Ketika pecah perang dunia ke II madrasah ini terpaksa ditutup, perang memang selalu membawa petaka (Palimai, 2010). Sebelum itu, di daerah Campalagian Mandar, menurut catatan, pendidikan dengan sistem tradisional telah bermula dari tahun 1913 di bawah asuhan H. Maddeppungeng Ta’dibuna, Vol. 7, No. 1, April 2018 67 Rasmin, Hafidhuddin, Husaini, Mujahidin yang pernah berguru di Mekah Saudi Arabia. Tempat ini menjadi pencetak kader-kader mubalig Islam di Sulawesi Selatan pada awal abad ke XX. Tempat pendidikan ini tidak membatasi usia para pelajarnya (Pawiloy, 1987). Di kerajaan Wajo ketika diperintah oleh La Mannang Toapamadeng Puangna Raden Galla, Arung Matoa ke-40 yang berkuasa pada tahun 1821-1825, beliau melakukan berbagai usaha dalam bidang pendidikan dan agama, seperti: memperluas dan menyempurnakan Masjid Jami’ Tosora; mendatangkan ulama dari Madinah, (biasa disebut oleh orang Wajo dengan Syekh Madinah); mengeluarkan perintah pada raja-raja bawahannya agar masjid yang ada dipelihara dan diperbaiki, dan yang belum memiliki masjid agar segera membangun supaya rakyat dapat salat secara berjamaah; pohon-pohon yang dikeramatkan agar ditebang; perempuan yang keluar rumah agar menggunakan tutup kepala dan kain sarung (baca: kerudung); dan dari segi pelaksanaan hukum, pemerintah memotong tangan bagi pencuri atas anjuran Syekh Madinah. Ketika La Oddang Datu Larompong, Arung Matoa Wajo ke-47, memerintah Wajo dari tahun 1926-1933. Beliau memiliki pengetahuan agama yang dalam, karena sejak kecil dididik oleh orang tuanya dalam masalah keagamaan. B. Pendidikan Kader Ulama Bugis g Karena kajian ini berhubungan dengan ulama Bugis, maka akan lebih baik jika membahas masalah kedudukan ulama Bugis yang sejatinya merupakan hasil pendidikan yang sangat sistematis dan terarah. Masyarakat Bugis menyebut ulama dengan panggilan anregurutta dan gurutta, dan pada saat seorang ulama sudah matang atau mencapai tingkat keahlian dalam bidang keagamaan dipandang tinggi serta berada pada posisi layak menjadi panutan disebabkan oleh perilakunya yang saleh, maka ia akan disebut sebagai seorang panrita (Abd Kadir, 2008). 2. Pendidikan Islam di Sulawesi Selatan Beliau disifatkan sering bergaul dengan para Ulama seperti, Haji Makkatu, seorang Ulama yang sangat tegas dalam memberantas segala kemungkaran dan merintis pengajian yang bersifat klasikal di Tosora, juga beliau dekat dengan Al-Bugisi, seorang Ulama Bugis yang lahir di Mekkah, ke Wajo pada tahun 1928 yang sangat berjasa dalam mengembangkan pendidikan Islam di Sulawesi Selatan dengan mencetak para ulama berkaliber nasional dan internasional, dia juga sebagai pelopor pemurnian ajaran agama Islam dan pembaruan sistem pendidikan Islam dengan Madrasah Arabiyah Islamiyah (MAI) yang berpusat di Sengkang (Palimai, 2010). Demikian pula di Kerajaan Bone, berkat bantuan Andi Mappanyukki alias Petta Mangkau Bone, pada tahun 1929 didirikan sebuah madrasah yang diberi nama “Madrasah Amirah” di Watampone. Pimpinannya ialah Abdul Aziz Asy Syimie berasal dari Mesir, pada tahun 1935 pimpinan madrasah beralih ke tangan Ustaz Abdul Hamid al Misyrie dan selanjutnya digantikan oleh Ustaz Mahmud al Jawad bekas Mufti Madinah al Munawarah yang sebelumnya pernah mengajar di Palopo, pada perkembangan selanjutnya tahun 1940 dibangunlah asrama para pelajar sebagai tempat tinggal dan juga dibangunkan gedung belajar yang teratur. Para pengasuh madrasah ini adalah para Ulama dari Bone sendiri yang pernah mukim dan belajar di Makkah dan Mesir (Palimai, 2010). Selanjutnya pada tahun 1932 atas inisiatif Raja Bone Andi Mappanyukki diadakan “Pertemuan Ulama se-Celebes Selatan” di Watampone, ibukota kerajaan Bone. Musyawarah tersebut dihadiri oleh dua puluh enam Ulama terkemuka dari seluruh Sulawesi Selatan termasuk Gurutta H. M. As’ad, dan membicarakan cara-cara pengelolaan pendidikan Islam bagi masyarakat umum (Palimai, 2010) Ta’dibuna, Vol. 7, No. 1, April 2018 68 Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said Ta’dibuna, Vol. 7, No. 1, April 2018 1. Epistemologi Ditilik dari segi bahasa panrita berasal dari kata ita yang berarti orang yang melihat, umum pula ditulis topanrita tambahan to adalah bahasa Bugis yang bermakna orang. Dalam perkembangannya, kata panrita bisa berarti keahlian teknis, seperti tercermin dari ungkapan panrita lopi (ahli pembuat perahu), panrita bola (ahli dalam membuat rumah), namun dalam pengertian lebih spesifik setelah terjadi islamisasi secara masif dan massal, termasuk merambah pada istilah bahasa, dan mengubah makna inti beberapa kata kunci termasuk panrita yang sebelumnya secara istilah adalah orang yang memiliki kemampuan khusus secara fisik dan metafisik. Setelah era islamisasi, panrita berubah arti menjadi ulama, sehingga dalam masyarakat Bugis panrita loppo berarti ulama besar, atau pendeta dalam istilah agama Kristen (Ide, 1977). Dari sinilah awal mulanya seorang pakar Bugis, Cristian Pelras yakin bahwa kata panrita diambil dari bahasa Sanskerta pandita yang berarti ‘pendeta atau pertapa.’ Dalam pengertian Pelras, panrita adalah ‘orang yang menguasai seluk beluk agama, bijaksana, saleh dan jujur (Christian, 2006)’ Makna topanrita yang lebih luas diberikan oleh Mochtar Pabottingi, seorang cendekiawan asal Sulawesi Selatan. Menurutnya, panrita adalah orang yang bersaksi, melihat dan menyimak atas suatu keadaan dan menyatakan keadaan sebenarnya. Di sini, panrita bukan saja berperan sebagai pengamat yang objektif atas keadaan di sekitarnya, tapi juga memberi penilaian, kritik dan pertimbangan atas suatu keadaan. Dengan makna ini, tidak berlebihan jika topanrita diidentikkan dengan konsep cendekiawan (intellectual) dalam terminologi modern. Lepas dari beragam pengertian di atas, topanrita juga merupakan salah satu dari empat kualitas ideal manusia Bugis, di luar kebangsawanan (arung), yang disebut dalam Lontara sebagai “sulapa’ eppa’” (segi empat). Keempat kualitas atau sifat tersebut merupakan modal yang harus dimiliki setiap pemimpin yang baik. Selain panrita (saleh), tiga sifat yang dimaksud adalah warani (berani), macca (cerdas) dan sugi (kaya), sebelum Islam datang, setelah hirarki golongan kaya, ada dua lagi lapisan masyarakat yang tersisa, maradeka dengan ata atau golongan merdeka dan hamba (Christian, 2006). Ta’dibuna, Vol. 7, No. 1, April 2018 69 Rasmin, Hafidhuddin, Husaini, Mujahidin Menurut Mattulada, keempat sosok ideal ini, yang dia sebut sebagai ‘golongan fungsional’, termasuk lapisan elite kedua dalam pelapisan masyarakat Bugis-Makassar periode Lontara (Mattudala, n.d.). Lapisan elite pertama adalah Arung yang terdiri atas anakarung, yaitu raja dengan lingkungan kerabat keluarga bangsawan yang menduduki jabatan-jabatan kepemimpinan politik pemerintahan secara turun-temurun, baik di pusat kerajaan maupun di daerah-daerah bawahannya. Anakarung menduduki status sosial tertinggi dalam masyarakat karena, secara genealogis, leluhur mereka dipercaya bersambung hingga ke sosok tomanurung. 1. Epistemologi Konsep tomanurung (orang yang turun dari langit) ini diadopsi oleh kerajaan-kerajaan utama di Sulawesi Selatan, khususnya Luwu, Gowa, Bone dan Soppeng. Untuk menjelaskan asal-usul kemunculannya seorang raja dan legitimasi tradisionalnya untuk menjadi pemimpin dalam masyarakatnya. Konsep ini sangat menentukan dalam proses pelapisan struktur sosial-politik dalam wilayah berbagai kerajaan Bugis-Makassar khususnya, dan wilayah Austronesia pada umumnya. Dalam konsep tomanurung, (Mattudala, n.d.) kemunculan atau kehadiran seorang raja pertama digambarkan secara mitologis sebagai berasal dari langit dan merupakan manusia setengah dewa. Mitos ini memberikan kepadanya kewibawaan yang tinggi sehingga mendapatkan kepatuhan dan penghormatan dari rakyatnya (Mattudala, n.d.). 2. Peranan dan Kedudukan Panrita Terkait peran dan kedudukan panrita sebelum Islam masuk secara resmi di Sulawesi Selatan dapat dilacak dari berbagai kerajaan, misalnya kerajaan Wajo pada abad ke-15, hiduplah salah satu panrita bernama La Tiringeng To Taba' (Abad ke-15 M) tak dapat dipisahkan dari perjanjian awal antara rakyat dan raja Wajo yang berhasil merumuskan prinsip-prinsip utama ketatanegaraan (konstitusi) kerajaan Wajo di abad ke-15. La Tiringeng mewujudkan diri sebagai sosok topanrita Bugis. Dalam berbagai Lontara Wajo, dia digambarkan sebagai seorang cendekiawan dan negarawan yang tidak saja bijaksana, cerdas dan mencintai rakyatnya, tetapi juga seorang ahli dan penegak hukum yang tegas, jujur dan tidak terbius oleh kekuasaan dan kekayaan (Patunru, 1965). Tidak aneh jika petuah-petuahnya lebih dari enam abad silam, menurut banyak pengamat budaya, masih relevan untuk ditelaah sebagai sumber inspirasi, landasan etika dan pedoman dalam menata kehidupan sosial, hukum, politik dan pemerintahan kontemporer, baik di tingkat lokal, regional maupun nasional (Halim & Sesember, 2012). Salah satu pesan La Tiringeng yang kerap kali dirujuk untuk menunjukkan aktualitas pesan itu dengan kondisi kontemporer adalah, “Napoallebirengngi to Wajoè, maradèkaè, na malempu, na mapaccing ri gau’ salaè, marèso mappalaong, na maparekki ri warang-paranna”. Orang Wajo mulia karena mereka memiliki kebebasan, kejujuran, kesucian dari perilaku buruk, kerajinan bekerja, dan memelihara harta benda (Noorduyn, 2000). Ada pun panggilan anregurutta hanya akan disematkan kepada seorang ahli agama namun tidak untuk yang ahli dalam ilmu pengetahuan lainnya, ilmu agama dimaksud adalah terminologi ilmu-ilmu fardu ‘ain dalam pandangan Al-Gazali dan ilmu naql 70 Ta’dibuna, Vol. 7, No. 1, April 2018 Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said menurut Ibn Khaldun. Karena itu, orang Bugis memahami ulama dengan pengertian khusus (Al-Jauziyah, 2012; Khaldun, 2010; Munawwir, 2006). Secara hierarkis, ulama Bugis juga bertingkat-tingkat, yang tertinggi adalah panrita, lalu menyusul anregurutta, kemudian gurutta, dan para ustaz, hanya saja, seorang panrita disebut juga anregurutta dan gurutta kedua gelar terakhir ini kerap berganti-ganti. Namun demikian, istilah kiai sebagaimana dipergunakan oleh masyarakat Jawa juga dipakai untuk menyebut golongan ulama, atau pimpinan pesantren dan mengajar kitab- kitab klasik (turâts) kepada para santrinya, dalam penelitian ini kata ‘kiai’ juga digunakan untuk menunjukkan kata ganti ulama, anregurutta, dan gururtta (Dhofier, 1994). Ta’dibuna, Vol. 7, No. 1, April 2018 2. Peranan dan Kedudukan Panrita Hanya saja, penggunaan istilah kiai dalam masyarakat Bugis bersifat umum untuk semua ahli agama, sementara istilah anregurutta selain menunjukkan kompetensi yang dimiliki juga merujuk pada perilaku kesehariannya (ampe-ampe madeceng) yang melambangkan dan menunjukkan kharisma dan kewibawaannya di tengah masyarakat, karena itulah, menurut Ahmad, anregurutta atau gurutta memang lebih spesifik untuk ulama Bugis. Dilihat dari sudut kompetensi, memang ulama Bugis, setidaknya merujuk pada tiga kompetensi dasar, meliputi, penguasaan ilmu agama, pengamalan agama, dan akhlak kepribadian (Abd Kadir, 2008). Tempat reproduksi ulama dalam masyarakat Bugis tidak berbeda dengan di Jawa, yaitu berasal dan berawal dari pesantren lalu diaplikasikan ilmunya, dan dimatangkan di tengah masyarakat. Pengetahuan yang selama ini bersifat teoritis di pesantren akan diuji di tengah masyarakat. Proses pematangan yang melibatkan ketiga komponen di atas akan menjadi penentu eksistensi seorang ulama Bugis. Posisi pesantren yang terbuka secara umum memungkinkan reproduksi ulama tetap terjaga, padahal awal-awal kedatangan Islam, umumnya ulama yang kelak akan menjadi panrita lebih didominasi kaum anakarung (bangsawan). Seiring berlalunya waktu, pondok pesantren dan madrasah membuka diri untuk semua kalangan yang ingin menjadi ulama menjadikan kaderisasi ulama berjalan secara berkesinambungan, dapat diakses oleh masyarakat luas (Abd Kadir, 2008). Jika di Jawa pola-pola kaderisasi ulama umumnya melibatkan jaringan antar- genealogi kekerabatan. Unsur keturunan dalam kalangan pesantren tradisional di Jawa memegang peranan penting dalam kaderisasi kiai. Seorang kiai tradisional mungkin anak dari kiai, atau kalau ayahnya bukan kiai, maka salah seorang familinya adalah kiai, atau memang kakeknya seorang kiai, begitulah seterusnya. Ini pula yang membedakan dengan ulama Bugis yang lebih cenderung bergerak di luar pola hubungan genealogi kekerabatan sehingga sulit menemukan adanya anak ulama melakukan hubungan pernikahan dengan ulama lainnya. Pola koneksi atas dasar genealogi keilmuan lebih diutamakan daripada Ta’dibuna, Vol. 7, No. 1, April 2018 71 Rasmin, Hafidhuddin, Husaini, Mujahidin kekerabatan. Ulama Bugis mereproduksi santri menjadi ulama lebih dari sekadar mereproduksi anak keturunan menjadi ulama. Pola regenerasi ulama khas Bugis juga dijumpai dalam penelitian ini yang mengungkap konsep kaderisasi ulama yang pernah diterapkan oleh Al-Bugisi yang merintis dan memimpin Madrasah Arabiah Islamiyah (MAI) yang berlokasi di Sengkang, lembaga pendidikan yang bertahan hingga hari ini telah terbukti menjadi alat reproduksi ulama Bugis. Para generasi pelanjut adalah terdiri dari para santri pilihan yang dianggap cakap menata dan mengatur lembaga serta memiliki keilmuan yang memadai lalu disempurnakan dengan pengamalan yang konsisten serta ketinggian adab yang dikenal dengan ampe-ampe madeceng (akhlak mulia). C. PERAN DAN PEMIKIRAN K.H. LANRE SAID 1. Tujuan Pendidikan Lanre Said j Lanre Said jika ditelusuri secara mendalam dapat dipahami tujuan pendidikan yang ia inginkan. Dalam berbagai kesempatan, sebagaimana yang dituturkan oleh muridnya, bahwa tujuan utama pendidikan yang ia inginkan adalah untuk mencetak generasi pelanjut yang mampu melahirkan manusia saleh, beradab, tangguh dalam berjuang, memiliki pemahaman keagamaan yang berdasarkan dengan apa yang telah ajarkan oleh Nabi Muhammad dan telah dilalui dan dipraktikkan bersama para sahabat-sahabatnya, serta para tabiin dan pengikut tabiin. Untuk mencapai target yang berat itu, maka para santri harus melalui proses dan perjalanan yang cukup panjang. Dimulai dengan penguasaan terhadap al-Qur’an lewat hafalan 30 juz, dipertajam dengan kemampuan bacaan yang baik dan benar sesuai kaidah-kaidah tajwid, pemahaman tentang al-Qur’an itu sendiri dengan terlebih dahulu mengajarkan dan memahamkan ilmu-ilmu alat yang berhubungan dengan al-Qur’an, seperti pelajaran Ulumul-Qur’an yang di dalamnya berbagai jenis ilmu, misalnya, nasikh wal mansukh, asbabun-nuzul, al-qira’ah assab’ah dan lain-lain. Penguasaan ilmu dua belas juga kerap ia tekankan, agar dapat memahami al-Qur’an dan ilmu-ilmu yang berhubungan dengan kitab suci. Yang dimaksud dengan ilmu dua belas nahwu, sharaf, balaghah yang meliputi, bayan, badi’, dan ma’ani. Tafsir, ulumul- tafsir, hadis, ulumul-hadits, fiqih, dan ushulul fiqih, dan mantik. Dengan penguasaan terhadap al-Qur’an dan perangkat-perangkatnya, maka secara perlahan seorang santri akan dapat mencapai gelar ulama, karena dibekali dengan hafalan Al-Quran. Tapi, selain kapasitas intelektual bagi Lanre Said juga mencontohkan langsung bagaimana seorang pimpinan yang hidup dengan sederhana dan bersahaja. Hidup apa adanya, dan tidak banyak menuntut. Karena baginya, hanya dengan hidup sederhana seorang ulama akan bisa berjuang dengan sungguh-sungguh. Ta’dibuna, Vol. 7, No. 1, April 2018 72 Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said Selain itu, Lanre Said, dalam berbagai ceramahnya selalu menyatakan bahwa tujuan didirikannya lembaga pendidikan Majelisul Qurra Wal Huffadh adalah untuk berlayar ke sebuah pulau yaitu pulau al-Qur’an dan hadis sahih yang telah dicontohkan oleh Nabi dan Para sahabat-sahabatnya, serta para tabiin dan pengikut para tabiin. Kalau para santri dan pelanjutnya kemudian tidak memahami tujuan pondok yang ia ibaratkan sebagai kapal itu, maka susah untuk mencapai tujuan mulia itu. Untuk itu, sejak awal Lanre Said telah menanamkan ilmu, ibadah, dan amal yang benar kepada segenap santrinya. Perkara-perkara bidah akidah dan ibadah telah ia tuangkan dengan baik melalui tulisan-tulisannya dalam kitab Adz-Dzikra. Dengan ilmu, ibadah, dan amal yang benar diharapkan dapat benar-benar melahirkan alumni yang siap untuk mengabdi pada umat. C. PERAN DAN PEMIKIRAN K.H. LANRE SAID 1. Tujuan Pendidikan Lanre Said Kecuali itu, Lanre Said juga menekankan bahwa saat ini adalah masa yang tersulit bagi umat Islam, terutama yang berada di pedalaman. Ini terjadi karena para ulama, cendekiawan, penyuluh agama berlomba-lomba masuk ke kota untuk mengejar kemewahan dan hidup senang. Di saat yang sama kegelapan menyelimuti orang-orang yang berada di pedalaman. Ia menulis bahwa akan datang suatu masa, di mana para ulama berlomba-lomba masuk kota mengejar kemewahan, dan pada saat yang sama para pendeta masuk kampung untuk menyesatkan umat. Dari sini dapat dikatakan bahwa tujuan pendidikan menurut Lanre Said adalah untuk mencetak manusia yang beriman, berilmu, beramal saleh, saling menasihati dalam kebenaran, dan kesabaran. Inilah hakikat tujuan manusia diciptakan sebagaimana tujuan pendidikan yang ideal dan selaras dengan firman Allah dalam Surat Al-Ashr, ayat 1-3. 2. Lembaga Pendidikan Lanre Said g Begitu Lanre Said pulang dari rantau, dengan persiapan yang matang, ia langsung mendirikan lembaga pendidikan dalam bentuk halaqah dengan fasilitas apa adanya. Santri dan kiai menyatu dalam satu atap. Dengan rumah panggung beratap daun rumbia menjadi saksi awal mula berdirinya lembaga pendidikan Majlisul Qurra’ Wal Huffadz atau MQWH, dimulai pada jam 07.00 Tanggal 7 Agustus 1975 berlokasi di kampung Tuju-tuju dan dimulai dengan 7 jumlah santri, langsung di pimpin oleh Lanre Said. Dengan fasilitas yang serba minim ini, tidak menyurutkan sama sekali semangat Lanre Said untuk maju ke depan mengejar cita-citanya yang sangat mulia. Secara logika untuk saat itu memang hampir tidak ada yang bakal percaya kalau MQWH bisa menjelma menjadi pondok pesantren seperti sekarang (Darul Huffadh), betapa tidak, tempat belajar saja pada awal mulanya hanya belajar di bawah kolong rumah kiai yang berukuran 32 Meter persegi, materi pelajarannya pun sangat sederhana, bahkan jumlah santrinya yang sama dengan jumlah hari dalam seminggu ini lebih banyak bermandi lumpur di empang ketimbang menimba ilmu di kelas. Ta’dibuna, Vol. 7, No. 1, April 2018 73 Rasmin, Hafidhuddin, Husaini, Mujahidin Rutinitas harian para santri lebih banyak tersita dipakai untuk membangun empang agar dapat dijadikan tambak udang dan ikan demi kepentingan jangka panjang, agar kelak pondok tidak terlalu susah memikirkan masalah lauk-pauk dan sebisa mungkin agar bisa menjadikan tambak di atas sebagai sumber usaha yang dikelola oleh pondok untuk kepentingan para santri-santrinya. Saat itu, lokasi yang akan dijadikan tambak masih berupa hutan bakau dan semak- belukar, jadi praktis, Lanre Said beserta para santrinya selama lima tahun bermula dari berdirinya MQWH pada tahun 1975-1980 hampir tidak pernah absen ke empang, bahkan tidak jarang ketika para santrinya sedang menghafal Al-Qur’an di sore hari beliau sendirian turun ke empang untuk melihat keadaan, jika ada yang bocor maka langsung ditambal agar bocornya tidak bertambah besar, dan beliau baru meninggalkan empang jika menjelang waktu Magrib. Nominal santrinya hanya tujuh orang anak-anak yang ke semunya lulusan Sekolah Dasar, mereka juga hanya berasal dari keluarga pimpinan dan istrinya yang hanya berasal dari dua daerah Tuju-tuju dan Sumbawa. 2. Lembaga Pendidikan Lanre Said Menurut Ilham Kadir, yang sekarang menetap di Daerah Batam Kepulauan Riau, bahwa pada dasarnya ada 8 kandidat santri yang akan diresmikan oleh Lanre Said, tetapi karena para calon santri di atas harus melewati ujian berupa harus menghafal satu juz dalam jangka masa satu bulan dan salah satu di antara para calon santri tersebut harus tereliminasi yaitu Mustaqim Yahya atau saudara dari dua santri perdana, Andi Lukman Yahya dan Andi Sudirman Yahya yang mana mereka ini adalah putra Andi Yahya, yang merupakan keluarga terdekat Lanre Said dan juga pendamping setia beliau dikala duka maupun suka, semenjak di DI/TII hingga Lanre Said lebih dulu pulang ke rahmatullah. Jumlah santri ini bertahan hingga tahun 1979, jadi selama empat tahun pertama berdirinya pondok ini hanya memiliki tujuh santri yang selalu setia taat dan patuh pada pimpinan, mereka semua ini begitu gigih bekerja demi kemaslahatan pondok, walaupun demikian mereka juga tak hilang identitasnya sebagai santri, setiap hari dan malam mereka tetap belajar dan menambah hafalan, jika mereka tidak dapat memenuhi ketentuan target hafalan yang harus dicapai dalam sehari sebanyak satu halaman, maka mereka juga dihukum dengan cara harus menghafal al Qur’an di rumah pimpinan dari Jam 07.00 pagi hingga 12.00 siang. Pada dasarnya sebelum Lanre Said secara formal membuka lembaga pendidikan pondok pesantren, dalam artian para santri harus tinggal bersama dengan kiai dan belajar, bekerja, serta beramal sesuai dengan ketentuan yang belaku di dalam pondok, sebenarnya Ustazah Siti Nur Hasanah atau Petta Cinnong Istri Lanre Said sudah eksis mengajar ilmu-ilmu agama fardu ‘ain dan mengaji, baik untuk pemula maupun sudah lanjutan untuk masyarakat Tuju-tuju. Ta’dibuna, Vol. 7, No. 1, April 2018 74 Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said Pelajaran untuk para pemula seperti biasanya belajar mengaji ala orang Bugis Bone, belajar membaca huruf hijaiah dengan menyebut, alefu ri ase’na a; alefu ri awana i; alefu dapenna u; A, I, U, dan ada pun yang sudah bisa membaca dengan baik sesuai dengan hukum-hukum tajwid dan makhraj-nya, maka mereka akan diajar tarannum atau mallagu karena memang Petta Cinnong memiliki suara yang indah dan bacaan yang bagus ditambah dengan penguasaannya terhadap berbagai macam jenis Qira’ah. Jadi dengan sendirinya orang-orang di Tuju-tuju atau yang berasal dari kampung sebelah berduyun- duyun datang ke rumah Petta Cinnong untuk belajar Tarannum. Ta’dibuna, Vol. 7, No. 1, April 2018 2. Lembaga Pendidikan Lanre Said Dikala MQWH sudah berdiri, Petta Cinnong tetap saja mengajar pada siapa saja yang ingin belajar baca tulis al Qur’an, cuma saja karena santri sudah ada, jadi secara langsung fokus utama istri pimpinan ini terkuras hampir seluruh waktunya untuk mengurus para santri ini, mulai dari memasak, mengarahkan, mendidik, mengajar, dan sebagainya. Ditambah lagi ke semua jumlah santri ini bermukim bersama Petta Cinnong, sedangkan istri yang satunya Petta Paccing belum dijadikan tempat tinggal para santri, mungkin karena pertimbangan jumlah putra-putri pimpinan yang lahir dari rahim Petta Paccing jauh lebih banyak, jadi waktunya secara langsung banyak tersita untuk mengurus anak sendiri, di samping karena memang Petta Paccing hanya ibu rumah tangga saja dalam artian tidak terlibat dalam hal belajar mengajar sebagaimana Petta Cinnong. Pada konsep awal berdirinya pondok ini memiliki nama Darul Ulum yang telah ditetapkan oleh Lare Said saat mengadakan pertemuan keluarga di Surabaya, saat itu di depan para keluarganya beliau sudah bulat tekadnya kembali ke Sulawesi untuk mendirikan lembaga pendidikan yang berbentuk pondok pesantren dan saat itu memilih nama Darul Ulum sebagai nama pondok pesantren yang akan segera dibuka di Sulawesi Selatan tepatnya di Daerah Tuju-tuju. Setelah kembali ke Tuju-tuju, ternyata rencana tidak selamanya berjalan dengan sesuai yang diharapkan, betapa tidak, masa lalunya sebagai Ketua Mahkamah Agung dimasa DI/TII membuat Lanre Said tetap mendapat pengawasan yang khusus di zaman itu, mulai dari pihak birokrasi seperti Lurah, Camat, Hingga Bupati atau pihak keamanan baik itu, polisi di bawah Kapolsek atau tentara di bawah Koramil, mereka tidak henti- hentinya mencurigai segala sepak terjang sang Ustaz ini. Dengan berbagai pertimbangan, setelah tiba di Tuju-tuju dan mulai merintis lembaga pendidikan yang bernuansa pondok ini, maka beliau menamakan tempat pendidikannya ini sebagai Majelis al Qurra’ wal Huffadh, Majelis jika diartikan menurut etimologi atau bahasa adalah berarti tempat duduk, namun bila diartikan menurut terminologi atau istilah maka kata ini memiliki makna yang sangat beragam, mulai dari yang politis hingga yang sangat umum di telinga masyarakat, yang politis misalnya kata “Majelis” dapat diartikan sebagai dewan atau rapat yang mengembang tugas-tugas kenegaraan yang Ta’dibuna, Vol. 7, No. 1, April 2018 75 Rasmin, Hafidhuddin, Husaini, Mujahidin tertentu dan terbatas, sedangkan yang umum biasa diketahui oleh masyarakat kita adalah suatu golongan atau kelompok terkoordinasi yang rutin mengadakan pertemuan keagamaan. Majelis al Qurra’ wal Huffadh dapat diartikan dengan makna yang sederhana adalah satu tempat untuk membaca, mempelajari, dan menghafal isi al Qur’an. 2. Lembaga Pendidikan Lanre Said Dan pada tanggal 11 November 1992 adalah perubahan nama dari Majelis al Qurra’ wal Huffadh menjadi Darul al Qurra’ wal Huffadh, yang pada saat itu dikonsep langsung oleh Muttaqien Said di Bantu dengan para guru-guru. Satu hal yang dapat kita ambil pelajaran dari sini adalah tingginya toleransi Lanre Said dalam menghadapi bentuk-bentuk yang bersifat teknis atau istilah tanpa mengubah substansi atau isi itu sendiri. Pada bulan Agustus 1989, merupakan babak baru dalam fase perkembangan MQWH, ini terjadi karena adanya eksodus besar-besaran alumni PM Gontor ke pondok ini, bahkan bisa dikatakan mungkin inilah pondok pesantren satu-satunya di Indonesia yang jumlah gurunya hampir separuh dari total jumlah santri secara keseluruhan pada periode 1989-1994. Para alumni PM Gontor ini menjadikan MQWH sebagai pilihan favorit untuk mengabdi, ataupun menjadikan tempat transit sebelum masuk ke bangku kuliah baik dalam maupun luar negeri ataupun bagi mereka yang ingin langsung mengabdi ke masyarakat. hal yang paling memikat hati mereka adalah dapat mengafal Al-Qur’an dengan durasi waktu yang cukup singkat, bahkan ada yang dapat khatam hanya dalam jangka masa 60 hari sambil berguru dengan Lanre Said, selain itu mereka dapat mengamalkan ilmu yang telah didapatkan di PM Gontor. Mereka inilah para agen of change di MQWH, yang sebelumnya lembaga pendidikan ini masih mengadopsi sistem halaqah, namun semua berubah dalam jangka masa yang singkat kepada sistem modern sebagaimana PM Gontor sendiri setelah para alumninya datang ke MQWH. Keahlian Lanre Said dalam mengatur para alumni PM Gontor ini juga harus diacungi jempol, mereka merasa diberi kebebasan sepenuhnya untuk berekspresi dalam berkarya, sama sekali tidak ada tekanan, selama untuk kebaikan dan kemajuan pondok tidak ada masalah, yang ditangani langsung oleh Lanre Said hanyalah persoalan ibadah inti saja. Adapun pengajaran dan sebagainya semua bebas ala Gontor. Para alumni PM Gontor yang datang ke MQWH pada periode 1989-1994, mereka adalah mayoritas alumni 1989-1993, pada tahun-tahun ini para tenaga pengajar murni monopoli alumni Gontor, namun pada tahun 1994 sudah mulai juga berdatangan dari pondok alumni atau cabang Gontor, seperti Darul Arafah dan Darul Hikmah Medan Sumatera Utara, Darul Muttaqin dan Darunnajah, Bogor Jabar, dan beberapa pondok pesantren yang mengadopsi sistem KMI. IV. KESIMPULAN IV. KESIMPULAN Ta’dibuna, Vol. 7, No. 1, April 2018 76 Pendidikan Islam dalam Perspektif Ulama Bugis K.H. Lanre Said Pemikiran pendidikan Islam sejatinya telah melahirkan gagasan dan aplikasi dalam regenerasi ulama, tidak terkecuali ulama Bugis. Christian, P. (2006). The Bugis. (H. dan N. S. diterjemahkan denganoleh Abdul Rahman Abu, Penerj.). Jakarta: Nalar dan Forum. V. DAFTAR PUSTAKA Al-Jauziyah, I. al-Q. (2012). Fawâ’id al-Fâwâa’id. (Muhtadi & A. Risdianto, Penerj.). Jakarta: Darus Sunnah Press. Ardiansyah, M., Hafidhuddin, D., Mujahidin, E., & Syafrin, N. (2017). The Concept of Adâb by Syed Muhammad Naquib al-Attas and Its Relevance to Education in Indonesia. International Journal of Islamic Education Ta’dibuna, 1(1), 53–64. B i M (1999) R k t K il I l d P litik Y k t Y B t 2. Lembaga Pendidikan Lanre Said H li W H & S b (2012) T i d A d l M k B i Dhofier, Z. (1994). Tradisi Pesantren: Studi tentang Pandangan Hidup Kiai. Jakarta: LP3ES. Halim, W. H., & Sesember. (2012). Topanrita dan Anregurutta dalam Masyarakat Bugis Dhofier, Z. (1994). Tradisi Pesantren: Studi tentang Pandangan Hidup Kiai. Jakarta: LP3ES. Halim, W. H., & Sesember. (2012). Topanrita dan Anregurutta dalam Masyarakat Bugis Abad XX. Jurnal Al- Ulum, 12(2). H b ll h (1999) S j h P didik I l I d i J k R j G i d P k , ( ) g g p J Halim, W. H., & Sesember. (2012). Topanrita dan Anregurutta dalam Masyarakat Bugis Abad XX. Jurnal Al- Ulum, 12(2). ( ) bullah. (1999). Sejarah Pendidikan Islam Indonesia. Jakarta: Raja Graindo Perkasa. Hasbullah. (1999). Sejarah Pendidikan Islam Indonesia. Jakarta: Raja Graindo Perkasa. Husaini, A. (2013). Fiilsafat Ilmu Perspective Barat dan Islam. Jakarta: Gema Insani Press. Ide, S. M. (1977). Kamus Bahasa Bugis-Indonesia. Jakarta: Departemen Pendidikan dan Kebudayaan RI ( ) j J j Husaini, A. (2013). Fiilsafat Ilmu Perspective Barat dan Islam. Jakarta: Gema Insani Press. Husaini, A. (2013). Fiilsafat Ilmu Perspective Barat dan Islam. Jakarta: Gema Insani Press. Ide S M (1977) Kamus Bahasa Bugis-Indonesia Jakarta: Departemen Pendidikan dan Husaini, A. (2013). Fiilsafat Ilmu Perspective Barat dan Islam. Jakarta: Gema Insani Press. Ide, S. M. (1977). Kamus Bahasa Bugis-Indonesia. Jakarta: Departemen Pendidikan dan b d Ide, S. M. (1977). Kamus Bahasa Bugis-Indonesia. Jakarta: Departemen Pendidikan dan Kebudayaan RI. Kadir, A. (2003). Metodologi Penelitian Kualitatif. In Indobis (Vol. 1, hal. 104–105). Makassar. adir, A. (2008). Ulama Bugis. Makassar: Balai Penelitian dan Pengembangan Agama ( ) g g g g Khaldun, I. (2010). Muqaddiman Ibn Khaldûn, Tahqîq Majdî Fathî al-Sayyed. Mesir: Dâr al- Taufiqiyah li al-Turâts. Mahlani, G. (2013). Sintesa Pendidikan Islam. Makassar: LSQ. Manti, B. B., Husaini, A., Mujahidin, E., & Hafidhuddin, D. (2016). Konsep Pendidikan Modern Mahmud Yunus dan Kontribusinya Bagi Lembaga Pendidikan Islam di Indonesia. Jurnal Ta’dibuna, 5(2), 153–185. ( ) Mattudala. (n.d.). “Manusia dan Kebudayaan Bugis-Makassar dan Kaili d Sulawesi” dalam Antropologi. Mujahidin, E. (2005). Pesantren Kilat: Alternatif Pendidikan Agama Di Luar Sekolah. Jakarta: Pustaka al-Kautsar. Muljana, S. (2008). Kesadaran Nasional, dari Kolonialisme sampai Kemerdekaan. Yogyakarta: LKiS. Munawwir, I. (2006). Mengenal pribadi 30 pendekar dan pemikir Islam dari masa ke semasa. Bina Ilmu. Noorduyn, J. (2000). 2. Lembaga Pendidikan Lanre Said Maka, tugas kita hari ini adalah menjadi mata rantai ulama dan intelektual yang telah melahirkan gagasan dan gerakan pendidikan lewat berbagai macam wadah. Baik melalui lembaga, yayasan, maupun organisasi massa bahkan kementerian agama dengan tujuan melestarikan pendidikan Islam. Pemikiran pendidikan terus berkembang untuk merespons masalah-masalah internal dan eksternal. Dari dalam para tokoh pendidikan berusaha mengembangkan pendidikan Islam dengan berbagai dinamika yang ada, menguatkan fondasi-fondasi peserta didik, khususnya terkait materi pengembangan akidah dan syariat agar mereka tetap kokoh memegang teguh keyakinan. Dari luar, berusaha beradaptasi dengan baik, menahan gempuran globalisasi, menyaring setiap sistem yang ada, lalu menyerap dan menerapkan jika dianggap perlu. Ulama Bugis telah membuktikan bahwa mereka tetap eksis dengan perubahan zaman yang begitu cepat. Merespons dengan baik setiap pergantian zaman, tetap menjadi bagian penting dalam membangun bangsa lewat dunia pendidikan dengan mencerdaskan kehidupan bangsa, membangun masyarakat beradab. Itulah di antara inti dari tujuan pemikiran pendidikan Islam dari zaman ke zaman. Di antara ulama terkemuka Bugis adalah Lanre Said. Beliau telah meletakkan dasar- dasar pemikiran pendidikan Islam di masyarakat Bugis, terutama pendidikan Tahfidzul Qur’an. Tujuan pendidikan tahfizhul qur’an bagi KH Lanre Said adalah untuk memasyarakatkan Al-Qur’an. Dengan hidupnya budaya hafal Al-Qur’an maka umat Islam tidak akan susah diajak berlayar ke pulau Al-Qur’an. Yang dimaksud Lanre Said dengan pulau Al-Qur’an adalah mewujudkan tatanan masyarakat yang hidup dengan Al-Qur’an. Masyarakat qur’ani adalah mereka yang menjadikan kitab suci sebagai ukuran benar salahnya sebuah tindakan. Pulau Al-Qur’an adalah negara yang merujuk pada Al-Qur’an dalam segala bentuk kehidupannya. Inilah tujuan tertinggi yang dikehendaki oleh Lanre Said. Wallahu A’lam! V. DAFTAR PUSTAKA Al-Jauziyah, I. al-Q. (2012). Fawâ’id al-Fâwâa’id. (Muhtadi & A. Risdianto, Penerj.). Jakarta: Darus Sunnah Press. Ardiansyah, M., Hafidhuddin, D., Mujahidin, E., & Syafrin, N. (2017). The Concept of Adâb by Syed Muhammad Naquib al-Attas and Its Relevance to Education in Indonesia. International Journal of Islamic Education Ta’dibuna, 1(1), 53–64. Bruinessen, M. van. (1999). Rakyat Kecil, Islam, dan Politik. Yogyakarta: Yayasan Bentang Budaya. Christian, P. (2006). The Bugis. (H. dan N. S. diterjemahkan denganoleh Abdul Rahman Abu, Penerj.). Jakarta: Nalar dan Forum. 77 Ta’dibuna, Vol. 7, No. 1, April 2018 Rasmin, Hafidhuddin, Husaini, Mujahidin Damanhuri, A., Mujahidin, E., & Hafidhuddin, D. (2013). Inovasi pengelolaan pesantren dalam menghadapi persaingan di era globalisasi. Jurnal Ta’dibuna, 2(1), 17–37. Dhofier, Z. (1994). Tradisi Pesantren: Studi tentang Pandangan Hidup Kiai. Jakarta: LP3ES. 2. Lembaga Pendidikan Lanre Said “The Wajorese Merchants’ Community in Makassar” dalam Roger Tol, Kees van Dijk dan. In G. Acciaioli (Ed.), Authority and Enterprise among the Peoples of South Sulawesi. Leiden: KITLV Press. Palimai, I. K. (2010). Jejak Dakwah KH Lanre Said: Ulama Pejuang dari DI/TII hingga Era Reformasi. Jogjakarta: Aynat Publishing. Patunru, A. D. (1965). Sedjarah Wajo. Makassar: YKSST. Pawiloy, S. (1987). Sejarah Perjuangan Angkatan 45 di Sulawesi Selatan. Ujungpandang: Dewan Harian Daerah Angkatan 45. Umar, H. (2000). Riset Sumber Daya Manusia dalam Organisasi (Vol. (Cet. III;). Jakarta: PT. Gramedia Pustaka Utama. Umar, H. (2000). Riset Sumber Daya Manusia dalam Organisasi (Vol. (Cet. III;). Jakarta: PT. Gramedia Pustaka Utama. Yunus, M. (1996). Sejarah Pendidikan Islam di Indonesia. Jakarta: PT. Hidkarya Agung. Yunus, M. (1996). Sejarah Pendidikan Islam di Indonesia. Jakarta: PT. Hidkarya Agung. Ta’dibuna, Vol. 7, No. 1, April 2018 78
https://openalex.org/W3115503739	https://www.researchsquare.com/article/rs-16579/latest.pdf	English	null	Over reliance on pesticides and poor handling practices characterize intensive vegetable farming: case of selected smallholders in southwestern Uganda	Research Square (Research Square)	2,020	cc-by	11,648	Over reliance on pesticides and poor handling practices characterize intensive vegetable farming: case of selected smallholders in southwestern Uganda Eziron Rwakipamba Uganda Martyrs University Faculty of Agriculture Godfrey Sseremba (  gsseremba16@gmail.com ) Uganda Martyrs University Faculty of Agriculture https://orcid.org/0000-0001-9625-0570 John Byalebeka Uganda Martyrs University Faculty of Agriculture Joseph Ssekandi Uganda Martyrs University Faculty of Agriculture Julius Mwine Uganda Martyrs University Faculty of Agriculture Eziron Rwakipamba Uganda Martyrs University Faculty of Agriculture Godfrey Sseremba (  gsseremba16@gmail.com ) Uganda Martyrs University Faculty of Agriculture https://orcid.org/0000-0001-9625-0570 John Byalebeka Uganda Martyrs University Faculty of Agriculture Joseph Ssekandi Uganda Martyrs University Faculty of Agriculture Julius Mwine Uganda Martyrs University Faculty of Agriculture Research article Keywords: Chemical spray frequency, intensive vegetable production, pesticide spillages, produce intoxication Posted Date: March 10th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-16579/v1 DOI: https://doi.org/10.21203/rs.3.rs-16579/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/36 Page 1/36 Page 1/36 Abstract This study investigated the extent of pesticide use and poor handling practices which characterize intensive vegetable farming, among smallholder vegetable farmers in south western Uganda. 100% of respondents were using pesticides for control of pest and diseases, and over 78% applied pesticides on a twice-a-week basis. Majority of farmers used pesticides in violation of recommended procedures/rules; use of unsafe storage facilities, ignore risks and safety instructions, and did not calibrate the spray equipment, and disposed of containers unsafely. Poor pesticide-handling practices were associated with farmers’ lack of knowledge and awareness in use and handling, inadequate extension services, low education levels, and the everyday context in which pesticides were bought and used. A careful approach to analyze pesticide use and handling practices to systematically identify ways to change the current farmers’ practices needs to be adopted or undertaken. Study area A case study was made in major vegetable producing areas of Kabarole district, in western Uganda. The major cultivated vegetable crops in the area include tomatoes, cabbages, onions, egg plants and green pepper. Before starting the study, ethical approval was sought from Office of the Dean, Faculty of Agriculture, Uganda Martyrs University. Background Exposures to toxic pesticides can result in health hazards in the form of acute or chronic illnesses (Maumbe and Swinton, 2003; Amuoh, 2011). It is critical therefore to be aware about the extent of use and handling practices of pesticides among smallholder farmers who dominate the majority in sub-Saharan African countries. This study intended at investigating the extent of pesticide use and poor handling practices in intensive vegetable farming areas by smallholder farmers. Background Applying pesticides on fields can affect animals and other living organisms which interact with the targeted pests. The reduction in these other organisms can result in changes in the biodiversity of an area and affect natural biological balances (Amuoh, 2011). biodiversity (Mahmood et al., 2016). Inappropriate use of pesticides leads to reduction of beneficial species such as bees, birds, soil and aquatic organisms. Applying pesticides on fields can affect animals and other living organisms which interact with the targeted pests. The reduction in these other organisms can result in changes in the biodiversity of an area and affect natural biological balances (Amuoh, 2011). Pesticides can affect other areas during application and can cause severe problems in different crops, livestock, waterways and the general environment; wildlife and fish are the most affected. Taking special note of weather conditions can reduce drift. The use of pesticides may lead to residues in human food and drinking water. This can either be by direct application onto the food, or by biomagnification along the food line and run off in domestic water. Not all levels are undesirable but unnecessary and dangerous levels must be avoided through knowledge of the dynamics of safe pesticide use and good agricultural practice. When the same pesticides are overused, the targeted pest can develop resistance to the pesticide. Excessive exposure to pesticides without safe handling procedures and wearing of protective clothing can lead to poisoning. Poisoning risks depend on dose, toxicity, duration of exposure accumulation levels and sensitivity. Farmers and farmworkers can get exposed to pesticides through four primary routes namely ingestion, inhalation, dermal absorption, and absorption through the eyes. Individuals in a farm situation can get exposed to pesticides in various ways (Okello and Swinton, 2010; Amuoh, 2011). Exposures to toxic pesticides can result in health hazards in the form of acute or chronic illnesses (Maumbe and Swinton, 2003; Amuoh, 2011). It is critical therefore to be aware about the extent of use and handling practices of pesticides among smallholder farmers who dominate the majority in sub-Saharan African countries. This study intended at investigating the extent of pesticide use and poor handling practices in intensive vegetable farming areas by smallholder farmers. Individuals in a farm situation can get exposed to pesticides in various ways (Okello and Swinton, 2010; Amuoh, 2011). Background Pesticides usage is gaining increased attention in the media for their beneficial effects in fighting pests and diseases. Pesticide use is now a commonly used practice by most farmers to control pests and diseases in their vegetable production systems. This is often at the expense of the environment and human health; the widespread use of pesticides has caused a growing concern about their effects on the ecosystem components. Poor pesticide handling practices have been recognized as a major threat to the ecosystem and biodiversity sustainability as well as a public health problem (resulting in increased cases of non-communicable diseases where new research results indicate that pesticides increase the risk of severe health problems like cancer, Parkinson, dementia, diabetes and other diseases, Eyhorn et al., 2015). However, there is still a lack of attention paid to the significant role that poor pesticide handling practices play in contributing to the problem. In many countries, water and food contamination by pesticides is considered an important method of human harm. While there are no accurate data available on acute pesticide poisoning due to occupational and accidental exposure, most estimates are in the range of several million cases per year, as this is in line with (Lah 2011; Hicks 2013; Mahmood et al., 2016). Acute pesticide poisoning is a serious problem in developing countries and emerging economies, where many farmers use highly hazardous products, often without adequate protective measures, using application practices that may lead to water, food/produce and environmental pollution (Eyhorn et al., 2015). Although pesticides enter the human body through ingestion, inhalation or penetration via skin; majority of people get affected via intake of pesticide contaminated food and water (Mahmood et al., 2016). Multiple pesticide residues are found in drinking water/surface water and food; unintentional pesticide poisoning and acute effects are also a serious problem in agricultural communities, of the low and middle-income countries (Jørs et al., 2018). It is critical therefore, to effectively investigate pesticide handling practices in respect to levels of farmers’ awareness in pesticide handling and application practices. Most studies about pesticides in Uganda have reported less on pesticide use and handling practices in respect to farmer’s knowledge. Excessive use of pesticides may lead to the destruction of Page 2/36 Page 2/36 biodiversity (Mahmood et al., 2016). Inappropriate use of pesticides leads to reduction of beneficial species such as bees, birds, soil and aquatic organisms. Study design and population A cross-sectional case study was conducted. The target population was the vegetable farmers involved in production of major vegetables (such as tomatoes, cabbages and onions). The number of farmers involved in vegetable production (Tomatoes, Cabbages, and Onions, among others) in the different villages of the 3 parishes of Kichwamba was not directly known and the study sample was established based a guide by Syed (2017) as detailed herein. Consultation of local council (LC1) chairpersons with Page 3/36 the aid of the respective parish chiefs in conjunction with the sub county agricultural officer was also made in order to access vegetable producers with farming experience of not less than one year. Sampling Sample size was determined according to Syed (2017) who asserted that, if the population is unknown, a minimum of 384 responses are sufficient for substantial data collection and analysis. The standard required sample size would be around 383 for a population with a 5% margin of error and a 95% confidence level. Using the formula for the representative sample- n: n = (Z-score)² * p(1-p) / (margin of error)² = (1.96)² 0.5(1-0.5) / (0.05)² = 3.8416 0.25 / 0.0025 = 384.16 n = (Z-score)² * p(1-p) / (margin of error)² = (1.96)² 0.5(1-0.5) / (0.05)² = 384.16 Note: (Z-score = 1.96 for a 95% confidence level) Note: (Z-score = 1.96 for a 95% confidence level) Then the standard sample computed above would then be adjusted to the specific research population using the following formula below; n-adjusted = (n) / 1 + [(n – 1) / N- population] n-adjusted = (n) / 1 + [(n – 1) / N- population] But our study population is 300 vegetable farmers, our margin of error = 7%, p- value = 0.5 and a confidence interval of 90% (1.645) was used for this research. Therefore, the sample size was determined as below; n = (Z-score)² p(1-p) / (margin of error)² = (1.645)² 0.5(1-0.5) / (0.07)² Page 4/36 Statistical analysis The data was then statistically analyzed while considering the following statistical variables; number of farmers using pesticides in their vegetable fields, frequency of application, and application interval at particular period of weather conditions. Descriptive statistics using percentages; of farmers using pesticides, farmers who apply pesticides to same crop a specific number of times in a given time period and farmers who apply pesticides in a particular time interval in respect to particular weather condition respectively, were used. = 2.706025 0.25 / 0.0049 = 2.706025 * 0.25 / 0.0049 = 2.706025 * 0.25 / 0.0049 = 138 NB (Z-score is 1.645 for a 90% confidence level) NB (Z-score is 1.645 for a 90% confidence level) NB (Z-score is 1.645 for a 90% confidence level) Therefore: sample n above was adjusted to the current research population (N =300) n adjusted. = (n) / 1 + [(n – 1) / N-population] Therefore: sample n above was adjusted to the current research population (N =300) n adjusted. = (n) / 1 + [(n – 1) / N-population] = (138) / 1+ [(138 – 1) / 300] = (138) / 1+ (137/300) = (138) / (1+ 0.457) = (138) / 1.457 = 138 / 1.457 = 94.715 = 95 Respondents Therefore: sample n above was adjusted to the current research population (N =300) = 95 Respondents The study was to be conducted on 95 respondents as per sample calculations above, however due to limited time, resource constraints; terrain of the area and other logistical issues, the study was able to involve 90 respondents. In his study snowball sampling technique was employed. One vegetable farmer who was introduced to the research team by the Local Council chairperson was interviewed, who then led the interviewer to the next farmer and the process continued until the sample population, above was covered. The number of respondents achieved per parish depended on population of vegetable farmers in that parish (Table 1). Table 1: showing number of respondents achieved per parish Table 1: showing number of respondents achieved per parish Page 5/36 Page 5/36 S/N Parishes number of respondents 1. Bwanika 46 1. Mabale 38 1. Kihondo 06 Total 90 S/N Parishes number of respondents Data collection The variables considered included age, gender, marital status and level of education, number of farmers using pesticides on vegetables, Frequency of application and Application interval at a period of weather conditions. Data was collected through a farmer’s survey by face-to-face interviews with farmers, field observations and pictures taken as well. One interviewed farmer led the interviewer to another farmer unti data was collected from all sampled farmers. A structured questionnaire (questionnaire developed and used for this study is included as supplementary material) was made and administered to collect the data on the extent of pesticide use by farmers and farmer’s’ awareness in the use and handling of pesticides. Observations in farmers’ fields were done and where possible pictures portraying relevant information were taken. Data collection Data was collected through a farmer’s survey by face-to-face interviews with farmers, field observations and pictures taken as well. A first interviewed farmer led the interviewer to another farmer until data was Page 6/36 collected from total sample of farmers. A structured questionnaire was made and administered to collect the data on farmers’ awareness in the use and handling of pesticides. Observations in farmers’ fields were done and where possible pictures portraying relevant information were taken. Data on the following variables was collected: trainings in pesticide use and handling, extension services, education level, calibration of the spray equipment, method/skill of application, disposal method, and method of pesticides storage and consultation for advice. Statistical analysis Descriptive statistics using percentages of farmers; who have received training in pesticide use and handling, farmers who received pesticide extension services, farmers of different education levels, farmers who are able to calibrate their spray equipment, farmers using a particular way of applying pesticides i.e. spot vs. routine application, farmers using a particular disposal method, farmers using a particular way of pesticides storage and farmers who consult on pesticide knowledge e.g. from other farmers respectively, were used. Inferential statistics were not appropriate since nonprobability sampling was used. Quantitative data was analyzed using frequency tables, and graphs. Analysis of qualitative data was by content familiarization involving reading through the interviews several times after collecting responses, which enabled the researcher to make notes and understand the data very well. This helped the researcher to locate bits of information that supported the interpretation and elaboration where the researcher explored the details of the data in order to obtain in-depth understanding of the research phenomenon. The margin of error of 7%, p- value of 0.5 and a confidence interval of 90% (1.645) was used for this research. Table 2: The Socio-Demographic Data Aspects n= (90) Variable Response Frequency Percent AGE 15-30 43 47.8 31-40 34 37.7 41-50 9 10 51 Above 4 4.5 RESPONDENT'S SEX Male 64 71.1 Female 26 28.9 Total 90 100 MARITAL STATUS Single 14 15.6 Married 72 80 Divorced 3 3.3 Widowed 1 1.1 Total 90 100 EDUCATION LEVEL None 8 8.9 Primary 51 56.7 Lower secondary 25 27.8 Higher secondary 3 3.3 Tertiary 3 3.3 Total 90 100 Social demographic information Basic information about the socio-economic characteristics farmers is presented in Table 2. Information about the social demographic background was obtained from both male and female individuals who use pesticides. The socio-demographic characteristics included sex, age, marital status, and level of education. A total of 90 farmers were contacted during the study (participation rate =100%). Majority of the study participants were between the age brackets of 15-30 (47.8%), ages 31-40 (37.7%), 41-50 (10%), and 4.5% were aged 51 and above. Most of these farmers were males (74.4%) whereas 28.9% were females. 80%, of the participants were married, 15.6% were single, the divorced and widowed were 3.3% and 1.1% respectively. 56.7% of participants were primary leavers, 27.8% O’ Level dropouts and 6.6% had high levels of education. Their average farm size was small, estimated between 0.5 -1.5 acres. Family size was about four persons per household, and average vegetable farming experience was 6 years. The high yield was the main motive behind using pesticides, followed by high profit. Page 7/36 Page 7/36 Table 2: The Socio-Demographic Data Aspects n= (90) Establishing the extent of pesticide use in the area In this study, majority of farmers had grown vegetables such as onions, tomatoes, cabbages eggplants and green pepper for a long time ranging from eight months to ten years. 93.3% for more than one year, while only 6.7% for a maximum of ten months. 100% of farmers reported a pest and disease problem Page 8/36 Page 8/36 affecting their crops seasonally, monthly; weekly and daily. More than 90% of the farmers confirmed presence of pests and or diseases by themselves, whereas only 4.4% by consulting other people. 95.6% of farmers try to manage pests and disease problem by themselves and 100% of farmers reported spraying pesticides to manage pests and diseases. More than 80% of farmers rely only on pesticide spraying for management and control of pests and diseases and less than 7% of the farmers use weed control and other options of pest and diseases control in addition to chemical spraying. affecting their crops seasonally, monthly; weekly and daily. More than 90% of the farmers confirmed presence of pests and or diseases by themselves, whereas only 4.4% by consulting other people. 95.6% of farmers try to manage pests and disease problem by themselves and 100% of farmers reported spraying pesticides to manage pests and diseases. More than 80% of farmers rely only on pesticide spraying for management and control of pests and diseases and less than 7% of the farmers use weed control and other options of pest and diseases control in addition to chemical spraying. Table 3: Number and percentage of farmers using pesticides (extent of pesticide use, table 1 of 4) Page 9/36 Variable Response Frequency Percent Period taken by farmer in vegetable growing Years 84 93.3 Months 6 6.7 Total 90 100 How often do you face pests and or disease problem Seasonally 59 65.6 Monthly 15 16.7 Weekly 14 15.6 Daily 2 2.2 Total 90 100 How do you confirm type of a particular pest or disease on a veg. Establishing the extent of pesticide use in the area crop Consultation 4 4.4 Self- observation 86 95.6 Total 90 100 Who determines the control method/ technique for pest or disease Self 86 95.6 Consulting 4 4.4 Total 90 100 How often do you apply pesticides on your farm Seasonally 15 16.7 Monthly 5 5.6 Weekly 70 77.8 Total 90 100 Pests and diseases managed by farmer Yes 90 100 No 00 000 Total 90 100 Farmer manages pests and diseases by chemical spraying Yes 90 100 No 00 000 Total 90 100 Farmer manages pests and diseases by weed control in addition to chemical control Yes 6 6.7 No 84 93.3 Total 90 100 Table 4: How farmers use different types of pesticides (extent of pesticide use, Table 2 of 4) Variable Variable Period taken by farmer in vegetable growing No 84 93.3 Total 90 100 Farmers use more than one type of pesticides on the same crop or field and most times mixed in one tank (table 4). Findings show that 71.1% of the farmers used herbicides to prepare their fields and only 28.9% used tillage methods. 100% of farmers used conventional insecticides and fungicides for control of insect pests and fungal diseases respectively. Onions farmers used herbicides most for field preparation followed by tomato farmers, the least users being green pepper and eggplant farmers respectively. 71.1% of all farmers used conventional insecticides to control insect pests; only 28.9% did not use insecticides. 88.9% of all farmers were onion farmers who used insecticides; where as 11.1% did not grow onions. Only 38.9% of all farmers were cabbage producers and all (100%) used insecticides; 61.1% did not grow cabbages; 70% of all farmers were tomatoes produces and all (100%) used insecticides; 30% did not grow tomatoes. Table 4: How farmers use different types of pesticides (extent of pesticide use, Table 2 of 4) Table 4: How farmers use different types of pesticides (extent of pesticide use, Table 2 of 4) Page 11/36 Variable Response Variable Response Frequency % Farmer uses different types of pesticides on the farm Yes 90 100 No 0 0 Total 90 100 Farmer uses herbicides in vegetable farm Yes 64 71.1 No 26 28.9 Total 90 100 Farmer uses herbicides in Onion fields Yes 60 66.7 No 30 33.3 Total 90 100 Farmer uses herbicides in Cabbage fields Yes 26 28.9 No 64 71.1 Total 90 100 Farmer uses herbicides in tomato fields Yes 42 46.7 No 48 53.3 Total 90 100 Farmer uses herbicides in Eggplant fields Yes 1 1.1 No 89 98.9 Total 90 100 Farmer uses herbicides in Green pepper fields Yes 4 4.4 No 86 95.6 Total 90 100 Farmer's purpose to use herbicides Kill weeds for seed bed preparation 65 72.2 Weeding the crop 25 27.8 Total 90 100 Farmer uses conventional insecticides in veg. Variable fields Yes 90 100 No 0 0 Total 90 100 fields No 10 11.1 Total 90 100 Farmer uses Conventional insecticides in cabbage fields Yes 35 38.9 No 55 61.1 Total 90 100 Farmer uses Conventional insecticides in Tomato fields Yes 63 70 No 27 30 Total 90 100 fields Findings in table 5 indicate 4.4% and 7.8% farmers were producing eggplants and green pepper respectively, and all (100%) applied insecticides to their fields; 95.6% and 92.2% did not growing eggplants and green pepper respectively. 100% of farmers use fungicides; 88.9% of all were onion farmers and all used conventional fungicides, 11.1% farmers did not grow onions. 36.7% were growing cabbages and all (100%) used fungicides in their fields, and 63.3% farmers did not grow cabbage use Table 5: How farmers use different types of pesticides (extent of pesticide use, Table 3 of 4) Table 5: How farmers use different types of pesticides (extent of pesticide use, Table 3 of 4) Page 13/36 Page 13/36 Variable Response Frequency Percent Farmer uses Conventional insecticides in Eggplant fields Yes 4 4.4 No 86 95.6 Total 90 100 Farmer uses Conventional insecticides in Green pepper fields Yes 7 7.8 No 83 92.2 Total 90 100 Farmer's Purpose to use conventional insecticides Kill insect pests damaging the crop 90 100 Reduce pests 0 0 Total 90 100 Farmer use Organic insecticides in veg. fields Yes 0 0 No 90 100 Total 90 100 Farmer uses Organic insecticides in Onion fields Yes 0 0 No 90 100 Total 90 100 Farmer uses Organic insecticides in cabbage fields Yes 0 0 No 90 100 Total 90 100 Farmer uses Organic insecticides in Tomato fields Yes 0 0 No 90 100 Total 90 100 Farmer uses Organic insecticides in Eggplant fields Yes 0 0 No 90 100 Total 90 100 Farmer uses Organic insecticides in Green pepper fields Yes 0 0 No 90 100 Total 90 100 Variable Frequency Percent Response 0 plant nutrients Not at all 90 100 Total 90 100 Farmer uses conventional fungicides in veg. Variable fields Yes 90 100 No 0 0 Total 90 100 Farmer uses conventional fungicides in Onion fields Yes 80 88.9 No 10 11.1 Total 90 100 Farmer uses conventional fungicides in Cabbage fields Yes 33 36.7 No 57 63.3 Total 90 100 Findings in Table 6 shows that 68.9% of farmers were tomato producers and all (100%) using conventional fungicides in their tomato fields; 31.1% of farmers did not grow tomatoes. 4.4% and 7.8% of farmers were producing eggplants and green pepper respectively and all (100%) use conventional fungicides in their fields; 95.6% and 92.2% were not growing eggplants and green pepper respectively. No organic pesticide was used by any farmer Table 6: How farmers use different types of pesticides (extent of pesticide use, Table 4 of 4) Page 15/36 Variable Response Frequency Percent Farmer uses conventional fungicides in Tomato fields Yes 62 68.9 No 28 31.1 Total 90 100 Farmer uses conventional fungicides in Eggplant fields Yes 4 4.4 No 86 95.6 Total 90 100 Farmer uses conventional fungicides in Green pepper fields Yes 7 7.8 No 83 92.2 Total 90 100 Farmer's purpose to use Conventional fungicides Control fungal diseases on crops 88 97.8 Control disease and keep env't safe 2 2.2 Total 90 100 Farmer uses Organic fungicides in veg. fields Yes 0 0 No 90 100 Total 90 100 Farmer use Organic fungicides in Onion fields Yes 0 0 No 90 100 Total 90 100 Farmer use Organic fungicides in Tomato fields Yes 0 0 No 90 100 Total 90 100 Farmer use Organic fungicides in Cabbage fields Yes 0 0 No 90 100 Total 90 100 Farmer use Organic fungicides in Eggplant fields Yes 0 0 No 90 100 Total 90 100 Frequency Percent Variable Response Farmer use Organic fungicides in Green pepper fields Yes 0 0 No 90 100 Total 90 100 Farmer's purpose to use Organic fungicides Not at all 90 100 Control disease & keep env’t safe 0 0 Total 90 100 Farmer uses other pesticide option types Yes 0 0 No 90 100 Total 90 100 Table 7 presents the level and extent of pesticide use in terms of frequency and interval of application by farmers in the study area. From the findings, 77.8% of farmers applied pesticides weekly, 5.6% and 16.7% apply pesticides monthly and seasonally respectively. Variable 98.9% of famers apply pesticides on crops routinely with or without pests and or diseases on crops; and only 1.1% use spot spraying only when they see pests and or diseases on crops. In the terms of different weather conditions, the frequency, intensity and interval of pesticide application keep changing. During rain seasons, 47.8% of farmers apply pesticides every 4 days; 28.9% apply pesticides weekly; 15.6% apply pesticides every 5 days; 5.6% apply pesticides every 3 days and only 2.2% apply pesticides above 7 days. In dry seasons 55.6% apply pesticides in an interval above 7 days; 43.3% apply pesticides weekly and only 1.1% apply pesticides every 4 days. Table 7 presents the level and extent of pesticide use in terms of frequency and interval of application by farmers in the study area. From the findings, 77.8% of farmers applied pesticides weekly, 5.6% and 16.7% apply pesticides monthly and seasonally respectively. 98.9% of famers apply pesticides on crops routinely with or without pests and or diseases on crops; and only 1.1% use spot spraying only when they see pests and or diseases on crops. In the terms of different weather conditions, the frequency, intensity and interval of pesticide application keep changing. During rain seasons, 47.8% of farmers apply pesticides every 4 days; 28.9% apply pesticides weekly; 15.6% apply pesticides every 5 days; 5.6% apply pesticides every 3 days and only 2.2% apply pesticides above 7 days. In dry seasons 55.6% apply pesticides in an interval above 7 days; 43.3% apply pesticides weekly and only 1.1% apply pesticides every 4 days. Table 7: Frequency of Pesticide application and application interval in particular weather conditions Page 17/36 Page 17/36 Page 17/36 Variable Response Frequency Percent How long have you been applying pesticides on your farm Years 85 94.4 Months 5 5.6 Total 90 100 By which method do you apply pesticides on your crop land Routine application 89 98.9 Spot application 1 1.1 Total 90 100 How often do you apply pesticides on the same crop during rainy season every 3 days 5 5.6 every 4 days 43 47.8 every 5 days 14 15.6 every 7 days 26 28.9 Above 7 days 2 2.2 Total 90 100 How often do you apply pesticides on the same crop during dry season every 4 days 1 1.1 every 7 days 39 43.3 Above 7 days 50 55.6 Total 90 100 Variable Information concerning farmer’s awareness in the use and handling of pesticides extension 1 1.1 Neighbours 1 1.1 Total 90 100 Have you ever received any training or knowledge about mixing pesticides Yes 7 7.8 No 83 92.2 Total 90 100 No 0 0 Total 90 100 How do you determine mixing rates Guessing 31 34.4 Reading instructions on the label 54 60 Consulting 4 4.4 Others (guessing) 1 1.1 Total 90 100 How do you determine mixing rates 70% of farmers interviewed (Table 9), determine the right pesticide application method by reading instructions on labels; 23.3% consult neighbors, 3.3% consult agro- dealers while 3.3% use guessing. For pesticide application frequency, 90% respondents use given sets of routine application frequencies (like twice a week, weekly, etc) with or with no pest or diseases appearing on crops; and 10% only determined application frequency by looking at presence or abundance of pests or diseases. 83.3% of farmers mix pesticides in their fields; 13.3% mixed pesticides at water sources and 3.3% mixed pesticides at their homes before application in the field. Mixing pesticides and applying them in the fields is done 100% by farmers themselves. Only 5.6% of farmers contacted accessed extension services and all the 94.4% never accessed extension services. 70% of farmers interviewed (Table 9), determine the right pesticide application method by reading instructions on labels; 23.3% consult neighbors, 3.3% consult agro- dealers while 3.3% use guessing. For pesticide application frequency, 90% respondents use given sets of routine application frequencies (like twice a week, weekly, etc) with or with no pest or diseases appearing on crops; and 10% only determined application frequency by looking at presence or abundance of pests or diseases. 83.3% of farmers mix pesticides in their fields; 13.3% mixed pesticides at water sources and 3.3% mixed pesticides at their homes before application in the field. Mixing pesticides and applying them in the fields is done 100% by farmers themselves. Only 5.6% of farmers contacted accessed extension services and all the 94.4% never accessed extension services. 84.4% (Table 9) bathe and clean spray equipments at home, 14.4% at water sources and 1.1% do it in the field, in the bush among others. 8.9% of the farmers contacted dispose pesticide containers by burying them, 28.9% by burning them, 2.2% by throwing them in water, 56.7% by throwing them in the bush and 3.3% by other means such as using them for home use or selling them. Information concerning farmer’s awareness in the use and handling of pesticides The level of farmer’s awareness in the use and handling of pesticides (Table 8) in the study area, indicate that; 91.1% of farmers apply pesticides on crops on a routine basis irrespective of the presence or absence of pest and or diseases; 8.9% applied pesticides when pests or diseases appear on the crops. Only 27.8% consult experts for knowledge on how to apply pesticides; 72.2% do not seek for any knowledge but rely on local experiences. 97.8% of farmers decide on their own to spray pesticides in their crop’s fields; 2.2% consult agriculture extension workers or neighbors regarding use of pesticides. Only 7.8% of farmers contacted received trainings in pesticide mixing and application knowledge; 92.2% do not receive any knowledge. Also 100% of the farmers mix pesticides themselves for application in their fields, irrespective of having been trained or not. Regarding mixing rates, 34.4% of farmers mixed pesticides by guessing; 60% by reading instructions on labels and 5.5% consult others (chemical dealers, extension workers, Neighbors and fellow farmers). Page 18/36 Page 18/36 Table 8: Farmer’s awareness in the use and handling of pesticides (1 of 3) Page 19/36 Variable Response Variable Response Frequency (#) Percent (%) Do you consult any expert on how to apply pesticides Yes 25 27.8 No 65 72.2 Total 90 100.0 Is Use of pesticides the only way/option to control pests and diseases Yes 80 88.9 No 10 11.1 Total 90 100 Do you use other pests control options other than pesticides Yes 10 11.1 No 80 88.9 Total 90 100 When do you always apply pesticides on your crops Routinely (seen or not seen the pests & or diseases on the crop) 82 91.1 Spot application (when insects or disease are seen on the crop) 8 8.9 Total 90 100 Do you consult any expert on how to apply pesticides Yes 25 27.8 No 65 72.2 Total 90 100 Who decides when and how to spray in regard to safe use of pesticides Self 88 97.8 Agric. Information concerning farmer’s awareness in the use and handling of pesticides Only 1.1% of famers calibrate spray equipment; all the 98.9% of the farmers do not calibrating their spray equipment. Table 9: Farmer’s awareness in the use and handling of pesticides (2 of 3) 9: Farmer’s awareness in the use and handling of pesticides (2 of 3) Table 9: Farmer’s awareness in the use and handling of pesticides (2 of 3) Page 21/36 Variable Variable Response Response Variable Response Frequency % How do you know the correct pesticide application method Self, by reading instructions on the labels 63 70 Consulting the agro dealers 3 3.3 Consulting neighbors 21 23.3 Others (guessing) 3 3.3 Total 90 100 How do you determine frequency of pesticide application Presence and abundance of pests and or disease 9 10 Routine with or no pest or disease present 81 90 Total 90 100 Where do you mix pesticides before spraying At home 3 3.3 At a water point or source 12 13.3 In the field 75 83.3 Total 90 100 Who sprays pesticides on your vegetable field Self 90 100 Special applicator 0 0 Total 90 100 Have you ever received pesticide extension services Yes 5 5.6 No 85 94.4 Total 90 100 Where do you bath, wash your clothes, and clean the equipments after spraying At home 76 84.4 At a water source 13 14.4 Else where 1 1.1 Total 90 100 How do you dispose of used containers of pesticides Bury them 8 8.9 Burn them 26 28.9 Throw them in water 2 2.2 Throw then a way in the bush 51 56.7 Throw them else where 3 3.3 How do you know the correct pesticide application method 3.3 Total 90 100 Where do you get pesticide equipment Own 40 44.4 Hiring 49 54.4 Other e.g. group equipment 1 1.1 Total 90 100 Do you calibrate your spray equipment before spraying Yes 1 1.1 No 89 98.9 Total 90 100 More than 95% of farmers determined application rates by guessing. 66.7% of farmers were able to know the expiry dates of the chemicals by reading the label, while 33.3% could not tell the expiry dates on labels. Only 5.6% of farmers had attended seminars and workshops regarding pesticide use; 94.4% did not. Although farmers used a lot of pesticides in their vegetable fields, (table 10), 75.6% of the farmers reported being aware that pesticides through runoffs could easily reach domestic water sources; only 24.4% were not aware. 83.3% of farmers had their fields located on hills; 16.7%, their vegetable fields were located in valleys where water sources were found. 100% of farmers reported all domestic water sources were located in valleys. Variable Table 10: Farmer’s awareness in the use and handling of pesticides (3 of 3) Table 10: Farmer’s awareness in the use and handling of pesticides (3 of 3 Page 23/36 Page 23/36 Variable Response Frequency (#) Percent (%) How do you determine the application rate of your equipment None 89 98.9 Consult agric. extension 1 1.1 Total 90 100 Where do you store the spray equipments and pesticides Separate store 2 2.2 In the house 83 92.2 Anywhere (in the field) 5 5.6 Total 90 100 Do you have any knowledge about expiry dates of pesticides Yes 60 66.7 No 30 33.3 Total 90 100 Have you attended seminars, Workshops, or Trainings on safe use of pesticides Yes 5 5.6 No 85 94.4 Total 90 100 Are you aware pesticides in farms can reach water sources through runoffs Yes 68 75.6 No 22 24.4 Total 90 100 Where is your vegetable field located On the hill 75 83.3 In the valley 15 16.7 Total 90 100 Where are your domestic water sources located In valleys 90 100 On hills 0 000 Total 90 100 Do you know some of the domestic water sources and where they are located Yes 90 100 No 0 000 Total 90 100 Discussion Since more than 80% of farmers relied only on pesticide spraying for management and control of pests and diseases and only less than 7% of them used other options in addition to pesticides spraying, imply that the rate of pesticide use in the study area of focus is so high; clearly reported by 100% of farmers who produce the different vegetable crops. This reveals that the overall levels of pesticide use in the area is so significant and the implication is that it could result in the residues accumulating in water sources and food chains, exceeding safe limits and causing overall ecosystem degradation. High reliance by farmers on chemical spraying as the major option for pest and diseases control, and use of numerous types of pesticides in their fields (Table 4), most times combined in one tank mix indicates there is a high rate and extent of pesticide use in the area; which puts a serious risk to the surrounding forms of biodiversity and entire ecosystem components, such as water resources by residual accumulation and pollution. Notwithstanding the risk of pest and disease resistance to pesticides, likely to result into more pesticides sprays due to continuous pest and disease reoccurrence which puts more risks of pesticide pollution to the environment and spillover effects through water movement, spray drifts and attachment to soil particles. High reliance by farmers on chemical spraying as the major option for pest and diseases control, and use of numerous types of pesticides in their fields (Table 4), most times combined in one tank mix indicates there is a high rate and extent of pesticide use in the area; which puts a serious risk to the surrounding forms of biodiversity and entire ecosystem components, such as water resources by residual accumulation and pollution. Notwithstanding the risk of pest and disease resistance to pesticides, likely accumulation and pollution. Notwithstanding the risk of pest and disease resistance to pesticides, likely to result into more pesticides sprays due to continuous pest and disease reoccurrence which puts more risks of pesticide pollution to the environment and spillover effects through water movement, spray drifts and attachment to soil particles. This study revealed that all farmers who were engaged in producing any vegetable crop did so with use of pesticides (insecticides, fungicides and herbicides) and in each case the usage rate was almost 100%. Variable Variable vegetable fields flowing to valleys during rains No 1 1.1 Total 90 100 vegetable fields flowing to valleys during rains 100 Discussion Results in Table 2 imply that respondents were able to give reasonable answers in regard to the issues asked under the survey, given categories of their education levels. A wide range of vegetables produced in the area of study focus indicated the level of diversity of vegetable crop production. The high average number of years of vegetable production indicated that farmers had vast knowledge and experience in vegetable farming. High yields motivated farmers for pesticides use, followed by high profit, implying that pesticides reduced risks of crop damage by insects and fungal diseases, the major threat to crop yield, quality and profit; therefore, farmers had a sound reason to use pesticides. In this study, 100% of farmers visited, faced a problem of pests and diseases in their fields and used pesticides to control them, implying that prevalence of pests and diseases was the driving force for farmers into pesticides use (Tables 3, 4, 5 and 6). Since majority of the farmers managed pests and disease problems by themselves imply that they used knowledge within their means, given that most of them did not receive trainings. Since more than 80% of farmers relied only on pesticide spraying for management and control of pests and diseases and only less than 7% of them used other options in addition to pesticides spraying, imply that the rate of pesticide use in the study area of focus is so high; clearly reported by 100% of farmers who produce the different vegetable crops. This reveals that the overall levels of pesticide use in the area is so significant and the implication is that it could result in the residues accumulating in water sources and food chains, exceeding safe limits and causing overall ecosystem degradation. In this study, 100% of farmers visited, faced a problem of pests and diseases in their fields and used pesticides to control them, implying that prevalence of pests and diseases was the driving force for farmers into pesticides use (Tables 3, 4, 5 and 6). Since majority of the farmers managed pests and disease problems by themselves imply that they used knowledge within their means, given that most of them did not receive trainings. Discussion The implication of this high rate of pesticide use therefore, is the high risk of pesticide residues accumulation of in water, air, soil particles and in the food chain, and high resistance of pests and diseases to pesticides which results into high prevalence of pests and diseases, resulting from mutant Page 25/36 Page 25/36 strains, causing changes and loses of biodiversity and alterations in ecosystem functions through changes in food webs and food chains. Farmers do not use any organic pesticides; it could be because they are not easily available on the market. Since the study revealed that some farmers were unable read pesticide labels to determine correct application rates, imply that excessive or lower rates were used by farmers. Common practices revealed were use of arbitrary measures like tablespoons and bottle tops. So, violation of pesticide application procedures was revealed, which results into high or low quantities of pesticides being applied. High pesticide quantities and application frequencies were a response to increased pest and disease problems, as in Okonya et al. (2015). Tomato was the heaviest consumer of pesticides at two routine sprays a week, which most farmers used. Farmers reported, they were not ready to give up the ‘insurance’ against losses, achieved by pesticide use, imply increased pesticide use in the area, moreover in Uganda, particular pesticides formulations were now within the purchasing power of more producers following the removal of the import tax on agricultural chemicals (Karungi et al., 2011). About 80% of farmers apply pesticides to their vegetable fields; weekly or twice a week means that most farmers apply pesticides more frequently and therefore, more pesticides were frequently pumped into the environment. So, effects of such pesticides (pollution, toxicity) were more likely to be exposed to the surrounding biodiversity and could be felt as both short- and long-term effects. Routine pesticide spraying practiced by more than 90% of the farmers, irrespective of pests and or disease presence, was an indication of incorrect application frequencies of pesticides (twice a week, so on), resulting in unnecessary excess amounts which potentially could increase pesticide residues and pollution in surrounding ecosystems such as water sources. Discussion It was therefore apparent that though spraying pesticides was necessary for increased productivity and quality, aspects of frequency, interval and intensity of pesticides use required desirable knowledge by farmers, since this was likely to cause havoc by accumulating pesticide residues in the different ecosystem components such as water sources, food chains, air among others, hence resulting in ecosystem and biodiversity imbalances. Water and food quality and safety are likely to be affected, and resistance to pesticides by pests and diseases duly likely to exist, if no strategies to regulate frequency, interval and intensity of pesticide application were put in place. The frequency, intensity and interval of pesticide application keep changing depending on the prevailing weather conditions. During the rainy seasons, different farmers apply pesticide at different intervals ranging from every 3 days, every 4 days, and every 5 days, weekly and above 7 days depending on the type of crop. In dry seasons application intervals ranging from every 4 days, weekly and above 7 days were reported by different farmers. Although both cases had some close intervals of pesticide application, rainy seasons had more pesticides applied to the fields than in the dry seasons. This means, in rainy seasons more pesticides residues accumulate in the ecosystem components such as water sources through run offs and percolation through soil particles, than in dry periods; meaning that more pesticide residues are most likely to be present in water during rainy seasons. Page 26/36 Given that tomatoes consumed more pesticides, in more close intervals both in wet and dry seasons, followed by onion and cabbages, meant that tomatoes, onions and cabbage contributed more pesticide residues to the ecosystem in the respective order. It was realized that synthetic pesticides were intensively used in vegetable farming (especially on Tomato, Onion and Cabbage). Use of pesticides in vegetable production in this study area of focus was dependent upon high pest infestations, prevalence of diseases, and the crop grown. Therefore the more the farmers apply pesticides more frequently (twice a week, weekly) imply that more pesticides are pumped into the environment, whose likely effects may include high residual accumulation and pollution in the ecosystem and serious biodiversity degradation which can have both short and long term effects to biotic elements. Discussion Other research findings have also shown heavy usage of pesticides on tomato in other countries in Africa; Ntow et al., (2006); Karungi 2011 working on tomato in Ghana indicated that farmers sprayed an average of 6–12 times a season, whereas it was 5–16 times or more per cropping season in Tanzania (Ngowi et al., 2007; Lekei et al., 2014; Karungi 2011. Such heavy use of pesticides implies that pesticide residues accumulation levels in the ecosystem components would raise higher, causing water quality loss, biodiversity imbalances, and frequent contact of farmers with pesticides, could lead to significant health problems. Fungicides are not easily observed to cause serious and acute damage to farmer's health but it has been reported that there is a long-term risk for cancer development and endocrine disruption resulting from farmer's exposure to fungicides containing mancozeb (Novikova et al., 2003; Mpala et al 2016), which can be by direct physical contact, contact through water and food. The dithiocarbamate family of fungicides is also suspected to have reproductive (Restrepo et al., 1990), and mutagenic effects in human cells exposed to it (Pretty and Waibel, 2012, Pretty, 2012). Findings also revealed the status quo of biotic constraints to vegetable production in the study area, was high and the easiest available pests and disease control measure was spraying synthetic pesticides. Fungicides were the most used because of fungal blights, especially Phytophthora, which were ever- present, and which would result in more than 75% crop losses if not checked; this was in line with Akemo et al. (2000) and Karungi et al. (2011). Farmers responded to the threat by effecting routine/calendar sprays with fungicides, majority of them spraying as often as twice a week. Although pesticides are useful in fostering the agriculture production, but when improperly used can pose dangerous risks to the entire ecosystem. Therefore, knowledge of the possible entry and effects of pesticides contaminants and pollution to the environment requires in-depth analysis of types of pesticides, application methods, and education levels of the users, pesticide handling and the trainings given to the users. Information concerning farmer’s awareness in the use and handling of pesticides Information concerning farmer’s awareness in the use and handling of pesticides Given that only 27.8% farmers seek for knowledge on how to apply pesticides, but majority farmers do not, coupled with inadequate trainings in pesticide use; implies that most farmers used pesticides Page 27/36 incorrectly, which increase risks of resultant excessive or inadequate pesticide quantities and might cause increased residual accumulation and pollution of the ecosystem or resistant strains of pests and diseases due to insufficient doses used. Self-decisions by farmers with less pesticide knowledge, implies incorrect use of pesticides resulting in contamination and pollution of the ecosystem components such as water sources. Farmers’ trainings in pesticide use and handling practices were so low compared to the high rate of pesticides use by farmers. However most farmers engaged in vegetable production attained some level of education (Table1), which would implies that, they are able to read instructions on pesticide labels to determine the right application methods, rates of application among others, this is not a guarantee as most farmers did not bother reading instructions while using pesticides. Bathing and cleansing of spray equipment in water by some farmers; imply a serious risk of reduced water quality due to contamination by pesticides which could build to toxic levels to cause short and or long-term effects. The implication of improper disposal of pesticide containers; for instance, when thrown in water, cause direct water contamination, whereas when thrown to bushes, may still find their way to different ecosystems including water causing contamination and accumulation of pesticide residues in water. . Some farmers were not able to identify expiry dates on pesticide labels, implying that they sometimes applied expired pesticides to their crops, a practice riskier to the crops, the environment and human health. This reflects that majority of farmers did not receive any knowledge from training workshop, seminar or awareness campaigns about pesticide use and handling practices implying high rate of incorrect pesticide usage in the study area, which translates into high accumulation of pesticides residues in the ecosystem, resulting into pollution of the environmental components. Some farmers were not able to identify expiry dates on pesticide labels, implying that they sometimes applied expired pesticides to their crops, a practice riskier to the crops, the environment and human health. Information concerning farmer’s awareness in the use and handling of pesticides and cleansing of the spray equipment in water sources. This is rel received pesticide extension services in respect to the high rate of and inadequate agricultural extension officers. It means, majority area apply pesticides with inadequate knowledge, hence improper used and may leads to excessive or inadequate quantities which m in ecosystem components like water and food chains to toxic leve pesticides which provoke farmers to spray excessively, inputting m environment. Majority of farmers used a lot of pesticides in their fields (Table 10), even when they knew that through runoffs pesticides easily reach water sources. This implies that something forced them to continuously use pesticide, perhaps monetary attachment or profit gains for livelihood. Since more than 80% of the farmers reported their vegetable gardens being located on the hill sides of the water sources and others had their vegetable fields located in the valleys where water sources were found; and 100% of them reported that water collecting sources were all located in the valleys, imply a high risk of contaminating water sources by pesticides from vegetable fields through runoffs, percolation through soil particles and spray drifts brought down by rain, hence increasing pesticide residue in water Farmers used arbitrary tank-mixtures of pesticides as a way to increase effectiveness or save on labour. A similar situation was reported from Tanzania where a study on pesticides usage in small-scale vegetable farms revealed that a third of the interviewed farmers applied pesticides in tank-mixtures (Ngowi et al., 2007; Jokha (2015). In all cases, there were no specific instructions either from the labels or extension workers regarding these tank mixtures. Mixing of pesticides by inexperienced farmers is not encouraged because the combinations used are indiscriminate. The practice defies some of the basic principles of pesticide management. For instance, Metcalfe et al., (2014) in his recommendation of strategies for pesticide management, states that the use of mixtures of pesticides must be avoided, since mixtures of pesticides generally result in the simultaneous development of resistance. Binhney (2001) working in Ghana attributed the increase in incidences of insect pest infestation of tomato after pesticide applications to indiscriminate combinations of pesticides, particularly of insecticides. Moreover, label instructions do not cover tank mixtures of pesticides and give no information on the compatibility of inert ingredients such as emulsifiers and wetting agents. It is riskier to mix two different types of formulations for example wettable powders with emulsifiable concentrates. Information concerning farmer’s awareness in the use and handling of pesticides This reflects that majority of farmers did not receive any knowledge from training workshop, seminar or awareness campaigns about pesticide use and handling practices implying high rate of incorrect pesticide usage in the study area, which translates into high accumulation of pesticides residues in the ecosystem, resulting into pollution of the environmental components. Majority farmers were not able to calibrate their spray equipment to determine right application rates; implying destruction of crops by excessive quantities or pest and or disease resistance to pesticides in case of low quantities which ultimately increases pest populations and continuous crop destruction by resistant disease causing agents. Since use of pesticides for pest management reported as the easiest farmer’s option, implied that more pesticides entered surrounding ecosystem components; however, actual amounts of pesticides used in Uganda were not known. In general, a significantly high use of pesticide on vegetables in the study area exists, implying that; increasing the acreage for vegetable growing potentially increases pesticide use in the area, which results in polluting the ecosystem. Farmers apply pesticides to crops sold to the market, improving quality for cash, and as vegetables increasingly become a staple crop in Uganda’s economy, farmers apply pesticides to safeguard themselves from yield loss. This high use of pesticides results in contamination of the environment including water resources. Since most farmers mixed pesticides in their fields which were reportedly on hills, with water sources in adjacent valleys, imply that water run offs during rains, from mixing grounds in the fields could end up in the nearby water sources posing a risk of water contamination by pesticides; but also those who reportedly mixed at the water sources, contaminated the water directly by spills during measurements Page 28/36 Page 28/36 and cleansing of the spray equipment in water sources. This is related to the low number of farmers who received pesticide extension services in respect to the high rate of pesticide use in the area (Table 18), and inadequate agricultural extension officers. It means, majority of the vegetable farmers in the study area apply pesticides with inadequate knowledge, hence improper application and handling practices used and may leads to excessive or inadequate quantities which may increase accumulation of residues in ecosystem components like water and food chains to toxic levels, or cause pest resistance to pesticides which provoke farmers to spray excessively, inputting more pesticide residues to the environment. Information concerning farmer’s awareness in the use and handling of pesticides Smit et al., (2002); Karungi et al., (2011), Nansen (2013) and Jokha (2015) observed that there was an interaction between fungicides, insecticides and water mineral content that influenced the efficacy of individual pesticide against fungal pathogens and insect mortality and some tank mixtures induced phytotoxicity on tomato. There is limited information on the reaction and effects of the mixtures being used in the case studies. In addition, farmers did not consider that unspecified tank mixing of pesticides could be less effective and cause adverse effects to their health or the environment. Instead, the tank mixing was carried out to save time, labour cost and with anticipation of high efficacy in pests and Page 29/36 Page 29/36 diseases control. Sherwood et al., (2005) also reported that potato farmers in Ecuador were mixing pesticides mainly to reduce costs associated with spraying. diseases control. Sherwood et al., (2005) also reported that potato farmers in Ecuador were mixing pesticides mainly to reduce costs associated with spraying. Findings in this study were also in agreement with findings from the case studies elsewhere in Sub Saharan Africa (Matthews et al., 2003; Ntow et al., 2006; Jokha 2015; Ngowi et al., 2007; Karungi et al., 2011) which show that internationally banned/restricted pesticides such as Carbofuran and Endosulfan are still being used by minimally educated farmers on horticultural crops. These pesticides pose a serious threat to the ecosystem. The problem is particularly widespread in sub-Saharan Africa, where the advent of liberalization of pesticides input markets has weakened quality control (FAO/WHO, 2001; Jokha 2015). Prior to liberalization, there were relatively few actors involved in pesticide provision, which made regulation and control simple. In Uganda, importation and distribution of pesticides and other agricultural inputs used to be conducted by the Government and its Parastatals, which had proper procedures for safe handling and distribution of pesticides. Entry of more firms into the market runs the risk of erosion of quality control and packaging standards, the breaching of national regulations and the unimpeded movement of banned or restricted chemicals across borders (Mudimu et al., 1995; Jokha 2015). It also raises concerns about the ability of regulatory agencies to control their activities, since it requires more vigorous scrutiny and screening of imports and monitoring of distribution and usage (Mudimu et al., 1995; Williamson, 2003; Jokha 2015) with huge financial and human resource implications for these agencies. Information concerning farmer’s awareness in the use and handling of pesticides Pesticide provision in a market-driven economy needs an effective regulatory framework in order to create full and fair competition, to protect the environment such as aquatic resources, to guarantee the quality of the products and to avoid the spread of pests and diseases (Shepherd and Farolfi, 1999; Jokha 2015). These are critical challenges for hard-pressed African regulators. Most of the pesticides on the Ugandan market in particular are pesticides that have been around for a long time within a limited range. On record in 1999, 190 pesticide formulations had been registered (includes insecticide, fungicides, and herbicides), a pittance if compared to developed countries like the United States of America; where over 50,000 had been registered by that period (Schaefers et al., 1999; Jokha 2015). This is a great disadvantage to pesticide users who have limited choice for safe pesticides. This discrepancy in accessibility to safer pesticides between countries at different levels of economic development poses a challenge in developing safer alternatives such as IPM systems in some developing countries. Safer pesticides are either inaccessible or outside the income bracket of small-scale farmers. For such countries to remain competitive in international export markets, the policy environment, particularly regarding registration of newer and safer agrochemicals must be more conducive. The improper disposal methods of the empty pesticide containers and plastic bags of pesticides imply poor handling. The containers and plastic bags being either thrown in the pit latrines, thrown away in the bush or in water bodies, left around the farms or open burned. This observation correlates with a study in Brazil which assess exposure to pesticides in which most respondents above 50% reported to leave empty pesticide containers within their fields (Araujo et al., 1999; Jokha 2015). Page 30/36 Page 30/36 Most pesticides have been classified as insecticides, acaricides, molluscides, nematicides, fungicides, rodenticides and herbicides. The common types include organophosphates (Bromophos, DDVP (Dichloro dimethyl vinyl phosphate), Diazinon, Dursban, Dimethoate, Malathion, Parathion), organochlorines (Aldrin, DDT (Dichloro diphenyl trichloroethane), Dieldrin, Lindane, Thiodan, Toxaphene), carbamates (Dithane M45, Dithane M22, Furadan), pyrethrins/pyrethroids (Ambush CY (Permethrin), Ripcord (Cypermethrin, Decamethrin), phenoxy acetic acid (2-4-D (Dichlorophenoxy acetic acid), 2-4-5-T (Trichlorophenoxy acetic acid), MCPA (Monochlorophenoxy acetic acid), inorganic metals (shell copper (copper oxide), lead arsenate arsenic trioxide, phenylmercuric acetate) and bipyridyls (Grammoxone (Paraquat), Weedol and Diquat). Vegetable farmers use a variety of these pesticides to control a host of pests, ranging from pre-harvest termites to post-harvest storage pests. Information concerning farmer’s awareness in the use and handling of pesticides While many are contact pesticides, systemic pesticides, which are potentially riskier because they penetrate living tissue, are also used. According to Jeyaratnam (1990) and Zare (2015), 80% of the Uganda’s population are involved in agriculture and a modest estimate of 3% of all agricultural workers in developing countries are affected by pesticide poisoning each year translating into over 700 000 cases in Uganda annually. Safety behaviors in pesticide use are considered the most important determinants of the adverse health effects among farmers, which meant that the risk of pesticide exposure was strongly associated with farmer’s behavior when working with pesticides (Sharifzadeh et al., 2019). However, there is limited understanding of farmer’s behavior and its determinants especially in developing countries like Uganda. The sampled farmers used different types of insecticides, and fungicides. Some of the farmers applied weedicides for field preparation for vegetables. Both insecticides and fungicides were used by all the farmers since insect and fungal attack was severe. Conclusion The study revealed that proper pesticides handling and application practices were not followed by most farmers in their vegetable fields. The consequence was accumulation of pesticide residues in water, crop produce and the general environment. This was triggered by lack of fundamental skills and knowledge of safe pesticide use, and safety equipment necessary for handling pesticides. The study also adduced that agricultural labourers are causing substantial harm to themselves, their communities and their environment, in their attempt to intensively grow different vegetables. The increasing use of pesticides demands for holistic measures towards preventing further accumulation of residue levels in the ecosystem, lest these become pollutants. Declarations Ethics approval and consent to participate Page 31/36 Ethics approval was sought from Office of the Dean, Faculty of Agriculture, Uganda Martyrs University. The approval notices also served as an introductory letter for seeking content to individual respondents and participants. The letter is included as supplementary material. Ethics approval was sought from Office of the Dean, Faculty of Agriculture, Uganda Martyrs University. The approval notices also served as an introductory letter for seeking content to individual respondents and participants. The letter is included as supplementary material. Funding We are grateful for the financial support provided by World Bank’s African Centre of Excellence in Agroecology and Livelihood Systems (ACALISE) at Faculty of Agriculture, Uganda Martyrs University. Availability of data and material Data will be availed on request. List of Abbreviations IPM – Integrated Pest Management DDVP - Dichloro dimethyl vinyl phosphate DDT --- Dichloro diphenyl trichloroethane MCPA -- Monochlorophenoxy acetic acid ACALISE -- African Centre of Excellence in Agroecology and Livelihood Systems Competing interests All authors declare that no competing interest exists. Consent for Publication Not applicable Authors' contributions ER: Conducted the study and drafted the manuscript Page 32/36 GS: Supervised the study, performed statistical analysis and co-drafted manuscript JB: Designed study tools and co-drafted manuscript GS: Supervised the study, performed statistical analysis and co-drafted manuscript JB: Designed study tools and co-drafted manuscript Acknowledgements We are grateful for the continuous and kind support, guidance and cooperation by the staff of the Faculty of Agriculture, Uganda Martyrs University, offered throughout the struggle, which led to the overall success of this study. References Akemo, M.C., Kyamanywa, S., Luther, G., Ssekyewa, C., Erbaugh, J.M. & Warren, H. 2000. Developing IPM systems for tomato in Central and Eastern Uganda. IPMCRSP sixth Annual Report. No. 6:117-121. Akemo, M.C., Kyamanywa, S., Luther, G., Ssekyewa, C., Erbaugh, J.M. & Warren, H. 2000. Developing IPM systems for tomato in Central and Eastern Uganda. IPMCRSP sixth Annual Report. No. 6:117-121. Amouh, C.N., 2011. A case study of health risk estimate for pesticide-users of fruits and vegetable farmers in Cameroon. Master in Bioscience Engineering, Ghent University, Belgium. Araujo, A.P., Telles, D.L., Gorni, R. and Lima, L.A. (1999). Endosulpfan residues in Brazilian tomatoes and their impact on public health and the environment. Bulletin of Environmental Contamination and Toxicology, 62:671-676. Binhney, P.M., 2001. Pesticide use pattern and insecticide residue levels in tomato (Lycopersicum esculentum) in some selected production systems in Ghana. MPhil Thesis, University of Ghana, Legon. Eyhorn, F., Roner, T. and Specking, H., 2015. Reducing pesticide use and risks-What action is needed?. Briefing Paper, Helvetas, pp.14-16 FAO/WHO (2001) Amount of poor-quality pesticides sold in developing countries alarmingly high. FAO/WHO press release, 1 February 2001. FAO/WHO (2001) Amount of poor-quality pesticides sold in developing countries alarmingly high. FAO/WHO press release, 1 February 2001. GFC (21 December 2015) “Road distance between Kampala and Kichwamba with map”. Globefeed.com (GFC). Retrieved 21 December 2015. GFC (21 December 2015) “Road distance between Kampala and Kichwamba with map”. Globefeed.com (GFC). Retrieved 21 December 2015. Google (December 2015) “Location of Uganda technical college Kichwamba at Google maps” (Map). Google Maps. Google. Retrieved 21 December 2015. Google (December 2015) “Location of Uganda technical college Kichwamba at Google maps” (Map). Google Maps. Google. Retrieved 21 December 2015. Page 33/36 Hicks B (2013) Agricultural pesticides and human health. In: National Association of Geoscience Teachers. Available from: http://serc.carleton.edu/NAGTWorkshops/health/case_studies/pesticides.html. Accessed Jan 13, 2014 Jeyaratnam, J., 1990. Acute pesticide poisoning: a major global health problem. World Health Statatistisc Quarterly, 43(3), pp.139-44. Jeyaratnam, J., 1990. Acute pesticide poisoning: a major global health problem. World Health Statatistisc Quarterly, 43(3), pp.139-44. Jokha, M.O.H.A.M.E.D., 2015. EFFECTS OF Agricultural pesticides and nutrients residue in Weruweru sub- catchment, Tanzania. Nairobi: Kenyatta University Jokha, M.O.H.A.M.E.D., 2015. EFFECTS OF Agricultural pesticides and nutrients residue in Weruweru sub- catchment, Tanzania. Nairobi: Kenyatta University Jørs, E., Neupane, D. and London, L., 2018. Pesticide poisonings in low-and middle-income countries. Jørs, E., Neupane, D. and London, L., 2018. Pesticide poisonings in low-and middle-income countries. Karungi, J., S. References Kyamanywa, E. Adipala, and M. Erbaugh. 2011. Pesticide utilization, regulation and future prospects in small scale horticultural crop production systems in a developing country, pesticides in the modern world - pesticides use and management, Dr. Margarita Stoytcheva (Ed.), ISBN: 978-953-307-459- 7, InTech, DOI: 10.5772/17170. Available at: http://www.intechopen.com/books/pesticides-in-the-modern- world-pesticides-use-andmanagement/pesticide-utilisation-regulation-and-future-prospects-in-small- scalehorticultural-crop-production-s [25 June 2014]. Karungi, J., S. Kyamanywa, E. Adipala, and M. Erbaugh. 2011. Pesticide utilization, regulation and future prospects in small scale horticultural crop production systems in a developing country, pesticides in the modern world - pesticides use and management, Dr. Margarita Stoytcheva (Ed.), ISBN: 978-953-307-459- 7, InTech, DOI: 10.5772/17170. Available at: http://www.intechopen.com/books/pesticides-in-the-modern- world-pesticides-use-andmanagement/pesticide-utilisation-regulation-and-future-prospects-in-small- scalehorticultural-crop-production-s [25 June 2014]. Lah K (2011) Effects of pesticides on human health. In: Toxipedia. Available from http://www.toxipedia.org/display/toxipedia/Effects+of+Pesticides+on+Human+Health. Accessed Jan 16, 2014 Lah K (2011) Effects of pesticides on human health. In: Toxipedia. Available from http://www.toxipedia.org/display/toxipedia/Effects+of+Pesticides+on+Human+Health. Accessed Jan 16, 2014 Mahmood, I., Imadi, S.R., Shazadi, K., Gul, A. and Hakeem, K.R., 2016. Effects of pesticides on environment. In Plant, soil and microbes (pp. 253-269). Springer, Cham. Mahmood, I., Imadi, S.R., Shazadi, K., Gul, A. and Hakeem, K.R., 2016. Effects of pesticides on environment. In Plant, soil and microbes (pp. 253-269). Springer, Cham. Matthews, G., Wiles, T., & Baleguel, P. 2003. A survey of pesticide application in Cameroon. Crop Protection 22: 707–714 Matthews, G., Wiles, T., & Baleguel, P. 2003. A survey of pesticide application in Cameroon. Crop Protection 22: 707–714 Maumbe, B.M. and Swinton, S.M., 2003. Hidden health costs of pesticide use in Zimbabwe's smallholder cotton growers. Social Science & Medicine, 57(9), pp.1559-1571. Mengistie, B.T., Mol, A.P., Oosterveer, P. and Simane, B., 2015. Information, motivation and resources: The missing elements in agricultural pesticide policy implementation in Ethiopia. International journal of agricultural sustainability, 13(3), pp.240-256. Metcalf RL, Changing role of insecticides in crop protection. Annu. Rev. Entomol. 25:119–256 (1980). Mudimu, G.D., Chigume, S. & Chikanda, M. 1995. Pesticide use and policies in Zimbabwe. Pesticide Policy Project Publication Series, No. 2. Hanover, Germany: Institut für Gartenbauökonomie, University of Hannover, Germany. Nansen, C. and Ridsdill-Smith, J.T., 2013. The performance of insecticides–a critical review. In Insecticides-Development of Safer and More Effective Technologies. IntechOpen. Page 34/36 Page 34/36 Page 34/36 Ngowi, A.V.F., Mbise, T.J., Ijani, A.S.M., London, L. and Ajayi, O.C., 2007. Pesticides use by smallholder farmers in vegetable production in Northern Tanzania. Crop Protection (Guildford, Surrey), 26(11), p.1617. Novikova, I.I., Litvinenko, A.I., Boikova, I.V., Yaroshenko, V.A. & Kalko, G.V. 2003. References How we can determine the sample size from an unknown population; University of Management and Technology (Pakistan) https://www.researchgate.net/profile/Syed_Ali284 Williamson, S. 2003. Pesticide provision in liberalised Africa: out of control? Agricultural Research and Extension Network. Network Paper No. 126. ISBN 0 85003 639 9. Zare, S., Behzadi, M., Tarzanan, M., Mohamadi, M.B., Omidi, L., Heydarabadi, A.B. and Kazemi, S., 2015. The impacts of pesticides on the health of farmers in Fasa, Iran. Electronic physician, 7(4), p.1168. References Biological activity of new microbiological preparations designed for plant protection against diseases of vegetable crops and potato, Mikol. Fitopatol. 37 (1) (2003), pp. 92–98 Ntow, W.J., Gijzen, H.J., Kelderman, P. and Drechsel, P., 2006. Farmer perceptions and pesticide use practices in vegetable production in Ghana. Pest Management Science: formerly Pesticide Science, 62(4), pp.356-365. Okello, J.J. and Swinton, S.M., 2010. From circle of poison to circle of virtue: pesticides, export standards and Kenya’s green bean farmers. Journal of agricultural economics, 61(2), pp.209-224. Okonya, J.S. and Kroschel, J., 2015. A cross-sectional study of pesticide use and knowledge of smallholder potato farmers in Uganda. BioMed research international, 2015. Pretty, J. and Waibel, H., 2012. Paying the price: the full cost of pesticides. In The pesticide detox (pp. 61- 76). Routledge. Pretty, J. ed., 2012. The pesticide detox: towards a more sustainable agriculture. Earthscan. Puz-y-Mino, C. Bustamente, G. Sanchez, M.E. & Leone, P.E. 2002. Cytogenetic monitoring in a population occupationally exposed to pesticides in Ecuador, Environ. Health Perspect. 110:1077–1080. Schaefers, G.A., Hedlund, R.C. & Kyamanywa, S. 1999. Report on pesticide use by strategic objective 1 partners and on Uganda’s pesticide regulatory status. Task Order No. 1648. Schaefers, G.A., Hedlund, R.C. & Kyamanywa, S. 1999. Report on pesticide use by strategic objective 1 partners and on Uganda’s pesticide regulatory status. Task Order No. 1648. Schaefers, G.A., Hedlund, R.C. & Kyamanywa, S. 1999. Report on pesticide use by strategic objective 1 partners and on Uganda’s pesticide regulatory status. Task Order No. 1648. Sharifzadeh, M.S., Abdollahzadeh, G., Damalas, C.A., Rezaei, R. and Ahmadyousefi, M., 2019. Determinants of pesticide safety behavior among Iranian rice farmers. Science of The Total Environment, 651, pp.2953-2960. Shepherd, A.W. & Farolfi, S. 1999. Export crop liberalization in Africa, FAO Agricultural Services Bulletin 135, Rome, 89pp. Sherwood, S. Cole, D., Crissman, C. & Paredes, M. 2005. From pesticides to people: improving ecosystem health in the northern Andes. In: J. Pretty, (Ed). The Pesticide Detox. towards a more sustainable agriculture, Earthscan, London, pp. 147–164. Page 35/36 Page 35/36 Page 35/36 Smit, Z.K., Indjic, D., Belic, S. & Miloradov, M., 2002. Effect of water quality on physical properties and biological activity of tank mix insecticide-fungicide spray, In: Paroussi, G., Voyiatzis, D., Paroussis, E. (Eds.), Proceedings of the Second Balkan Symposium on Vegetables and Potatoes (579), International Society Horticultural Science, Leuven, Belgium, pp., 551–556. Syed Ahmad Ali, 2017. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. QuestionnaireEziron30May2018.docx QuestionnaireEziron30May2018.docx QuestionnaireEziron30May2018.docx QuestionnaireEziron30May2018.docx Page 36/36
W4221061852.txt	https://hal-insu.archives-ouvertes.fr/insu-03810458v1/file/EGU22-9645-print.pdf	en		Variability of dissolved organic carbon (DOC) in the 6 largest Arctic rivers estimated using high resolution Sentinel-2 and Landsat-8 imageries over the 2013-2021 period.	null	2,022	cc-by	531	EGU22-9645 https://doi.org/10.5194/egusphere-egu22-9645 EGU General Assembly 2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Variability of dissolved organic carbon (DOC) in the 6 largest Arctic rivers estimated using high resolution Sentinel-2 and Landsat-8 imageries over the 2013-2021 period. Fabrice Jégou1, Gaëtane Jallais2, Elodie Salmon3, Bertrand Guenet4, Pierre-Alexis Herrault5, Sébastien Gogo6, Laure Gandois7, Christophe Guimbaud8, Fatima Laggoun-Defarge9, Nathalie Moulard11, Roman Teisserenc10, and Jean-Sébastien Moquet12 1 CNRS / Université d'Orléans, LPC2E, Orléans, France (fabrice.jegou@cnrs-orleans.fr) 2 LPC2E, Université d'Orléans, CNRS, CNES, Orléans, France (fabrice.jegou@cnrs-orleans.fr) 3 Laboratoire de Géologie, ENS, Paris, France (elodie.salmon@gmail.com) 4 Laboratoire de Géologie, ENS, Paris, France (guenet@biotite.ens.fr) 5 LIVE, Université de Strasbourg,France, CNRS (Pierre-alexis.herrault@live-cnrs.unistra.fr) 6 ECOBIO, Université Rennes 1, CNRS, Rennes, France (sebastien.gogo@univ-rennes1.fr) 7 Laboratoire d’Écologie Fonctionnelle et Environnement, CNRS, Castanet Tolosan, France (laure.gandois@ensat.fr) 8 LPC2E, Université d'Orléans, CNRS, CNES, Orléans, France (christophe.guimbaud@cnrs-orleans.fr) 9 ISTO, Université d'Orléans, CNRS, Orléans, France (Fatima.Laggoun-Defarge@univ-orleans.fr) Laboratoire d’Écologie Fonctionnelle et Environnement, CNRS, Castanet Tolosan, France (roman.teisserenc@toulouse- 10 inp.fr) 11 LPC2E, Université d'Orléans, CNRS, CNES, Orléans, France (nathalie.moulard@cnrs-orleans.fr) 12 ISTO, Université d'Orléans, CNRS, Orléans, France (jean-sebastien.moquet@cnrs-orleans.fr) Climate warming with permafrost thaw will modify lateral carbon export, from terrestrial to aquatic ecosystems with a potential huge impact on the Arctic rivers, draining organic-rich soils and in fine into the Arctic Ocean. The majority of annual DOC fluxes by Arctic rivers are transported during the snowmelt break-up period, which makes field measurements of DOC difficult. Passive spatial remote sensing is a very relevant tool to increase the spatial and temporal coverage of these observed values. In the framework of the French CNES DOC-Rivers project we proposed to apply the approach consisting in analyzing satellite imageries to evaluate DOC concentrations in the 6 great Arctic Rivers: Lena, Ob’, Yenisey, Yukon, MacKenzie, Kolyma. The algorithm, first, establishes a multilinear relationship between ground-based chromatic dissolved organic matter (CDOM) observations and specific satellite color bands to construct a complete satellite CDOM database. Another linear regression is used afterward with in-situ data from the Arctic Great Rivers Observatory (ArcticGRO) initiative to correlate CDOM and DOC observations. Using this second linear regression, we can predict the DOC content from the previous construct satellite CDOM database. River discharge measurements from the ArcticGRO database also enable to estimate the evolution of DOC export to the Arctic Ocean from satellite data. We applied this approach to high-resolution satellite data issued from Sentinel 2 (A 2015-2022, B 2017-2022) and Landsat 8 (2013-2022) to create a multi-instrumental synergy. This new database provides an unprecedented source of information for understanding DOC dynamics of in Arctic rivers and assessing its transfer from large catchments to the Arctic Ocean. This database provides information on the variability of DOC during the whole ice-free season and serve to locate areas with higher concentrations and fluxes during the 2013-2021 period. We plan to complement our database on future period with data from new satellite missions (Landsat 9, Sentinel 2C), on the present time with data from on-going missions (Sentinel 3, MODIS) and on past period with data from low resolution observations as Landsat 5 and Landsat 7. This extension of the database over a longer period of time will furnish insight in response to climate warming. Powered by TCPDF (www.tcpdf.org)
https://openalex.org/W4309212269	https://www.frontiersin.org/articles/10.3389/fncel.2022.998408/pdf	English	null	Single nuclei RNA sequencing investigation of the Purkinje cell and glial changes in the cerebellum of transgenic Spinocerebellar ataxia type 1 mice	Frontiers in cellular neuroscience	2,022	cc-by	17,761	Single nuclei RNA sequencing investigation of the Purkinje cell and glial changes in the cerebellum of transgenic Spinocerebellar ataxia type 1 mice OPEN ACCESS EDITED BY Marco Martina, Northwestern University, United States REVIEWED BY Josef P. Kapfhammer, University of Basel, Switzerland Giorgio Grasselli, University of Genoa, Italy CORRESPONDENCE Marija Cvetanovic mcvetano@umn.edu †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Cellular Neurophysiology, a section of the journal Frontiers in Cellular Neuroscience RECEIVED 01 August 2022 ACCEPTED 27 October 2022 PUBLISHED 15 November 2022 CITATION Ella Borgenheimer1†, Katherine Hamel1†, Carrie Sheeler1†, Francisco Labrada Moncada1, Kaelin Sbrocco1, Ying Zhang1,2 and Marija Cvetanovic1,3 Ella Borgenheimer1†, Katherine Hamel1†, Carrie Sheeler1†, Francisco Labrada Moncada1, Kaelin Sbrocco1, Ying Zhang1,2 and Marija Cvetanovic1,3* 1Department of Neuroscience, University of Minnesota, Minneapolis, MN, United States, 2Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN, United States, 3Institute for Translational Neuroscience, University of Minnesota, Minneapolis, MN, United States Glial cells constitute half the population of the human brain and are essential for normal brain function. Most, if not all, brain diseases are characterized by reactive gliosis, a process by which glial cells respond and contribute to neuronal pathology. Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disease characterized by a severe degeneration of cerebellar Purkinje cells (PCs) and cerebellar gliosis. SCA1 is caused by an abnormal expansion of CAG repeats in the gene Ataxin1 (ATXN1). While several studies reported the effects of mutant ATXN1 in Purkinje cells, it remains unclear how cerebellar glia respond to dysfunctional Purkinje cells in SCA1. To address this question, we performed single nuclei RNA sequencing (snRNA seq) on cerebella of early stage Pcp2-ATXN1[82Q] mice, a transgenic SCA1 mouse model expressing mutant ATXN1 only in Purkinje cells. We found no changes in neuronal and glial proportions in the SCA1 cerebellum at this early disease stage compared to wild-type controls. Importantly, we observed profound non-cell autonomous and potentially neuroprotective reactive gene and pathway alterations in Bergmann glia, velate astrocytes, and oligodendrocytes in response to Purkinje cell dysfunction. Borgenheimer E, Hamel K, Sheeler C, Moncada FL, Sbrocco K, Zhang Y and Cvetanovic M (2022) Single nuclei RNA sequencing investigation of the Purkinje cell and glial changes in the cerebellum of transgenic Spinocerebellar ataxia type 1 mice. Front. Cell. Neurosci. 16:998408. doi: 10.3389/fncel.2022.998408 © 2022 Borgenheimer, Hamel, Sheeler, Moncada, Sbrocco, Zhang and Cvetanovic. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). TYPE Original Research PUBLISHED 15 November 2022 DOI 10.3389/fncel.2022.998408 TYPE Original Research PUBLISHED 15 November 2022 DOI 10.3389/fncel.2022.998408 KEYWORDS cerebellum, SCA1, Purkinje cells, Bergmann glia, oligodendrocytes, velate astrocytes cerebellum, SCA1, Purkinje cells, Bergmann glia, oligodendrocytes, velate astrocytes KEYWORDS Introduction that glia undergo reactive gliosis in SCA1 mice and contribute to disease pathogenesis in a stage-of-disease dependent manner. Speciﬁcally, we found that glia are neuroprotective early and harmful late in SCA1 disease progression, thus suggesting that glial function could be a key therapeutic target for SCA1 (Kim et al., 2018). Our aim in this work was to investigate the underlying changes occurring in glia across the SCA1 cerebellum and, in doing so, to highlight key pathways that may be important for glial responses to PC dysfunction. To accomplish this, we used the transgenic SCA1 mouse model, Pcp2- ATXN1[82Q] line which expresses mutant ATXN1 only the cerebellar Purkinje cells (Burright et al., 1995). This allows us to speciﬁcally identify the non-cell autonomous pathways activated in glia by dysfunctional Purkinje cells without the confounding factor of gene expression changes driven by mutant ATXN1 expression within glial cells.” Glial cells play key roles required for normal brain function (Barres, 2008). These roles include maintaining homeostasis of ions, neurotransmitters, and water, providing neurotrophic and energy support, removal of unused synapses, and fast propagation of neuronal potentials (Kofuji and Newman, 2004; Parkhurst et al., 2013; Perea et al., 2014; Verkhratsky and Nedergaard , 2018). In most neurodegenerative diseases, glial cells undergo reactive gliosis: a process that includes gene expression, morphological, and functional changes (Burda and Sofroniew, 2014; Escartin et al., 2021). These glial changes have shown an active role in the pathogenesis of neurodegenerative diseases (Lobsiger and Cleveland, 2007; Sheeler et al., 2020). In some situations, reactive gliosis was beneﬁcial by delaying and ameliorating pathogenesis, but reactive glia may also be harmful and can exacerbate neuronal dysfunction and disease pathogenesis (Heneka et al., 2014; Pekny et al., 2014; Kim et al., 2018). The neuroprotective or harmful nature of reactive glia likely depends on many factors including the stage of disease progression, the signaling that triggers gliosis, and glial gene expression changes. To investigate gene expression changes at the cell-type level we used single-nuclei RNA sequencing. Bulk RNA sequencing has been useful for determining cerebellum-wide changes in the gene expression in SCA1 mouse models (Serra et al., 2004; Ingram et al., 2016a; Driessen et al., 2018). However, bulk RNA sequencing precludes the detection of transcriptional changes at the single-cell level and is also confounded by the possible change in the proportion of cell types (Klein et al., 2015; Lacar et al., 2016; Habib et al., 2017). Single nuclei RNA sequencing investigation of the Purkinje cell and glial changes in the cerebellum of transgenic Spinocerebellar ataxia type 1 mice The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Cellular Neuroscience frontiersin.org 01 10.3389/fncel.2022.998408 Borgenheimer et al. Frontiers in Cellular Neuroscience Introduction Previous studies have shown advantages of single nuclei over single cell RNAsequencing. These include preservation of a larger number of cells across multiple subtypes, while minimizing the eﬀects of cell dissociation on gene expression, and enriching for transcripts that are being actively transcribed in vivo (Lacar et al., 2016; Habib et al., 2017). Thus, we isolated nuclei from cerebella of SCA1 mice and wild-type controls, and used rigorous analysis to investigate gene expression changes in cerebellar Purkinje cells, and three types of cerebellar glia: Bergmann glia, velate astrocytes, and oligodendrocytes. Glia can become reactive in response to external and/or internal signaling. For instance, in all diseases, glia respond to neuronal dysfunction via external/non-cell autonomous signaling. Additionally, in some neurodegenerative diseases, mutant proteins are expressed within glial cells and can contribute to reactive changes in internal/cell-autonomous manner. Investigating internal (cell–autonomous) and external (non-cell-autonomous) reactive glial gene expression changes and their eﬀect on neurons will lead to a better understanding of the pathogenesis of neurodegenerative diseases. Spinocerebellar ataxia type-1 (SCA1) is a dominantly inherited neurodegenerative disorder caused by the abnormal expansion of CAG repeats in the Ataxin-1 (ATXN1) gene (Orr et al., 1993). Expansion of 39 or more CAG repeats drives severe pathology of cerebellar Purkinje cells and cerebellar gliosis. SCA1 symptoms include loss of balance and coordination, swallowing and speech diﬃculties, impairments in cognition and mood, and premature death (Orr and Zoghbi, 2007; Jacobi et al., 2013; Rüb et al., 2013; Moriarty et al., 2016; Diallo et al., 2017, 2019). While ATXN1-targeting antisense oligonucleotides (ASOs) show promise in pre-clinical trials (Friedrich et al., 2018), there are currently no disease-modifying treatments available for SCA1. The lack of available treatment options highlights the need for increased understanding of SCA1 pathogenesis. This is particularly relevant when considering early disease stages where the development of eﬀective therapies may lead to the delayed onset, reversal, or slowing of disease phenotypes. Nuclei isolation Nuclei for RNA sequencing were isolated using detergent mechanical lysis protocol as previously described (Matson et al., 2018). Brieﬂy, frozen or fresh whole cerebellum was placed into 1.5mL tube with 500uL pre-chilled detergent lysis buﬀer [low sucrose buﬀer (LSB) (sucrose 0.32M, 10 mM HEPES, 5mM CaCl2, 3mM MgAc, 0.1mM EDTA, 1mM DTT) with 0.1% Triton-X] and the tissue was homogenized using a mechanical homogenizer. A 40um strainer was placed over a pre-chilled 50mL tube and pre-wetted with 1ml LSB. 1mL of LSB was added to the tube containing the crude nuclei in the lysis buﬀer and mixed gently by pipetting (2-3 times). Crude nuclei prep was next passed over the 40uM strainer into the pre-chilled 50mL tube, washed with 1 ml LSB, and centrifuged at 3,200 g for 10 min at 4C. For identiﬁed cell types of Bergmann Cells, Velate Astrocytes, Granule Neurons, Purkinje Neurons, and Oligodendrocytes, scran (v 1.20.1) was used to normalize the single cell expression matrix, followed by limma-trend (v 3.48.3) to test for diﬀerential gene expression. Sample batches were considered as an independent factor in the design matrix of the statistical test (design = ∼genotype + batch). P values were adjusted using Benjamini-Hockberg method. Diﬀerential gene expression was determined by an adjusted p-values of 0.05. The lists of diﬀerentially expressed genes from diﬀerent cell types were further analyzed for GO term enrichment using ClusterProﬁler (v 4.0.5). Detailed information on the code we used can be found at https://github.com/yingzhang121/snSeq_SCA1. The pellet was resuspended in 3mL of LSB while gently swirling to remove the pellet from the wall, facilitating resuspension, and left on ice for 2 min. The suspension was transferred to an Oak Ridge tube and nuclei were homogenized in LSB for 15-30 s, all while keeping the sample on ice. Using a serological pipette, 12.5mL of density sucrose buﬀer (sucrose 1M, 10 mM HEPES, 3mM MgAc, 1mM DTT) was layered underneath the LSB homogenate, taking care not to create a bubble which would disrupt the density layer. Samples were then centrifuged at 3,200 g for 20 min at 4C and pelleted nuclei were resuspended in the resuspension solution (PBS, 0.4 mg/ml BSA, 0.2 U/µl RNAse inhibitor). Nuclei were ﬁltered through a 40um pore-size strainer followed by a 30um and 20um pore-size strainers. Mice The creation of the Pcp2-ATXN1[82Q] mice was previously described (Burright et al., 1995). We have backcrossed these mice onto the C57BL/6 background for 10 generations. As CAG repeats are unstable and tend to shrink in mice, we periodically sequence the CAG region to evaluate number of repeats (Gatchel and Zoghbi, 2005). At the time of experiments the average number of CAG repeats in our colony was 71. We used 12 week old Pcp2-ATXN1[82Q] mice and their littermate wild-type control mice. Animal experimentation was approved by the Institutional Animal Care and Use Committee (IACUC) of University of Minnesota and was conducted in accordance with the National Institutes of Health’s (NIH) Previous studies used mouse models of SCA1 to demonstrate that mutant ATXN1 causes gene expression changes in Purkinje cells. In addition, we have previously shown 02 frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. Principles of Laboratory Animal Care (86–23, revised 1985), and the American Physiological Society’s Guiding Principles in the Use of Animals (National Research Council (Us) Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011). platform, using 1 full lane of S4 chip each time. Sequencing depth ranges from ∼41,000 to ∼120,000 reads per nuclei. Raw, demultiplexed fastq ﬁles were analyzed by CellRanger (v5.0.1) using reference genome mm10 (refdata-gex-mm10- 2020-A, with the addition of human ATXN1 gene locus), with the option that allows alignment to un-spliced pre-mRNAs (– include-introns). The raw UMI count matrix was cleaned via DIEM algorithm (Alvarez et al., 2020). In summary, DIEM models cell debris using cells with less than 1,000 UMI detected (min_counts = 1,000). In case of overﬁtting, we increased the threshold to 3,000 (only for the two samples sequenced in the ﬁrst NovaSeq run). Then the debris scores were manually inspected to exclude true debris. The cleaned gene count table per donor was analyzed with R (v4.1.0) package Seurat (v 4.0.4) for cell type clustering and visualization. Further cell type identiﬁcation was accomplished with SingleR (v 1.6.1) using DropViz (Saunders et al., 2018). Cerebellum MetaCells reference. We assigned cell types by requiring the same Seurat cluster and the same SingleR annotation. Immunoﬂuorescent staining IF was performed on a minimum of six diﬀerent ﬂoating 45- µm-thick brain slices from each mouse (six technical replicates per mouse per region or antibody of interest) as we have previously described (Sheeler et al., 2021). We used primary antibody against DNER (Invitrogen, PA5-99872). Confocal images were acquired using a confocal microscope (Olympus FV1000, Leica Stellaris 8) using a 20X oil objective. Z-stacks consisting of twenty non-overlapping 1-µm-thick slices were Nuclei isolation A small sample of nuclei was pelleted and added to a slide with VectaShield with DAPI to verify single nuclei isolation under ﬂuorescent microscope (Supplementary Figure 1). The nuclear suspensions were processed by the Genomic Core at the University of Minnesota using 10X Chromium 3′ GEX Capture to Library Preparation (Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 with Single Cell 3′ v3.1 Gel Beads and Chromium Next GEM Chip G). Visualization of the results was generated using various R packages, including Seurat, ClusterProﬁler, enrichplot (v 1.12.2), and ggplot2 (v 3.3.5). Data is deposited at https://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE215336. To review GEO accession GSE215336 before it becomes public (when results are accepted for publication) please enter token ibqzeqsivrodhkr into the box. Frontiers in Cellular Neuroscience Sequencing and analysis 47,894 high-quality, snRNAseq proﬁles were imported into Seurat for clustering analysis and nuclei were classiﬁed into the cell types with SingleR (v 1.6.1) using DropViz Cerebellum MetaCells reference. (C) Relative proportions of cell types in WT and SCA1 mice. Average percentages of Purkinje cells (PC), Bergman Glia (BG), velate astrocytes (VA), and oligodendrocytes in Pcp2- ATXN1[82Q] mice and wild-type littermate controls (N = 3 of each) were calculated as percentage of total cells. Data is presented as mean ± SEM with average values for each mouse represented by a dot. Two-way ANOVA with Sidak’s multiple comparison did not detect any signiﬁcant difference between Pcp2- ATXN1[82Q] mice and wild-type littermate controls (Supplementary Table 2). (D) Total number of differentially expressed genes (DEG) in each cell type. P values were adjusted using Benjamini-Hockberg method. Signiﬁcant differential gene expression was determined by an adjusted p-values ≤0.05. Sequencing and analysis Library quality control was performed using the MiSeq system to estimate average numbers of nuclei per donor mouse. Then all nuclei from 6 donors were multiplexed and sequenced on two independent runs on the Illumina NovaSeq Frontiers in Cellular Neuroscience 03 frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. FIGURE 1 Experimental schematics, cell composition and DEGs per cell type in Pcp2-ATXN1[82Q] mice. (A) Schematic pipeline of SCA1 snRNA-seq and analysis of mouse (N = 6 mice) cerebellum (WT N = 3, Pcp2- ATXN1[82Q] SCA1 N = 3). Individual nuclei were isolated from the cerebella of 12 weeks old Pcp2- ATXN1[82Q] mice and wild-type littermate controls (N = 3 of each). After passing library QC nuclei were sequenced on Illumina NovaSeq platform. (B) Normalized violin plots showing expression of cell type-speciﬁc marker genes for each mouse cluster to evaluate cell type annotation. 47,894 high-quality, snRNAseq proﬁles were imported into Seurat for clustering analysis and nuclei were classiﬁed into the cell types with SingleR (v 1.6.1) using DropViz Cerebellum MetaCells reference. (C) Relative proportions of cell types in WT and SCA1 mice. Average percentages of Purkinje cells (PC), Bergman Glia (BG), velate astrocytes (VA), and oligodendrocytes in Pcp2- ATXN1[82Q] mice and wild-type littermate controls (N = 3 of each) were calculated as percentage of total cells. Data is presented as mean ± SEM with average values for each mouse represented by a dot. Two-way ANOVA with Sidak’s multiple comparison did not detect any signiﬁcant difference between Pcp2- ATXN1[82Q] mice and wild-type littermate controls (Supplementary Table 2). (D) Total number of differentially expressed genes (DEG) in each cell type. P values were adjusted using Benjamini-Hockberg method. Signiﬁcant differential gene expression was determined by an adjusted p-values ≤0.05. FIGURE 1 Experimental schematics, cell composition and DEGs per cell type in Pcp2-ATXN1[82Q] mice. (A) Schematic pipeline of SCA1 snRNA-seq and analysis of mouse (N = 6 mice) cerebellum (WT N = 3, Pcp2- ATXN1[82Q] SCA1 N = 3). Individual nuclei were isolated from the cerebella of 12 weeks old Pcp2- ATXN1[82Q] mice and wild-type littermate controls (N = 3 of each). After passing library QC nuclei were sequenced on Illumina NovaSeq platform. (B) Normalized violin plots showing expression of cell type-speciﬁc marker genes for each mouse cluster to evaluate cell type annotation. Frontiers in Cellular Neuroscience Statistics Wherever possible, sample sizes were calculated using power analyses based on the standard deviations from our previous studies, signiﬁcance level of 5%, and power of 90%. For RNA sequencing data, limma-trend (v 3.48.3) was used to test for diﬀerential gene expression. Sample batches were considered as an independent factor in the design matrix of the statistical test (design = ∼genotype + batch). P values were adjusted using Benjamini-Hockberg method. Diﬀerential gene expression was determined by an adjusted p-values of 0.05. For all other results, statistical tests were performed with GraphPad Prism 7.0 (GraphPad Software Inc.). Data was analyzed using unpaired Student’s t-test with Welch’s correction (RTqPCR of cerebellar mRNA from wild-type and Pcp2-ATXN1[82Q] mice in Supplementary Figures 2, 4A, 6B, and intensity of DNER staining in Supplementary Figure 4D), and two-way ANOVA (cell type and genotype) followed by the Sidak’s multiple comparison test (relative abundance of each cell type in wild-type and Pcp2-ATXN1[82Q] mice, Figure 1C) or Tukey’s multiple comparison test (percentage of cerebellar cell types expressing endogenous mouse Atxn1[2Q] or transgenic human ATXN1[82Q] in wild-type and Pcp2-ATXN1[82Q] mice, Supplementary Figure 3). Outliers were determined using GraphPad PRISM’s Robust regression and Outlier removal (ROUT) with a Q = 1% for non-biased selection. Raw data and exact p values are provided in Supplementary Tables 1–4. To investigate the gene expression changes attributable to speciﬁc cell types, we isolated individual nuclei from the cerebella of six 12 week old SCA1 and littermate control mice (Figure 1A and Supplementary Figure 2). Nuclei were captured and barcoded followed by single nuclei RNA sequencing (snRNAseq, Figure 1A; Klein et al., 2015; Zilionis et al., 2017). After removing debris nuclei using semi-supervised machine learning classiﬁer Debris Identiﬁcation using Expectation Maximization (DIEM) (Alvarez et al., 2020), we identiﬁed a total of 47,894 high-quality, snRNAseq proﬁles. They were imported into Seurat for clustering analysis and visualization. We then classiﬁed nuclei into the cell types with SingleR (v 1.6.1) using DropViz Cerebellum MetaCells reference (Saunders et al., 2018; Figure 1B). First, we wanted to determine whether cellular composition of the cerebellum is altered in SCA1, e.g., whether expression of mutant ATXN1 in Purkinje cells leads to the relative increase or decrease in the numbers of the main cerebellar cell types, including Purkinje cells (PCs), Bergmann glia (BG), velate astrocytes (VA) and oligodendrocytes (OL). Reverse transcription and quantitative polymerase chain reaction (RTqPCR) taken of each stained brain slice per brain region (i.e., six z-stacks per mouse, each taken from a diﬀerent brain slice). The laser power and detector gain were standardized and ﬁxed between mice, and all images for mice within a cohort were acquired in a single imaging session to allow for quantitative comparison. To quantify relative intensity of staining we measured the average signal intensity in the region of interest that included PCs soma and dendrites. Total RNA was extracted from dissected mouse cerebella, medulla, and hippocampus using TRIzol (Life Technologies), and RT-qPCR was performed as described previously (Lobsiger and Cleveland, 2007). We used IDT Primetime primers for the following genes: Ptch2, Dner and Gli. Relative mRNA levels were 04 frontiersin.org 10.3389/fncel.2022.998408 10.3389/fncel.2022.998408 Borgenheimer et al. calculated using 18S RNA as a control and wild-type mice as a reference using 2−11Ct as previously described (Lobsiger and Cleveland, 2007). Previous bulk RNA sequencing studies showed signiﬁcant gene expression changes underlying PC dysfunction in the cerebella at 12 weeks, but no detectable PC death (Ingram et al., 2016b; Mellesmoen et al., 2018). We have conﬁrmed that many of these genes, which are considered representative of Purkinje cell pathology in SCA1 by the ﬁeld, are changed in cerebella of our mice at this time point. These include Calbindin 1(Calb1), Purkinje cell protein 4 (Pcp4), Regulator of G protein signaling (Rgs8), inositol 1,4,5-trisphosphate receptor (ITPR), inositol polyphosphate-5-phosphatase (Inpp5) and GTPase Activating Rap/RanGAP Domain Like 3 (Garnl3) (Ingram et al., 2016b). All of these genes were signiﬁcantly downregulated in our 12 week old mice, indicating that molecular aspects of mutant ATXN1 on PCs were easily detectable in bulk tissue (Supplementary Figure 2 and Supplementary Table 1). Statistics We calculated the percentage of each of these cell types by dividing the number of nuclei identiﬁed as a speciﬁc cell type by the total number of nuclei. Comparing Pcp2-ATXN1[82Q] and wild-type mice, we have not found statistically signiﬁcant diﬀerence in the relative percentages of Purkinje cells (0.55 ± 0.068 vs. 0.53 ± 0.052), Bergmann glia (2.39 ± 0.16 vs. 2.52 ± 0.43), velate astrocytes (2.26 ± 0.28 vs. 1.95 ± 0.19), nor oligodendrocytes (2.91 ± 0.12 vs. 2.85 ± 0.35) indicating that at this early disease stage we cannot detect a change in the cerebellar cell type composition in Pcp2-ATXN1[82Q] mice (Figure 1C and Supplementary Table 2). Frontiers in Cellular Neuroscience Selective expression of mutant ATXN1 in Purkinje cells causes signiﬁcant gene expression changes in cerebellar glia Most gene expression studies to date have focused on understanding how mutant ATXN1 aﬀects Purkinje cells. Here we conﬁrmed that many of the genes considered representative of Purkinje cell-speciﬁc pathology are indeed uniquely changed in Purkinje cells. In addition, we investigated gene expression changes in BG, VA and OL in response to PC pathology. Using diﬀerential gene expression (DEG) analysis, we identiﬁed genes altered in each of these four cell types. We found that each cell type is uniquely aﬀected by ATXN1-driven PC dysfunction and demonstrate diﬀerent numbers of DEGs (Figure 1D). Remarkably, BG and PCs had a comparable number of DEGs (575 and 406, respectively) at 12 weeks despite the fact that mutant ATXN1 is expressed only in PCs in these mice. These signiﬁcant non-cell autonomous transcriptional alterations in BG may suggest that BG are highly sensitive to perturbation in PCs. Additionally, VA and OL had a notable number of DEGs (157 and 155, respectively, Figure 1D). These results indicate that BG, VA and OL all respond to mutant ATXN1 induced dysfunction in PCs. We also found increased expression of synaptic vesicle genes encoding for vacuolar V-type ATP-ase subunits (Atp6v0c, Atpgv0a1, and Atp6v1h) that provide energy in the form of ATP to fuel transport of neurotransmitters into synaptic vesicles and Solute carrier family 32a1 (Slc32a1), encoding vesicular GABA transporter VGAT (Vesicular GABA and Glycine Transporter). Increased expression of genes providing energy for GABA transport into vesicles as well as increased expression of vesicular GABA transporter could potentially result in higher GABA content per vesicle and increased GABA release from SCA1 PCs. Previous work has demonstrated marked changes in PC ﬁring in SCA1 which indicate dysfunctions in signaling and cellular physiology (Bushart et al., 2021). In keeping with this, we observed a proportion of gene expression changes suggestive of PCs reduced ability to respond to stimuli. Mutant ATXN1 expression in Purkinje cells does not alter proportions of cerebellar cells In this study, we sought to determine the non-cell autonomous reactive gene expression changes in cerebellar glia in response to mutant ATXN1-driven Purkinje cell dysfunction. To ensure that glial cells are aﬀected solely in response to neuronal dysfunction and not because of cell-autonomous expression of expanded ATXN1, we have used a Purkinje cell speciﬁc transgenic mouse model of SCA1, Pcp2-ATXN1[82Q] mice (Burright et al., 1995). In these mice, mutant ATXN1 with 82 CAG repeats is selectively expressed under Purkinje cell protein 2 (Pcp2) promoter in cerebellar Purkinje cells. We chose to examine glial changes at 12 weeks of age to capture an early stage of SCA1 pathology when disease is still reversible (Zu et al., 2004) and as such therapeutically noteworthy. Additionally, we determined which cerebellar cells express wild-type mouse and mutant human ATXN1. We found that endogenous wild-type mouse Atxn1[2Q] is expressed in PCs, BG, VA, and OL (Supplementary Figure 3A and Supplementary Table 3). Furthermore, mutant human ATXN1[82Q] is expressed only in PC cells in Pcp2- ATXN1[82Q] mice, conﬁrming the Purkinje cell expression speciﬁcity of this Frontiers in Cellular Neuroscience 05 frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. line (Supplementary Figure 3B and Supplementary Table 4). Additionally, no mutant human ATXN1 expression was found in any cell type in wild-type littermate control mice. line (Supplementary Figure 3B and Supplementary Table 4). Additionally, no mutant human ATXN1 expression was found in any cell type in wild-type littermate control mice. prevalence of downregulated genes is consistent with previous results and is generally thought to reﬂect the direct repressive eﬀect of mutant ATXN1 on PC gene transcription (Ingram et al., 2016b). Among the top ten upregulated genes were genes known for their roles in neurodevelopment including Heparan Sulfate 6-O-Sulfotransferase 3 (Hs6st3), Adhesion G Protein-Coupled Receptor L3 (Adgrl3), Contactin 5 (Cntn5), and Neuregulin 1 (Nrg1) (Mei and Xiong, 2008; Cocchi et al., 2016; El Hajj et al., 2017; Moon and Zhao, 2021). In addition, SCA1 PCs had increased expression of RALY RNA Binding Protein-like (Ralyl),which is known as an Alzheimer’s disease cognitive reserve gene (Zhang et al., 2020) (Figures 2A–C). Frontiers in Cellular Neuroscience Selective expression of mutant ATXN1 in Purkinje cells causes signiﬁcant gene expression changes in cerebellar glia This is indicated by the downregulated expression of genes encoding extracellular matrix proteins (Mucin3a (Muc3a), Collagen type 5 alpha1 (Col5a1), Collagen Type 8 Alpha 1 (Col18a) (Tsai et al., 2021), proteins involved in Wnt and GPCR signaling (leucine rich repeat containing G protein-coupled receptor 5 (Lgr5) that enhances Wnt signaling and phosphodiesterase 10 (Pde10) that plays a role in signal transduction by regulating the intracellular concentration of cyclic nucleotides cAMP and cGMP) (Carmon et al., 2012), signaling scaﬀolding protein Breast cancer anti-estrogen resistance protein 1 (Bcar1) (Furuichi et al., 2011; Maria del Pilar et al., 2017), and small brain speciﬁc non-coding RNA BC1 that regulates dendritic translation (Zhong et al., 2009; Robeck et al., 2016). This perturbed communication of SCA1 PCs is further supported by altered expression of genes involved in calcium and glutamatergic signaling. These downregulated genes encode for calcium voltage gated channels Cav2.1 and Cav3.1 (calcium voltage- gated channel subunit alpha1 A (Cacna1a), Cacna2d2, and Cacna1g), calcium endoplasmic reticulum pumps SERCA1 and SERCA2 (ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2 (Atp2a2) and Atp2a3), ionotropic glutamate receptors (glutamate ionotropic receptor AMPA type subunit 2 (Gria2), Gria3, glutamate Ionotropic Receptor Delta Type Subunit 2 (Grid2), Grid2 interacting protein(Grid2ip), Single-nuclei analysis conﬁrms previously identiﬁed gene expression changes in Purkinje cells and identiﬁes novel and potentially compensatory genes Single-nuclei analysis conﬁrms previously identiﬁed gene expression changes in Purkinje cells and identiﬁes novel and potentially compensatory genes Previous studies described diﬀerent sub-populations of PCs based on gene expression proﬁles and cellular physiology (Zhou et al., 2014; Kozareva et al., 2020). Moreover, several studies have indicated that SCA1 pathology is not uniform across the cerebellum (White et al., 2021). However, most of our understanding of mutant ATXN1 gene expression changes in Purkinje cells is derived from bulk RNA sequencing analysis. This prevents any identiﬁcation of altered pathology in diﬀerent sub-types of Purkinje cells which may be more vulnerable or disease resistant (Serra et al., 2004; Ingram et al., 2016b). To address this, we analyzed gene expression changes in PCs and compared the results to previously reported transcriptional alterations in SCA1. We identiﬁed 575 DEGs in Purkinje cells (false discovery rate adjusted p value p.adj/q < 0.05), 56% of which were downregulated and 44% upregulated (Figure 2A). This 06 frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. FIGURE 2 Single nuclei analysis identiﬁes SCA1 disease associated transcriptional changes in Purkinje cells. For identiﬁed Purkinje Neurons, limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in PCs. (B) Volcano plot showing DEGs of PC cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated PC DEGs in wild-type and Pcp2-ATXN1[82Q] samples determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] Purkinje cells. (E) Dot plot of the differential expression of Magenta genes in Purkinje cells, Bergmann glia, velate astrocytes and oligodendrocytes with color of dot depicting expression and size of the dot depicting percentage of cells. All selected genes had adjusted p-value ≤0.05. (F) Pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] PCs at 12 weeks of age. The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). FIGURE 2 Single nuclei analysis identiﬁes SCA1 disease associated transcriptional changes in Purkinje cells. For identiﬁed Purkinje Neurons, limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Single-nuclei analysis conﬁrms previously identiﬁed gene expression changes in Purkinje cells and identiﬁes novel and potentially compensatory genes Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in PCs. (B) Volcano plot showing DEGs of PC cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated PC DEGs in wild-type and Pcp2-ATXN1[82Q] samples determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] Purkinje cells. (E) Dot plot of the differential expression of Magenta genes in Purkinje cells, Bergmann glia, velate astrocytes and oligodendrocytes with color of dot depicting expression and size of the dot depicting percentage of cells. All selected genes had adjusted p-value ≤0.05. (F) Pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] PCs at 12 weeks of age. The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). FIGURE 2 Single nuclei analysis identiﬁes SCA1 disease associated transcriptional changes in Purkinje cells. For identiﬁed Purkinje Neurons, limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in PCs. (B) Volcano plot showing DEGs of PC cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated PC DEGs in wild-type and Pcp2-ATXN1[82Q] samples determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] Purkinje cells. (E) Dot plot of the differential expression of Magenta genes in Purkinje cells, Bergmann glia, velate astrocytes and oligodendrocytes with color of dot depicting expression and size of the dot depicting percentage of cells. All selected genes had adjusted p-value ≤0.05. (F) Pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] PCs at 12 weeks of age. The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). Single nuclei analysis identiﬁes SCA1 disease associated transcriptional changes in Purkinje cells. For identiﬁed Purkinje Neurons, limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. Frontiers in Cellular Neuroscience Single-nuclei analysis conﬁrms previously identiﬁed gene expression changes in Purkinje cells and identiﬁes novel and potentially compensatory genes N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in PCs. (B) Volcano plot showing DEGs of PC cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated PC DEGs in wild-type and Pcp2-ATXN1[82Q] samples determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] Purkinje cells. (E) Dot plot of the differential expression of Magenta genes in Purkinje cells, Bergmann glia, velate astrocytes and oligodendrocytes with color of dot depicting expression and size of the dot depicting percentage of cells. All selected genes had adjusted p-value ≤0.05. (F) Pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] PCs at 12 weeks of age. The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). 07 Frontiers in Cellular Neuroscience frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. RT-qPCR, we conﬁrmed decrease in Dner mRNA expression in SCA1 cerebella (41% decrease compared to WT controls, Supplementary Figure 4A and Supplementary Table 1). Furthermore, using immunohistochemistry, we found reduced levels of DNER protein in soma and dendrites of Purkinje cells in Pcp2-ATXN1[82Q] mice (0.679 ± 0.006 vs. 0.553 ± 0.041, ∼19% decrease compared to WT controls, Supplementary Figures 4B–D and Supplementary Table 1). glutamate ionotropic receptor NMDA type subunit 2A (Grin2a), and potassium channels (potassium Voltage-Gated Channel Subfamily C Member 3 (Kcnc3), associated with SCA13, and potassium voltage-gated channel subfamily A regulatory beta subunit 1 (Kcnab1) (Matsuda et al., 2006; Figures 2C–D). These results are consistent with previous studies implicating ion channel, calcium and potassium dysregulation and synaptic dysfunction in SCA1 (Bushart and Shakkottai, 2018). Notably, several of these genes have also been associated with other ataxias including Spinocerebellar ataxia autosomal recessive 18 (Grid2) and Episodic Ataxia Type 1 (Kcnab1). Using hierarchical enriched pathways analysis of up and downregulated genes in PCs we identiﬁed pathways involved in synapse function, including synapse organization, cell junction assembly, regulation of synapse structure or activity, synapse assembly, and regulation of neurotransmitter levels (Figure 2F). Single-nuclei analysis conﬁrms previously identiﬁed gene expression changes in Purkinje cells and identiﬁes novel and potentially compensatory genes Among top Gene ontology (GO) pathways associated with the upregulated genes were synapse assembly (q = 3.42 × 10−4), synaptic membrane adhesion (q = 6.1 × 10−4), cell junction assembly (q = 8.8 × 10−4), synapse organization (q = 3.2 × 10−3) and cell junction organization (q = 8.1 × 10−3). Kyoto Encyclopedia of Genes and Genomes (KEGG) identiﬁed axon guidance (q = 1.4 × 10−5) and synaptic vesicle cycle (q = 2.9 × 10−3) as altered pathways. GO pathway analysis of downregulated genes identiﬁed calcium binding (q = 9.8 × 10−3), and dendrite and dendritic tree (q values of 1.45 and 1.48 × 10−5, respectively), while top KEGG pathways of downregulated genes were long-term depression (q = 4.9 × 10−4), cholinergic (q = 3.2 × 10−3), glutamatergic (q = 3.3 × 10−3), serotoninergic (q = 5.2 × 10−3) and dopaminergic (q = 5.5 × 10−3) synapse and retrograde endocananbinoid signaling (q = 7.1 × 10−3). Alterations in calcium and glutamatergic signaling are consistent with previous results from bulk RNAseq in SCA1 cerebella. Our results build upon those previous studies by conﬁrming that ATXN1 is driving these changes speciﬁcally in PCs. Moreover, our results indicate upregulation of pathways associated with synapse formation as potential compensatory pathways for signaling disruptions in SCA1 PCs. SCA1 is a progressive disease worsening with aging. Previous studies used weighted Gene Co-expression Network Analysis (WGCNA) of cerebellar bulk RNA sequencing data from Pcp2-ATXN1[82Q] mice to identify a speciﬁc module (the Magenta Module) as a gene network signiﬁcantly correlated with SCA1 disease progression in Purkinje cells (Ingram et al., 2016b). Notably, of the 342 genes identiﬁed in the magenta module, the Allen brain atlas suggested that 94 (27%) are PC enriched, 175 genes (51%) are PC exclusive, and 31 genes (9%) belong to multiple cell types (Ingram et al., 2016b). Using single nuclei gene expression analysis, we determined that 103 (30.1%) of the previously identiﬁed Magenta Module genes are altered at 12 weeks of age in Pcp2-ATXN1(O’Leary et al., 2020) mice. This is consistent with previous bulk RNA sequencing studies that showed that not all of the Magenta Module genes were found to be signiﬁcantly altered at 12 weeks in Pcp2- ATXN1(O’Leary et al., 2020) mice (Ingram et al., 2016b). Of these genes, 74 were exclusively altered in PCs (71.8%), while 29 (28.2%) were also changed in two or more cell types, including BG, VAs and OLs. Frontiers in Cellular Neuroscience Single-nuclei analysis conﬁrms previously identiﬁed gene expression changes in Purkinje cells and identiﬁes novel and potentially compensatory genes We have also identiﬁed 531 DEGs in hATXN1+ PCs and 370 DEG in hATXN1- PC cells compared to WT. Notably, 76.5% of the DEG in hATXN1+ PCs were upregulated, e.g., out of 531 DEGs 406 genes were upregulated (Supplementary Figure 5 and Supplementary Table 5). All of the 125 downregulated hATXN1+ PCs genes were also downregulated in hATXN1- PCs. However, out of the 406 genes that were upregulated in hATXN1+ PCs, only 47 were upregulated in hATXN1- PCs. Among shared upregulated and downregulated genes were previously mentioned top DEGs (Figures 2B,C), but the relative fold change in their expression was larger in hATXN1+ PCs (Supplementary Figure 5B and Supplementary Table 5). For instance, log2FC for Hs6st3, and Ralyl were 2.4 and 1.7 in hATXN1+ PCs, and 1.2 and 1.08 in hATXN1- PCs. Among upregulated genes that were unique to hATXN1+ PCs was Gabrg2 (gamma-aminobutyric acid type A receptor subunit gamma2), which encodes for ionotropic GABA receptor A subunit. most 1.6 fold change. We have also identiﬁed 531 DEGs in hATXN1+ PCs and 370 DEG in hATXN1- PC cells compared to WT. Notably, 76.5% of the DEG in hATXN1+ PCs were upregulated, e.g., out of 531 DEGs 406 genes were upregulated (Supplementary Figure 5 and Supplementary Table 5). All of the 125 downregulated hATXN1+ PCs genes were also downregulated in hATXN1- PCs. However, out of the 406 genes that were upregulated in hATXN1+ PCs, only 47 were upregulated in hATXN1- PCs. Among shared upregulated and downregulated genes were previously mentioned top DEGs (Figures 2B,C), but the relative fold change in their expression was larger in hATXN1+ PCs (Supplementary Figure 5B and Supplementary Table 5). For instance, log2FC for Hs6st3, and Ralyl were 2.4 and 1.7 in hATXN1+ PCs, and 1.2 and 1.08 in hATXN1- PCs. Among upregulated genes that were unique to hATXN1+ PCs was Gabrg2 (gamma-aminobutyric acid type A receptor subunit gamma2), which encodes for ionotropic GABA receptor A subunit. Djukic et al., 2007; Miyazaki et al., 2017). Similarly, PCs signaling is important for the ongoing function of BG. In particular, sonic hedgehog (Shh) signaling from PCs to BG drives the development and maintenance of Bergmann glia character (Eiraku et al., 2005; Farmer et al., 2016). With such close interactions, BG are perfectly poised to sense and respond to PC dysfunction in SCA1. Single-nuclei analysis conﬁrms previously identiﬁed gene expression changes in Purkinje cells and identiﬁes novel and potentially compensatory genes Indeed, we have previously shown both that BG undergo reactive gliosis in patients with SCA1 and in SCA1 mouse models (Cvetanovic et al., 2015; Rosa et al., 2021) and that they contribute to disease pathogenesis in SCA1 mice. Thus, we next wanted to investigate molecular changes in BG that underlie their response to PC dysfunction early in SCA1 progression. We identiﬁed 406 DEGs (p.adj < = 0.05) in Bergmann glia. Out of these 151 (37.2%) were downregulated and 255 (62.8%) upregulated (Figure 3A). Glial ﬁbrillary acidic protein (Gfap) expression was increased, indicating a reactive glial phenotype similar to what we have found in previous work. In addition, we have found increased expression of several genes that have been attributed neuroprotective roles including secreted protein acidic and rich in cysteine like 1 (Sparcl1), ﬁbroblast growth factor 12 (Fgf12), platelet derived growth factor D (Pdgfd), and Cystatin 3 (Cst3) (Figures 3B,C). SPARCL1 promotes excitatory synapse formation. One of the early signs of SCA1 is loss of excitatory VGLUT2+ synapses on PCs (Kim et al., 2018). Increased Sparcl1 expression in SCA1 BG could thus be a compensatory mechanism to promote formation of new excitatory synapses as these signaling structures are lost. Expression of growth factors, such as Fibroblast growth factor (Fgf) 12, and Platelet Derived Growth Factor D (PdgfD) was also increased in SCA1 BG. Fibroblast growth factors (FGFs) via their receptors (FGFRs) modulate important cellular processes such as cell proliferation, and death. FGF12 interacts with all four major FGFR and protects cells from apoptosis (Sochacka et al., 2020). In response to injury or various stresses, PDGFs modulate neuronal excitability by aﬀecting ion channels, and stimulate survival signals rescuing cells from apoptosis (Funa and Sasahara, 2014). Increased expression of Fgf12 and PdgfD in BG is likely to be neuroprotective and delay dysfunction and cell death of PCs (Chopra et al., 2018). In addition, BG may be serving a neuroprotective role by upregulating the expression of the Cystatin 3 (Cst3), the most abundant extracellular inhibitor of cysteine proteases. Cysteine proteases are upregulated in neurodegenerative and neuroinﬂammatory conditions and can lead to ECM breakdown and cell death (Hook et al., 2015). Inhibitors of cysteine proteases including Cst3 have been shown to be neuroprotective in neurodegenerative diseases (Kaur and Levy, 2012; Zou et al., 2017). Thus, increase in Cst3 in BG could be neuroprotective by preventing deleterious ECM changes. Single-nuclei analysis conﬁrms previously identiﬁed gene expression changes in Purkinje cells and identiﬁes novel and potentially compensatory genes Together, increased expression of these neuro-supportive genes indicates potential mechanisms by which reactive SCA1 BG exert beneﬁcial eﬀects during the early stages of disease. In conclusion, we conﬁrmed changes in PC gene expression previously found using bulk RNA sequencing. These included a number of genes underlying pathways associated with changes in ion transport and synapse function in SCA1 Purkinje cells. We also identiﬁed novel genes that deepen our understanding of altered signaling responsiveness of SCA1 PCs. Moreover, we identiﬁed novel genes that are upregulated in PCs early in disease progression. These genes may provide compensatory roles, including added support to extracellular matrix underlying synapse structures and added support to signaling strength, such as the increased expression of synaptic vesicle gene Slc32a1 which may restore inhibitory/excitatory balance in the cerebellar network. Intriguingly, we showed that PCs diﬀer in the expression of mutant ATXN1 and that PCs expressing the highest, and therefore most detectable, levels of mutant ATXN1 show a high degree of gene upregulation. This is in stark contrast to the hATXN1- PCs from SCA1 cerebella with undetectable levels of mutant ATXN1 or total PCs in which most DEGs are downregulated. Frontiers in Cellular Neuroscience Single-nuclei analysis conﬁrms previously identiﬁed gene expression changes in Purkinje cells and identiﬁes novel and potentially compensatory genes Most intriguing were 13 Magenta Module genes that were changed in all four cell types (Figure 2E). For instance, expression of Magenta genes Itpr1, Pcp4 and Gng13 was signiﬁcantly suppressed in PCs and, to a lesser extent, in BG, VAs and OLs. As these genes, respectively, encode for proteins regulating calcium and GPCR signaling (such as inositol 3 phosphate receptor 1 (ITPR1) that regulates calcium release from the ER, Purkinje Cell Protein 4 (PCP4) that regulates calcium binding to calmodulin, and G Protein Subunit Gamma 13 (Gng13) that regulates G protein coupled receptor signaling), this indicates that calcium and G protein signaling are perturbed not only in PCs, as previously described, but also in BG, VAs, and OLs. Notably, most of the observed gene expression changes seem to be present only in a portion of PCs (Figures 2C,D). Consistent with previous studies that suggested intracerebellar diﬀerences in the expression of hATXN1 and in PCs pathology in SCA1 mice (Chopra et al., 2020; Kozareva et al., 2020; White et al., 2021), our analysis indicated that only portion of PCs (∼ 25%) express detectable levels of mutant human ATXN1 mRNA in this line (Supplementary Figure 3B). We named these cells hATXN1+ PCs, while the cells in which we did not detect human ATXN1 we named hATXN1- PCs. It is possible that our analysis may underestimate the number of hATXN1+ PCs by not detecting low human ATXN1 expression. With that caveat in mind, we next analyzed gene expression changes in SCA1 hATXN1+ PCs (31 cells) and hATXN1- PCs (110 cells) compared to PCs from wild-type mice (132 cells). Among the genes altered in PCs, but not changed in BG, VAs, or OLs, was Delta/Notch like EGF Repeat containing (Dner). Dner has been previously found to be an important component of PC-BG communication whereby cell surface expression of DNER in Purkinje cells induces morphological diﬀerentiation and functional maturation of Bergmann glia. Moreover, loss of Dner impairs mouse motor behavior implicating its importance for cerebellar function (Eiraku et al., 2005; Tohgo et al., 2006). Reduced Dner expression could be a mechanism by which SCA1 PCs signal changes to BG. Using In this limited analysis, we could only identify a handful of DEGs (35) between hATXN1+ and hATXN1- PCs with at 08 frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. most 1.6 fold change. Disrupted Bergmann glia-Purkinje cell signaling via sonic hedgehog in Spinocerebellar ataxia type 1 (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] Bergmann glia. (E) Dot plot of the pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] Bergmann glia at 12 weeks of age. The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). FIGURE 3 Mutant ATXN1 causes signiﬁcant non-cell autonomous gene expression perturbations in SCA1 Bergmann glia. (A) For identiﬁed Bergmann glia, limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in BG. (B) Volcano plot showing DEGs of BG cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated DEGs in wild-type and Pcp2-ATXN1[82Q] Bergmann glia determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] Bergmann glia. (E) Dot plot of the pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] Bergmann glia at 12 weeks of age. The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). Mutant ATXN1 causes signiﬁcant non-cell autonomous gene expression perturbations in SCA1 Bergmann glia. (A) For identiﬁed Bergmann glia, limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in BG. (B) Volcano plot showing DEGs of BG cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated DEGs in wild-type and Pcp2-ATXN1[82Q] Bergmann glia determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] Bergmann glia. (E) Dot plot of the pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] Bergmann glia at 12 weeks of age. Disrupted Bergmann glia-Purkinje cell signaling via sonic hedgehog in Spinocerebellar ataxia type 1 Bergmann glia are a special type of radial astrocytes which are intimately connected with Purkinje cells, both structurally and functionally. BG cell bodies surround the somas of PCs and their radial processes envelop PCs synapses in the molecular layer. This close morphological interaction is mirrored in the functional interdependence of BG and PCs. BG are essential for PCs function and viability through their many roles including maintenance of potassium homeostasis, removal of synaptic glutamate and provision of neurotrophic support (Rothstein et al., 1996; Grosche et al., 1999; Custer et al., 2006; 09 frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. FIGURE 3 Mutant ATXN1 causes signiﬁcant non-cell autonomous gene expression perturbations in SCA1 Bergmann glia. (A) For identiﬁed Bergmann glia, limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in BG. (B) Volcano plot showing DEGs of BG cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated DEGs in wild-type and Pcp2-ATXN1[82Q] Bergmann glia determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] Bergmann glia. (E) Dot plot of the pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] Bergmann glia at 12 weeks of age. The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). FIGURE 3 Mutant ATXN1 causes signiﬁcant non-cell autonomous gene expression perturbations in SCA1 Bergmann glia. (A) For identiﬁed Bergmann glia, limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in BG. (B) Volcano plot showing DEGs of BG cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated DEGs in wild-type and Pcp2-ATXN1[82Q] Bergmann glia determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. Disrupted Bergmann glia-Purkinje cell signaling via sonic hedgehog in Spinocerebellar ataxia type 1 The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). 10 Frontiers in Cellular Neuroscience frontiersin.org Borgenheimer et al. 10.3389/fncel.2022.998408 Borgenheimer et al. We have also observed changes in the expression of homeostatic BG genes responsible for regulating neuronal function. We found that expression of Kcnj10 (Figure 3D), encoding the potassium inward rectiﬁer Kir4.1, is decreased in BG at this stage of disease. Kir4.1 regulates extracellular potassium ion levels and, subsequently, PC excitability and ﬁring rate. Previous work has shown that the ﬁring rate of PCs is decreased early in SCA1 (Dell’Orco et al., 2015). This observed decrease in Kcnj10 expression is expected to moderate PC ﬁring rate (Djukic et al., 2007). BG may also moderate PC synaptic signaling via reduced expression of Solute carrier family 1 Member 3 (Slc1a3). Slc1a3 encodes for the plasma membrane glutamate aspartate transporter (GLAST) that is responsible for synaptic glutamate removal. Decreased Slc1a3 expression may result in slower and less eﬃcient glutamate removal, thus prolonging glutamate signaling on PCs. increased expression of Adcy8 in BG that may moderate their pro-inﬂammatory response in SCA1. Sox2 is a transcriptional regulator of BG reactivity. The long non-coding RNA (lncRNA) Rmst is known for its role in facilitating activation of Sox2. We observed increased expression of Rmst that may play a role in facilitating Sox2-regulation of reactive glial response in SCA1 BG (Ng et al., 2013; Cerrato et al., 2018). Hierarchical enriched pathways analysis of the DEGs in BG identiﬁed pathways involved in development, including cell junction assembly, positive regulation of cell projection organization, regulation of neurogenesis, axonogenesis, gliogenesis, small molecule metabolic processes and neuron apoptotic processes (Figure 3E). Among top GO pathways associated with the upregulated genes were nervous system development (q = 1.2 × 10−9), regulation of biological quality (q = 1.5 × 10−9), cell adhesion (q = 9.8 × 10−9), organonitrogen compound metabolic processes (q = 3.8 × 10−7) and cell projection organization (q = 5 × 10−8). KEGG identiﬁed circadian entrainment (q = 3.6 × 10−2) as dysregulated pathway in BG. This is very intriguing considering the role the cerebellum has in sleep and sleep disturbances in SCA patients (Pedroso et al., 2011; DelRosso and Hoque, 2014). Disrupted Bergmann glia-Purkinje cell signaling via sonic hedgehog in Spinocerebellar ataxia type 1 GO pathway analysis of downregulated genes identiﬁed transmembrane transporter binding (q = 7 × 10−3), smoothened binding and hedgehog receptor activity (q = 4.9 × 10−2), cell to cell signaling (q = 6.4 × 10−7), apoptotic processes (q = 1.3 × 10−2) and synapse (q = 5 × 10−14). KEGG analysis of downregulated genes identiﬁed retrograde endocannabinoid signaling (q = 1 × 10−3), glutamatergic (q = 2 × 10−3) and dopaminergic synapses (q = 4.3 × 10−2), long-term depression (q = 8.1 × 10−3) and Spinocerebellar ataxias (q = 8.4 × 10−3). Our results also provide insight into how are BG genes altered in SCA1. Previous studies have found that PCs regulate BG character via Sonic hedgehog (Shh) signaling (Farmer et al., 2016). For instance, Shh signaling from PCs regulates higher expression of Kcnj10 and lower expression of aquaporin 4 (Aqp4) in BG compared to velate astrocytes. Interestingly, we found that expression of Shh receptors Patched 1 and 2 (Ptch1, Ptch2) (Adolphe et al., 2014) is decreased in BG in Pcp2- ATXN1[82Q] cerebella indicating one critical way in which PC-BG communication is impacted in SCA1. Loss of PC-BG Shh signaling could aﬀect BG function in several ways, including decreased expression of Kcnj10 and increased expression of Aqp4, both changes that we indeed observed in SCA1 BG. Expression of Shh downstream transcriptional activator Gli1 is also decreased, further supporting dysfunctional Shh PC- BG signaling in SCA1 (Figure 3D). We investigated whether these changes in Shh signaling occur widely or only in the subset of BG, such as the ones residing next to hATXN1+ PCs. Most BG observed in this study showed decreased expression of Ptch1, Ptch2, and Gli1 (Supplementary Figure 6A). We conﬁrmed reduced expression of Ptch2 and Gli1 using RT-qPCR of whole Pcp2-ATXN1[82Q] cerebellar extracts (Supplementary Figure 6B and Supplementary Table 1). As the most logical cause of this reduced BG expression is decreased expression of ligand Shh from PCs, we assessed Shh expression in identiﬁed PC populations. However, we found no signiﬁcant change in Shh expression in total PCs, hATXN1+, or hATXN1- PCs (Supplementary Figure 6C and Supplementary Table 5). This may suggest either altered post-transcriptional regulation of Shh in PCs or broader changes in PC signaling capabilities that are disrupting this crucial mechanism. Our analysis supports the hypothesis that SCA1 BG increase their neuroprotective support in response to PC dysfunction. Frontiers in Cellular Neuroscience Disrupted Bergmann glia-Purkinje cell signaling via sonic hedgehog in Spinocerebellar ataxia type 1 It conﬁrms the reactive SCA1 phenotype previously observed through the upregulation of Gfap and suggests an ongoing regulation of neuroinﬂammatory response through the expression of Adcy8 and Rmst. More importantly, these results implicate altered PC-BG Shh signaling as one of the mechanisms by which BG function is perturbed in SCA1. Increased expression of genes that promote neurogenesis, gliogenesis, and synaptogenesis indicates compensatory gene expression changes in velate astrocytes Further, GO pathway analysis of the downregulated genes identiﬁed calcium ion binding (q = 3.1 × 10−3), vesicle mediated transport in synapse (q = 3.5 × 10−2), negative regulation of neuronal death (q = 4.1 × 10−2), glutamatergic synapse (q = 4.5 × 10−4), Spinocerebellar ataxia (q = 1.3 × 10−3), retrograde endocannabinoid signaling (q = 2.4 × 10−2), and long-term depression (q = 2.7 × 10−2). A previous study identiﬁed two diﬀerent types of reactive astrocytes that were termed “A1” and “A2,” respectively (Liddelow et al., 2017). A1 astrocytes are thought to be harmful as they up-regulate classical complement cascade genes previously shown to be destructive to synapses, while A2 astrocytes are thought to be neuroprotective due to increased expression of neurotrophic factors. To determine whether reactive BG and VAs are more like A1 or A2 astrocytes, we examined the expression of A1 and A2 astrocyte genes in VA and BG. While we found increased expression of panreactive genes Gfap, Vim, and CD44, we found no clear pattern or distinction in A1 or A2 type gene expression in BG or VAs (Figure 4E). Together, these results indicate that VAs exhibit a reactive astrocyte phenotype in SCA1 that is characterized by the expression of genes regulating synapse structure and maintenance. Furthermore, reduction in Vegf expression in VAs seems to implicate VAs as an important contributor to reduced VEGF expression in the SCA1 cerebellum, suggesting that they may play a role in PC pathology. As this model represents a non-cell autonomous response of these cells to PC dysfunction this could indicate a negative feedback mechanism that could contribute to progressive worsening of cellular pathology over time. Among the top upregulated VA genes were genes involved in neurogenesis, gliogenesis, and synapse maintenance including Neural Cell adhesion molecule 2 (Ncam2), Sema6D, Quaking (Qk), Serpine E2, Clusterin (Clu) and CUB And Sushi Multiple Domains 1 (Csmd1). NCAM2 is involved in many roles in the brain including neurogenesis, neuronal migration, neuronal diﬀerentiation, synaptogenesis, calcium signaling, and maintenance of presynaptic and postsynaptic compartments in adult brains. In addition, it has been proposed that a decrease in NCAM2 levels is associated with loss of synaptic structure in the early stages of neurodegenerative diseases (Parcerisas et al., 2021). Quaking (Qk) is an RNA binding protein residing in astrocyte processes and promotes astrocyte maturation (Sakers et al., 2021). Increased expression of genes that promote neurogenesis, gliogenesis, and synaptogenesis indicates compensatory gene expression changes in velate astrocytes Increased expression of genes that promote neurogenesis, gliogenesis, and synaptogenesis indicates compensatory gene expression changes in velate astrocytes Velate astrocytes (VAs) are a type of cerebellar astrocytes that reside in the granule cell layer. As such, they are surrounded by the most numerous neurons in the brain and are the only astrocytes in the brain that are largely outnumbered by neurons they are associated with (Herculano-Houzel, 2014). However, very little is known about the response of velate We further investigated the expression of genes that may regulate BG response to PC dysfunction. Calcium regulated adenylate cyclase (Adcy8) controls neuroinﬂammation by increasing the production of cyclic adenosine monophosphate (cAMP), an important negative regulator of inﬂammation (Wieczorek et al., 2012; Wang et al., 2020). We have found 11 frontiersin.org Borgenheimer et al. 10.3389/fncel.2022.998408 astrocytes to cerebellar pathology, including SCA1. Therefore, we next investigated genes and pathways altered in SCA1 velate astrocytes. VEGF is a neurotrophic factor whose decrease was previously shown to contribute to Purkinje cell pathology in SCA1 (Cvetanovic et al., 2011). We found a reduced expression of vascular endothelial growth factor (VEGF) suggesting that velate astrocytes contribute to a VEGF reduction in SCA1 (Figure 4D). We have identiﬁed 157 DEGs (p.adj < = 0.05) in SCA1 velate astrocytes compared to those of their wild-type littermate controls. A majority (114 or 72.6%) of the identiﬁed DEGs were upregulated (Figures 4A–D). Notably, one of genes with increased expression was Vimentin (Vim). Vim encodes for the intermediate ﬁlament predominantly found in astrocytes. Vimentin is upregulated in reactive astrogliosis (Janeczko, 1993; O’Leary et al., 2020), indicating that VAs, like BG, undergo reactive gliosis in SCA1 (Figure 4D). Apolipoprotein E (ApoE) also plays a role in reactive astrogliosis (Ophir et al., 2003; Sen et al., 2017). Increased expression of ApoE in VAs further indicates reactive velate astrogliosis in SCA1. Pathway analysis of all VA DEGs identiﬁed developmental pathways including regulation of neurogenesis, gliogenesis, positive regulation of projection organization, regulation of nervous system development, and regulation of protein catabolic process (Figure 4F). Breaking this down into pathways unique to up and downregulated genes, we found that GO pathway analysis of only upregulated genes highlighted many developmental pathways including cell morphogenesis, regulation of neuron projection development, positive regulation of gliogenesis, and glial diﬀerentiation (q values of 3.2 × 10−6, 1.5 × 10−5, 3.9 × 10−5, and 4.5 × 10−5, respectively). Increased expression of genes that promote neurogenesis, gliogenesis, and synaptogenesis indicates compensatory gene expression changes in velate astrocytes Astrocyte secreted Clu co- localizes with presynaptic puncta of excitatory neurons and loss of Clu led to impaired presynaptic function and reduced spine density in vivo (Chen et al., 2021). On the other hand, overexpression of Clu in astrocytes reduced pathology and restored synaptic function in mouse model of Alzheimer’s disease (Chen et al., 2021). Recently, CUB And Sushi Multiple Domains 1(Csmd1) was suggested to oppose the complement cascade that facilitates synaptic loss in neurodegeneration (Baum et al., 2020). Therefore, it is reasonable to assume that increased expression of Ncam2, Qk, Clu and Csmd1 in velate astrocytes may be neuroprotective in SCA1 by promoting astrocyte maturation and synaptic function. Gene expression analysis indicates reactive activation in Spinocerebellar ataxia type 1 oligodendrocytes (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] VAs. (E) Heatplot of normalized expression of astrocyte A1/A2 genes in BG and VA at 12 weeks of age. Color indicates logFC. (F) Dot plot of the pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] VA at 12 weeks of age. The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). We found increased expression of Cryab and ApoE indicating protective reactive oligodendrogliosis in SCA1.Notably, we observed increased expression of transcriptional factor JunD, which is a functional component of activated protein 1 (AP1) complex. The AP1 complex regulates many OL inﬂammatory and immune genes, suggesting that AP1 may modulate a non- cell autonomous reactive OL response in SCA1 pathogenesis (Behmoaras et al., 2008). expression of mature oligodendrocyte genes, including Myelin Oligodendrocyte Glycoprotein (Mog) in a knock-in mouse model of SCA1, Atxn1154Q/2Q mice (Tejwani et al., 2021). In Atxn1154Q/2Q mice mutant Atxn1 is expressed widely, including in oligodendrocyte precursor cells (OPC) and OLs. To gain insight into which of these gene expression changes are induced in response to PC pathogenesis alone, we examined genes and pathways altered in oligodendrocyte nuclei from Pcp2-ATXN1[82Q] mice compared to littermate controls. We found 154 DEGs in oligodendrocytes in Pcp2-ATXN1[82Q] cerebella, with the majority (94 DEG or 61%) of these genes being upregulated (Figure 5A). While the percentage of OLs identiﬁed in our SCA1 samples is not signiﬁcantly diﬀerent from WT controls, the expression of several oligodendrocyte marker genes including Transferrin (Trf), Mog, Claudin 11 (Cldn11) and Reticulon 4 (Rtn4) was increased (Figures 5B–D; Bradl and Lassmann, 2010). We also found altered expression of many genes important for oligodendrocyte function (Figure 5E). Among them is increased expression of Glutamate-Ammonia Ligase (Glul) encoding for glutamine synthetase (GS) This molecule serves as a key glutamate-catabolizing enzyme that catalyzes the conversion of glutamate to glutamine. Previous studies demonstrated that OLs support neuronal glutamatergic transmission via expression of GS (Xin et al., 2019). Moreover, Glul expression in OLs is increased in chronic pathological conditions including amyotrophic lateral sclerosis (ALS) and MS (Ben Haim et al., 2021). As glutamate signaling is perturbed in SCA1, it is possible that OLs increase GS expression to compensate for these changes in glutamate metabolism in SCA1. Gene expression analysis indicates reactive activation in Spinocerebellar ataxia type 1 oligodendrocytes Oligodendrocytes (OL) envelop axons with myelin and maintain long-term axonal integrity (Bradl and Lassmann, 2010). Cells of the oligodendrocyte lineage are important for cerebellar development and motor learning (Mathis et al., 2003; McKenzie et al., 2014). Previous studies have found signiﬁcant changes in the cerebellar white matter in SCA1 patients and in Pcp2-ATXN1[82Q] mice, indicating that oligodendrocytes (OLs) may be aﬀected in SCA1 (Liu et al., 2018; Park et al., 2020). Moreover, a recent study found a decrease in oligodendrocyte numbers in human post-mortem samples from patients with SCA1 (Tejwani et al., 2021) and a decreased Frontiers in Cellular Neuroscience 12 frontiersin.org Borgenheimer et al. 10.3389/fncel.2022.998408 FIGURE 4 Velate astrocytes gene expression changes in SCA1. For identiﬁed velate astrocytes limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in VA. (B) Volcano plot showing DEGs of VA cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated DEGs in wild-type and Pcp2-ATXN1[82Q] VA determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] VAs. (E) Heatplot of normalized expression of astrocyte A1/A2 genes in BG and VA at 12 weeks of age. Color indicates logFC. (F) Dot plot of the pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] VA at 12 weeks of age. The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). FIGURE 4 Velate astrocytes gene expression changes in SCA1. For identiﬁed velate astrocytes limma was used to test differential gene abundance between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in VA. (B) Volcano plot showing DEGs of VA cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated DEGs in wild-type and Pcp2-ATXN1[82Q] VA determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. Gene expression analysis indicates reactive activation in Spinocerebellar ataxia type 1 oligodendrocytes The color of dots depicts the adjusted p values, radius of the dot depicts gene counts (number of genes in the enriched pathway). Genes downregulated in SCA1 OLs included Atp2a2 and Atp2a3 which encode for the calcium endoplasmic reticulum pumps SERCA1 and SERCA2, respectively. This implicates a reduced ability of OLs endoplasmic reticula (ER) to buﬀer calcium. In addition, reduced expression of Atp2b2 that encodes for Plasma Membrane Calcium-Transporting ATPase is expected to reduce plasma membrane calcium buﬀering. Together these results indicate perturbed calcium homeostasis as a feature of reactive SCA1 OLs (Figures 5B-D). mutation was restricted to PCs, we aimed to provide insight into how these pathogenic processes within PCs aﬀect non- cell autonomous gene expression and signaling pathways within other cerebellar cells. To accomplish this, we have employed single nuclei RNA sequencing to investigate transcriptional changes in individual cerebellar cell types in “early stage” 12 week old animals, a time point which represents a phase of SCA1 in which symptoms are beginning to become apparent but before the progressive cell death of PCs within the cerebellum. Intriguingly, the top two pathways of OLs DEGs identiﬁed were cognition and locomotor behavior, implicating that molecular pathway alterations in SCA1 oligodendrocytes could be associated with the broader symptomology of SCA1. Pathway analysis of all OLs DEGs also identiﬁed regulation of neurotransmitter levels, positive regulation of synaptic transmission, vesicle-mediated transport in synapses and neurotransmitter secretion, potentially implicating OL in modulating synaptic transmission in SCA1 (Figure 5E). There are several important ﬁndings from our study. First, mutant ATXN1 expression in Purkinje cells causes profound transcriptional alterations in cerebellar glia in a non-cell autonomous manner. Mutant ATXN1 expression in PCs alone caused altered gene expression in every glial subtype analyzed, often driving an increase in gene expression (Bergmann glia (62.8%), velate astrocytes (72.6%) and oligodendrocytes (61%). As at this early disease stage there is no detectable Purkinje cell loss in Pcp2-ATXN1[82Q] mice, we propose that these glial transcriptome changes are in direct response to ATXN1-driven Purkinje cell dysfunction (Bell et al., 2010; Ingram et al., 2016b). Gene expression analysis indicates reactive activation in Spinocerebellar ataxia type 1 oligodendrocytes Increased expressions of Crystallin Alpha B (Cryab) and apolipoprotein E (ApoE) have been associated with protective reactive gliosis in neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Multiple Sclerosis (MS) (Ophir et al., 2003; Ousman et al., 2007; Liu et al., 2015; Kuipers et al., 2017; D’Agostino et al., 2019). Frontiers in Cellular Neuroscience 13 frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. FIGURE 5 Gene expression changes in SCA1 oligodendrocytes. Limma was used to test differential gene abundance in oligodendrocytes between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (Continued) FIGURE 5 Gene expression changes in SCA1 oligodendrocytes. Limma was used to test differential gene abundance in oligodendrocytes between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (Continue FIGURE 5 Gene expression changes in SCA1 oligodendrocytes. Limma was used to test differential gene abundance in oligodendrocytes between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (Continued FIGURE 5 Gene expression changes in SCA1 oligodendrocytes. Limma was used to test differential gene abundance in oligodendrocytes between control and Pcp2-ATXN1[82Q] samples. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values of 0.05. (A) Number of upregulated and downregulated differentially expressed genes (Continued 14 Frontiers in Cellular Neuroscience frontiersin.org Borgenheimer et al. 10.3389/fncel.2022.998408 FIGURE 5 (Continued) (DEGs) in OL. (B) Volcano plot showing DEGs of OL cluster in Pcp2-ATXN1[82Q] SCA1. (C) Heatplot displaying expression proﬁles of top 25 highest upregulated and downregulated DEGs in wild-type and Pcp2-ATXN1[82Q] oligodendrocytes determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. (D) Ridgeplot showing distribution of expression of selected genes in wild-type and Pcp2-ATXN1[82Q] OL. (E) Dot plot of the pathway analysis of total DEGs between WT and Pcp2-ATXN1[82Q] OL at 12 weeks of age. Frontiers in Cellular Neuroscience Gene expression analysis indicates reactive activation in Spinocerebellar ataxia type 1 oligodendrocytes Breaking this analysis down into pathways unique to up and downregulated genes, we found that GO pathway analysis of only downregulated genes identiﬁed pathways involved in transport and binding of ions including inorganic cation transmembrane transporter activity (q = 7.5 × 10−6), calcium binding (q = 9.3 × 10−6), P-type calcium transporter activity (q = 4.2 × 10−4), synaptic signaling (q = 1.6 × 10−7), modulation of chemical synaptic transmission (q = 5.4 × 10−6), glutamatergic synapse (q = 6.4 × 10−6), and Spinocerebellar ataxia (q = 2.9 × 10−5). Moreover, pathway analysis of OLs upregulated DEGs identiﬁed movement of cell or subcellular component (q = 9.4 × 10−5), cell migration (q = 8.8 × 10−4), myelin sheath (q = 2.4 × 10−7), and synapse (q = 1.4 × 10−4). Second, we identiﬁed novel transcriptional changes in PCs that may be relevant to disease pathogenesis. These potentially compensatory gene expression changes include increased expression of Ralyl, Atp6v0c, Atpgv0a1, Atp6v1h, Atp6v and Slc32a1. Ralyl is an RNA binding protein known for its neuroprotective role in Alzheimer’s (Zhang et al., 2020). Increased expression of three vacuolar V-type ATP-ase subunits (Atp6v0c, Atpgv0a1, and Atp6v1h) that provide electrochemical gradient to ﬁll synaptic vesicles with GABA, and Slc32a1, encoding vesicular GABA and Glycine Transporter (VGAT) that transports GABA in the vesicles may suggest increased GABA content in PCs synaptic vesicle. As spontaneous ﬁring rate of PCs is decreased in Pcp2-ATXN1[82Q] mice (Dell’Orco et al., 2015), an increase in GABA loading per vesicle may represent compensatory change to restore or ameliorate synaptic transmission by increasing the total amount of GABA released at PC terminals. Together, these analyses indicate that oligodendrocytes respond to PC dysfunction in SCA1 by becoming reactive. Reactive response of SCA1 OLs is characterized by increased expression of Cryab, ApoE, and Glul, and may be regulated by transcriptional factor AP1. While this may allow for increased neuroprotection, such as compensatory glutamate buﬀering, reactive OLs response may impact their calcium homeostasis. Third, because previous work in this mouse model suggested altered levels of mutant ATXN1 in PCs across the cerebellum, we assessed the relative expression of human ATXN1 within our SCA1 PCs. In doing so, we identiﬁed two populations- those that were expressing detectable ATXN1 (∼25%) and those that had no detectable ATXN1. It is likely that we underestimated the number of mutant ATXN1 expressing PCs Discussion Through this work we sought to increase our understanding of the cell-type speciﬁc changes during the early stages of SCA1. Furthermore, by using a model in which the pathological 15 frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. due to the rigor of our analysis. It is unclear whether PCs with undetectable mutant hATXN1 have reduced production of mutant ATXN1 mRNA or are more eﬃcient in transporting it out of the nucleus and/or degrading it. Intriguingly, in contrast to prevalent gene downregulation in total PCs and hATXN1- PCs, majority of DEGs were upregulated in hATXN1+ cells. It is possible that these upregulated genes provide resistance to SCA1, and it will be important for future studies to distinguish whether hATXN1+ or – cells are more aﬀected in SCA1. This result also indicates that some of the ATXN1 –induced gene expression changes may be missed when comparing total SCA1 to WT PCs. Furthermore, out of 370 DEGs in hATXN1- PCs, 355 were also present when comparing total SCA1 to WT PCs. Thus majority (64%) of DEGs identiﬁed in comparison of total SCA1 and WT PCs are genes with altered expression in PCs in which we could not detect mutant hATXN1. Mog, Cldn11, Rtn4, Glul, Cryab and ApoE. These results indicate neuroprotective eﬀects of reactive cerebellar OLs in SCA1. Finally, we identiﬁed shared DEGs and pathways that are altered in all four analyzed cell types in SCA1 cerebella and as such are promising candidates for future therapies. Transthyretin (Ttr) is a protein that binds and distributes retinol and thyroid hormones. Retinol is important for cerebellar function (Tafti and Ghyselinck, 2007) where it binds retinoic acid receptor-related orphan receptors (ROR). Previous studies identiﬁed the importance of retinol in SCA1 (Serra et al., 2006), by demonstrating reduced expression of RORa–regulated genes in cerebella of SCA1 mice, and that partial loss of RORa enhances PCs pathology (Serra et al., 2006). Recent studies found that Ttr plays important roles in other cells such as OPC, cerebellar granule neurons and astrocytes. For example, Ttr regulates proliferation and survival of OPCs (Alshehri et al., 2020), δ-GABAA-R expression in cerebellar granule neurons (Zhou et al., 2019), and glycolysis in astrocytes (Zawi¸slak et al., 2017). We found that Ttr expression is increased in SCA1 PCs, BG, VA and OL. Discussion It is likely that this increase in Ttr expression is beneﬁcial for PCs, OLs, astrocytes and granule neurons, and compensates for reduced RORa function in SCA1. It will be important to understand the mechanism of Ttr upregulation, and whether exogenous Ttr is therapeutically beneﬁcial in SCA1. Fourth, our results support perturbed Shh signaling as one of contributors to BG molecular alterations in SCA1. Sonic hedgehog (Shh) signaling is one of the key communication pathways by which PCs regulate BG character (Farmer et al., 2016). We found that the expression of Shh receptors Patched 1 and 2 and Shh signaling downstream transcription factor Gli1 are reduced in SCA1 BG. Previous studies have shown that Shh-Ptch2 communication regulates expression of homeostatic genes Slc1a3, Kcnj10, and Aqp4 in Bergmann glia (Farmer et al., 2016). We also validated a previously reported decrease in the expression of glutamate transporter Slc1a3 in Bergmann glia (Cvetanovic, 2015), and identiﬁed additional perturbations in the expression of homeostatic genes such as Kcnj10 and Aqp4 that are critical for PC function (Djukic et al., 2007; Nicita et al., 2018). Therefore, we propose that decreased Shh signaling contributes to reduced expression of Slc1a3 and Kcnj10 and increased expression of Aqp4 that we found in SCA1 BG. In addition, several Magenta module genes, including genes involved in calcium homeostasis, such as Atp2a2 and Itpr1 were reduced in all four cell types, indicating that restoring calcium homeostasis may be another good target for SCA1 therapeutic approaches. Selective neuronal vulnerability is a feature shared by many neurodegenerative diseases. In the case of SCA1, although mutant ATXN1 is expressed throughout the brain, cerebellar Purkinje cells (PCs) are most aﬀected (Servadio et al., 1995). Severe vulnerability of PCs in SCA1 is likely brought about by the combination of the toxic eﬀects of mutant ATXN1 within PCs and the changes in PCs microenvironment, including glial cell alterations. In this respect, it is important to note that glial cells in the cerebellum are found to have distinct transcriptomes compared to the glial cells in other brain regions (Grabert et al., 2016; Soreq et al., 2017; Boisvert et al., 2018). Uniqueness of cerebellar glia gets even more pronounced during aging (Grabert et al., 2016; Boisvert et al., 2018; Clarke et al., 2018). Frontiers in Cellular Neuroscience Discussion Yet, while neurodegeneration associated glial gene expression changes have been studied intensively in the other brain regions, less is known about gene expression changes in cerebellar glia during neurodegeneration. Our results provide much needed insight into gene expression changes of cerebellar Bergmann glia, velate astrocytes and oligodendrocytes in response to PC dysfunction. Fifth, we provide evidence that reactive response in velate astrocytes includes increased expression of several neuroprotective genes such as ApoE, Ncam2, Clu and Csmd1. ApoE, among other roles, contributes to astrocyte activation and increases BDNF secretion. Increased ApoE in SCA1 VAs may contribute to increased BDNF that is neuroprotective in SCA1 (Ophir et al., 2003; Sen et al., 2017; Mellesmoen et al., 2018; Sheeler et al., 2021). Ncam2, Clu and Csmd1 are protective against loss of synapses (Baum et al., 2020; Parcerisas et al., 2021), and their increased expression in SCA1 VAs may compensate for loss of synapses seen in SCA1. These results also increase our limited understanding of VAs reactive gliosis in cerebellar disease. Six, we identiﬁed intriguing transcriptional changes in SCA1 oligodendrocytes indicative of reactive oligodendrogliosis that may be regulated by increased expression of JunD. While OLs numbers do not seem to be altered at 12 weeks in Pcp2- ATXN1[82Q] cerebellum, we have found increased expression of OL marker genes and neurosupportive genes including Trf, It is likely that some of the molecular changes we identiﬁed are compensatory, allowing for continued cerebellar function, and that some may be pathogenic, promoting disease progression. 16 frontiersin.org Borgenheimer et al. 10.3389/fncel.2022.998408 SUPPLEMENTARY FIGURE 4 Decreased expression of Dner mRNA and protein in Purkinje Cells. (A). Quantitative RT-PCR of bulk cerebellar lysates from 12 weeks old wild-type and Pcp2-ATXN1[82Q] mice was used to evaluate expression of Dner. Data is presented as mean ± SEM with average values for each mouse represented by a dot. N = 7, ∗p < 0.05 unpaired t test with Welch’s correction. (B,C) Cerebellar slices from 12 week old wild-type and Pcp2-ATXN1[82Q] mice were stained with DNER antibody. (D) Confocal images and Image J were used to quantify DNER expression in soma and dendrites of PCs (right). Data is presented as mean ± SEM with values for each mouse analyzed represented by a dot. N = 3 wild-type and N = 5 Pcp2-ATXN1[82Q] mice∗p < 0.05 unpaired t-test with Welch’s correction. Scale bars = 50 µm. This work was supported by a National Institute of Health NINDS awards (R01 NS107387 and NS109077 to MC). Publisher’s note Our results provide a framework to investigate how individual pathogenic processes contribute to the sequence of progressive cerebellar dysfunctions in SCA1. Our results are freely available and we hope that they will stimulate future studies to causally investigate how these gene and pathway perturbations contribute to SCA1 pathogenesis as well as facilitate development of novel therapeutic approaches. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. SUPPLEMENTARY FIGURE 5 Gene expression changes in hATXN1 + and hATXN1- Purkinje cells. For identiﬁed hATXN1 + and hATXN1- Purkinje cells limma was used to test differential expression between control and Pcp2-ATXN1[82Q] samples. (A) Number of upregulated and downregulated differentially expressed genes (DEGs) in hATXN1 + and hATXN1- PCs. N = 3 mice of each genotype. P values were adjusted using Benjamini-Hockberg method. Differential gene expression was determined by an adjusted p-values ≤0.05. (B) Heatplot displaying expression proﬁles of selected upregulated and downregulated DEGs in wild-type PCs and hATXN1 + and hATXN1- PCs from Pcp2-ATXN1[82Q] cerebella determined by logFC values with adjusted p-values ≤0.05. N = 3 mice of each genotype. Frontiers in Cellular Neuroscience SUPPLEMENTARY FIGURE 2 Reduced expression of Purkinje cell genes in Pcp2-ATXN1[82Q] mice. Quantitative RT-PCR of bulk cerebellar lysates was used to evaluate expression of Purkinje cell genes Calb1, Pcp4, Homer3, Rgs8, ITPR, Inpp5 and Garnl3. Data is presented as mean ± SEM with average values for each mouse represented by a dot. N = 6 -7 mice per genotype (WT and Pcp2-ATXN1[82Q]) ∗p < 0.05 unpaired t test with W l h’ ti SUPPLEMENTARY FIGURE 3 MC conceptualized the study. EB, CS, KH, FLM, and KS performed the experiments. YZ and MC analyzed the data. MC, EB, CS, KH, FLM, KS, and YZ wrote the manuscript. All authors contributed to the article and approved the submitted version. Expression of endogenous mouse Atxn1[2Q] and mutant human ATXN1[82Q] in cerebellar cells. (A) Percentage of cerebellar cells expressing endogenous mouse Atxn1[2Q] in wild-type and Pcp2-ATXN1[82Q] mice. (B) Percentage of cerebellar cells expressing mutant human hATXN1[82Q] in wild-type and Pcp2-ATXN1[82Q] mice. Data is presented as mean ± SEM with average values for each mouse represented by a dot. N = 3. ∗P < 0.05 Two way ANOVA with Tukey’s multiple comparisons tests. Ethics statement Example of isolated nuclei showing absence of clumping and spherical, non-damaged nuclei. Nuclei for RNA sequencing were isolated from the mouse cerebella using detergent mechanical lysis protocol, and stained with DAPI. Scale bar = 100 µm. Animal experimentation was approved by Institutional Animal Care and Use Committee (IACUC) of University of Minnesota. Acknowledgments We acknowledge all the members of Orr and Cvetanovic laboratories for thoughtful discussions and feedback on the study. Work in this study was aided by the Single Cell Services of the University of Minnesota Genomics Center. Supplementary material All the data from this study are available from the authors. RNA sequencing data are deposited at https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE215336. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ fncel.2022.998408/full#supplementary-material Borgenheimer et al. References Chen, F., Swartzlander, D. B., Ghosh, A., Fryer, J. D., Wang, B., and Zheng, H. (2021). Clusterin secreted from astrocyte promotes excitatory synaptic transmission and ameliorates Alzheimer’s disease neuropathology. Mol. Neurodegener. 16:5. doi: 10.1186/s13024-021-00426-7 Adolphe, C., Nieuwenhuis, E., Villani, R., Li, Z. J., Kaur, P., Hui, C. C., et al. (2014). Patched 1 and patched 2 redundancy has a key role in regulating epidermal diﬀerentiation. J. Invest. Dermatol. 134, 1981–1990. doi: 10.1038/jid.2014.63 Alshehri, B., Pagnin, M., Lee, J. Y., Petratos, S., and Richardson, S. J. (2020). The Role of Transthyretin in Oligodendrocyte Development. Sci. Rep. 10:4189. Chopra, R., Bushart, D. D., and Shakkottai, V. G. (2018). Dendritic potassium channel dysfunction may contribute to dendrite degeneration in spinocerebellar ataxia type 1. PLoS One 13:e0198040. doi: 10.1371/journal.pone.0198040 Alvarez, M., Rahmani, E., Jew, B., Garske, K. M., Miao, Z., Benhammou, J. N., et al. (2020). Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classiﬁer DIEM. Sci. Rep. 10:11019. doi: 10.1038/s41598-020-67513-5 Chopra, R., Bushart, D. D., Cooper, J. P., Yellajoshyula, D., Morrison, L. M., Huang, H., et al. (2020). Altered Capicua expression drives regional Purkinje neuron vulnerability through ion channel gene dysregulation in spinocerebellar ataxia type 1. Hum. Mol. Genet. 29, 3249–3265. doi: 10.1093/hmg/ddaa212 Barres, B. A. (2008). The Mystery and Magic of Glia: A Perspective on Their Roles in Health and Disease. Neuron 60, 430–440. doi: 10.1016/j.neuron.2008.10. 013 Clarke, L. E., Liddelow, S. A., Chakraborty, C., Münch, A. E., and Heiman, M. (2018). Normal aging induces A1-like astrocyte reactivity. Proc. Natl. Acad. Sci. U. S. A. 115, E1896–E1905. Baum, M. L., Wilton, D. K., Muthukumar, A., Fox, R. G., Carey, A., Crotty, W., et al. (2020). CUB and Sushi Multiple Domains 1 (CSMD1) opposes the complement cascade in neural tissues. Biorxiv [Preprint]. doi: 10.1101/2020.09.11. 291427 Cocchi, E., Fabbri, C., Han, C., Lee, S. J., Patkar, A. A., Masand, P. S., et al. (2016). Genome-wide association study of antidepressant response: Involvement of the inorganic cation transmembrane transporter activity pathway. BMC Psychiatry 16:106. doi: 10.1186/s12888-016-0813-x Behmoaras, J., Bhangal, G., Smith, J., McDonald, K., Mutch, B., Lai, P. C., et al. (2008). Jund is a determinant of macrophage activation and is associated with glomerulonephritis susceptibility. Nat. Genet. 40, 553–559. Custer, S. K., Garden, G. A., Gill, N., Rueb, U., Libby, R. T., Schultz, C., et al. (2006). Bergmann glia expression of polyglutamine-expanded ataxin-7 produces neurodegeneration by impairing glutamate transport. Nat. Neurosci. 9, 1302–1311. References doi: 10.1038/nn1750 Bell, J. J. L., Lordkipanidze, T., and Cobb, N. (2010). Bergmann glial ensheathment of dendritic spines regulates synapse number without aﬀecting spine motility. Neuron Glia Biol. 6, 193–200. doi: 10.1017/S1740925X1000 0165 Cvetanovic, M. (2015). Decreased Expression of Glutamate Transporter GLAST in Bergmann Glia Is Associated with the Loss of Purkinje Neurons in the Spinocerebellar Ataxia Type 1. Cerebellum 14, 8–11. doi: 10.1007/s12311-014- 0605-0 Ben Haim, L., Schirmer, L., Zulji, A., Sabeur, K., Tiret, B., Ribon, M., et al. (2021). Evidence for glutamine synthetase function in mouse spinal cord oligodendrocytes. Glia 69, 2812–2827. Cvetanovic, M., Ingram, M., Orr, H., and Opal, P. (2015). Early activation of microglia and astrocytes in mouse models of spinocerebellar ataxia type 1. Neuroscience 289, 289–299. Boisvert, M. M., Erikson, G. A., Shokhirev, M. N., and Allen, N. J. (2018). The Aging Astrocyte Transcriptome from Multiple Regions of the Mouse Brain. Cell Rep. 22, 269–285. Cvetanovic, M., Patel, J. M., Marti, H. H., Kini, A. R., and Opal, P. (2011). Vascular endothelial growth factor ameliorates the ataxic phenotype in a mouse model of spinocerebellar ataxia type 1. Nat. Med. 17, 1445–1447. doi: 10.1038/nm. 2494 Bradl, M., and Lassmann, H. (2010). Oligodendrocytes: Biology and pathology. Acta Neuropathol. 119, 37–53. Burda, J. E., and Sofroniew, M. V. (2014). Reactive gliosis and the multicellular response to CNS damage and disease. Neuron 81, 229–248. doi: 10.1016/j.neuron. 2013.12.034 D’Agostino, M., Scerra, G., Cannata Serio, M., Caporaso, M. G., Bonatti, S., and Renna, M. (2019). Unconventional secretion of α-Crystallin B requires the Autophagic pathway and is controlled by phosphorylation of its serine 59 residue. Sci. Rep. 9:16892. doi: 10.1038/s41598-019-53226-x Burright, E. N., Clark, B. H., Servadio, A., Matilla, T., Feddersen, R. M., Yunis, W. S., et al. (1995). SCA1 transgenic mice: A model for neurodegeneration caused by an expanded CAG trinucleotide repeat. Cell 82, 937–948. doi: 10.1016/0092- 8674(95)90273-2 Dell’Orco, J. M., Wasserman, A. H., Chopra, R., Ingram, M. A. C., Hu, Y. S., Singh, V., et al. (2015). Neuronal Atrophy Early in Degenerative Ataxia Is a Compensatory Mechanism to Regulate Membrane Excitability. J. Neurosci. 35, 11292–11307. doi: 10.1523/JNEUROSCI.1357-15.2015 Bushart, D. D., and Shakkottai, V. G. (2018). Ion channel dysfunction in cerebellar ataxia. Neurosci. Lett. 688, 41–48. doi: 10.1016/j.neulet.2018.02.005688: 41-48 DelRosso, L. M., and Hoque, R. (2014). The cerebellum and sleep. Neurol. Clin. 32, 893–900. doi: 10.1016/j.ncl.2014.07.003 Bushart, D. D., Huang, H., Man, L. J., Morrison, L. M., and Shakkottai, V. G. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 17 frontiersin.org frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. SUPPLEMENTARY TABLE 1 Raw data from RTqPCR of PC and BG Genes, DNER intensity. SUPPLEMENTARY FIGURE 6 Changes in Shh signaling in Bergmann glia. (A) Ridgeplots showing distribution of expression of Ptch1, Ptch2, and Gli1 in BG population in wild-type and Pcp2-ATXN1[82Q] mice (N = 3 of each). P values adjusted using Benjamini-Hockberg method ≤0.05 for all three genes. (B) Quantitative RT-PCR of bulk cerebellar lysates from 12 weeks old wild-type and Pcp2-ATXN1[82Q] mice was used to evaluate expression of Ptch2 and Gli1 mRNA. Data is presented as mean ± SEM with average values for each mouse represented by a dot. N = 7, ∗p < 0.05 unpaired t test with Welch’s correction. (C) Ridgeplot showing distribution of Shh expression in hATXN1 + and hATXN1- PCs. P value adjusted using Benjamini-Hockberg method ≥0.05. DEG in PCs with detectable and not detectable expression of mutant human ATXN1. DEG in PCs with detectable and not detectable expression of mutant human ATXN1. References Microglial brain region -dependent diversity and selective regional sensitivities to aging. Nat. Neurosci. 19:504. doi: 10.1038/nn. 4222 Maria del Pilar, C. L., Marianna, S., Giusy, T., Shana, C., Deﬁllipi, P., and Cabodi, S. (2017). P130CAS/BCAR1 scaﬀold protein in tissue homeostasis and pathogenesis. Physiol. Behav. 176, 139–148. Grosche, J., Matyash, V., Möller, T., Verkhratsky, A., Reichenbach, A., and Kettenmann, H. (1999). Microdomains for neuron–glia interaction?: Parallel ﬁber signaling to Bergmann glial cells. Nat. Neurosci. 2, 139–143. doi: 10.1038/ 5692 Mathis, C., Collin, L., and Borrelli, E. (2003). Oligodendrocyte ablation impairs cerebellum development. Development 130, 4709–4718. Matson, K. J. E., Sathyamurthy, A., Johnson, K. R., Kelly, M. C., Kelley, M. W., and Levine, A. J. (2018). Isolation of adult spinal cord nuclei for massively parallel single-nucleus RNA sequencing. J. Vis. Exp. 2018:58413. doi: 10.3791/58413 Habib, N., Avraham-Davidi, I., Basu, A., Burks, T., Shekhar, K., Hofree, M., et al. (2017). Massively parallel single- nucleus RNA-seq with DroNc-seq. Nat. Methods 14, 955–958. doi: 10.1038/nmeth.4407 Matsuda, K., Matsuda, S., Gladding, C. M., and Yuzaki, M. (2006). Characterization of the δ2 glutamate receptor- binding protein delphilin: Splicing variants with diﬀerential palmitoylation and an additional PDZ domain. J. Biol. Chem. 281, 25577–25587. doi: 10.1074/jbc.M602044200 Heneka, M. T., Kummer, M. P., and Latz, E. (2014). Innate immune activation in neurodegenerative disease. Nat. Rev. Immunol. 14, 463–477. Herculano-Houzel, S. (2014). The Glia / Neuron Ratio?: How it Varies Uniformly Across Brain Structures and Species and What that Means for Brain Physiology and Evolution. Glia 62, 1377–1391. doi: 10.1002/glia.22683 McKenzie, I. A., Ohayon, D., Li, H., De Faria, J. P., Emery, B., Tohyama, K., et al. (2014). Motor skill learning requires active central myelination. Science 346, 318–322. doi: 10.1126/science.1254960 Hook, G., Jacobsen, J. S., Grabstein, K., Kindy, M., and Hook, V. (2015). Cathepsin B is a new drug target for traumatic brain injury therapeutics: Evidence for E64d as a promising lead drug candidate. Front. Neurol. 6:178. doi: 10.3389/ fneur.2015.00178 Mei, L., and Xiong, W. C. (2008). Neuregulin 1 in neural development, synaptic plasticity and schizophrenia. Nat. Rev. Neurosci. 9, 437–452. Mellesmoen, A., Sheeler, C., Ferro, A., Rainwater, O., and Cvetanovic, M. (2018). Brain derived neurotrophic factor (BDNF) delays onset of pathogenesis in transgenic mouse model of spinocerebellar ataxia type 1 (SCA1). Front. Cell. Neurosci. 12:509. doi: 10.3389/fncel.2018.00509 Ingram, M., Wozniak, E. A. L., Duvick, L., Yang, R., Bergmann, P., Carson, R., et al. (2016a). References A. (2021). Chlorzoxazone-Baclofen Combination Improves Cerebellar Impairment in Spinocerebellar Ataxia Type 1. Mov. Disord. 36, 622–631. doi: 10.1002/mds. 28355 Diallo, A., Jacobi, H., Cook, A., Giunti, P., Parkinson, M. H., Labrum, R., et al. (2019). Prediction of Survival With Long- Term Disease Progression in Most Common Spinocerebellar Ataxia. Mov. Disord. 34, 1220–1227. doi: 10.1002/mds. 27739 Carmon, K. S., Lin, Q., Gong, X., Thomas, A., and Liu, Q. (2012). LGR5 Interacts and Cointernalizes with Wnt Receptors To Modulate Wnt/β-Catenin Signaling. Mol. Cell. Biol. 32, 2054–2064. Diallo, A., Jacobi, H., Schmitz-Hubsch, T., Cook, A., Labrum, R., Durr, A., et al. (2017). Body Mass Index Decline Is Related to Spinocerebellar Ataxia Disease Progression. Mov. Disord. 4, 689–697. doi: 10.1002/mdc3.12522 Cerrato, V., Mercurio, S., Leto, K., Fucà, E., Hoxha, E., Bottes, S., et al. (2018). Sox2 conditional mutation in mouse causes ataxic symptoms, cerebellar vermis hypoplasia, and postnatal defects of Bergmann glia. Glia 66, 1929–1946. doi: 10.1002/glia.23448 Djukic, B., Casper, K. B., Philpot, B. D., Chin, L. S., and McCarthy, K. D. (2007). Conditional Knock-Out of Kir4.1 Leads to Glial Membrane Depolarization, Inhibition of Potassium and Glutamate Uptake, and Enhanced Short- Term Frontiers in Cellular Neuroscience 18 frontiersin.org 10.3389/fncel.2022.998408 Borgenheimer et al. in a biphasic, stage-of-disease speciﬁc manner. Glia 66, 1972–1987. doi: 10.1002/ glia.23451 Synaptic Potentiation. J. Neurosci. 27, 11354–11365. doi: 10.1523/JNEUROSCI. 0723-07.2007 Driessen, T. M., Lee, P. J., and Lim, J. (2018). Molecular pathway analysis towards understanding tissue vulnerability in spinocerebellar ataxia type 1. Elife. 7:e39981. doi: 10.7554/eLife.39981 Klein, A. M., Mazutis, L., Akartuna, I., Tallapragada, N., Veres, A., Li, V., et al. (2015). Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187–1201. doi: 10.1016/j.cell.2015.04.044 Eiraku, M., Tohgo, A., Ono, K., Kaneko, M., Fujishima, K., Hirano, T., et al. (2005). DNER acts as a neuron-speciﬁc Notch ligand during Bergmann glial development. Nat. Neurosci. 8, 873–880. doi: 10.1038/nn1492 Kofuji, P., and Newman, E. A. (2004). Potassium buﬀering in the central nervous system. Neuroscience 129, 1045–1056. Kozareva, V., Martin, C., Osorno, T., Rudolph, S., Guo, C., Vanderburg, C., et al. (2020). A transcriptomic atlas of the mouse cerebellum reveals regional specializations and novel cell types. Biorxiv [Preprint]. doi: 10.1101/2020.03.04. 976407 El Hajj, N., Dittrich, M., and Haaf, T. (2017). Epigenetic dysregulation of protocadherins in human disease. Semin. Cell Dev. Biol. 69, 172–182. doi: 10.1016/ j.semcdb.2017.07.007 Escartin, C., Galea, E., Lakatos, A., O’Callaghan, J. P., Petzold, G. References C., Serrano- Pozo, A., et al. (2021). Reactive astrocyte nomenclature, deﬁnitions, and future directions. Nat. Neurosci. 24, 312–325. doi: 10.1038/s41593-020-00783-4 Kuipers, H. F., Yoon, J., Van Horssen, J., Han, M. H., Bollyky, P. L., Palmer, T. D., et al. (2017). Phosphorylation of αB- crystallin supports reactive astrogliosis in demyelination. Proc. Natl. Acad. Sci. U. S. A. 114, E1745–E1754. doi: 10.1073/ pnas.1621314114 Farmer, W. T., Chierzi, S., Lui, C., Zaelzer, C., Jones, E. V., Bally, B. P., et al. (2016). Neurons diversify astrocytes in the adult brain through sonic hedgehog signaling. Science 351, 849–854. doi: 10.1126/science.aab3103 Lacar, B., Linker, S. B., Jaeger, B. N., Krishnaswami, S., Barron, J., Kelder, M., et al. (2016). Nuclear RNA-seq of single neurons reveals molecular signatures of activation. Nat. Commun. 7:11022. Friedrich, J., Henzler, C., Orr, H. T., Friedrich, J., Kordasiewicz, H. B., Callaghan, B. O., et al. (2018). Antisense oligonucleotide–mediated ataxin-1 reduction prolongs survival in SCA1 mice and reveals disease- associated transcriptome proﬁles. J. Clin. Investig. 3:e123193. doi: 10.1172/jci.insight.123193 Liddelow, S. A., Guttenplan, K. A., Clarke, L. E., Bennett, F. C., Bohlen, C. J., Schirmer, L., et al. (2017). Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487. doi: 10.1038/nature21029 Funa, K., and Sasahara, M. (2014). The roles of PDGF in development and during neurogenesis in the normal and diseased nervous system. J. Neuroimmune Pharmacol. 9, 168–181. doi: 10.1007/s11481-013-9479-z Liu, C. J., Rainwater, O., Clark, H. B., Orr, H. T., and Akkin, T. (2018). Polarization-sensitive optical coherence tomography reveals gray matter and white matter atrophy in SCA1 mouse models. Neurobiol. Dis. 116, 69–77. doi: 10.1016/j.nbd.2018.05.003 Furuichi, T., Shiraishi-Yamaguchi, Y., Sato, A., Sadakata, T., Huang, J., Shinoda, Y., et al. (2011). Systematizing and cloning of genes involved in the cerebellar cortex circuit development. Neurochem. Res. 36, 1241–1252. doi: 10.1007/s11064- 011-0398-1 Liu, Y., Zhou, Q., Tang, M., Fu, N., Shao, W., Zhang, S., et al. (2015). Upregulation of alphaB-crystallin expression in the substantia nigra of patients with Parkinson’s disease. Neurobiol. Aging 36, 1686–1691. doi: 10.1016/j. neurobiolaging.2015.01.015 Gatchel, J. R., and Zoghbi, H. Y. (2005). Diseases of unstable repeat expansion: Mechanisms and common principles. Nat. Rev. Genet. 6, 743–755. doi: 10.1038/ nrg1691 Lobsiger, C. S., and Cleveland, D. W. (2007). Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease. Nat. Neurosci. 10, 1355–1360. doi: 10.1038/nn1988 Grabert, K., Michoel, T., Karavolos, M. H., Clohisey, S., Baillie, J. K., Stevens, M. P., et al. (2016). References G., Nguyen, B., Luttik, K., Lee, C., Gionco, J., et al. (2021). Longitudinal single-cell transcriptional dynamics throughout neurodegeneration in SCA1. Biorxiv [Preprint]. doi: 10.1101/2021.10.22.465444 Tohgo, A., Eiraku, M., Miyazaki, T., Miura, E., Kawaguchi, S. Y., Nishi, M., et al. (2006). Impaired cerebellar functions in mutant mice lacking DNER. Mol. Cell. Neurosci. 31, 326–333. doi: 10.1016/j.mcn.2005.10.003 Park, Y. W., Joers, J. M., Guo, B., Hutter, D., Bushara, K., Adanyeguh, I. M., et al. (2020). Assessment of Cerebral and Cerebellar White Matter Microstructure in Spinocerebellar Ataxias 1, 2, 3, and 6 Using Diﬀusion MRI. Front. Neurol. 11:411. doi: 10.3389/fneur.2020.00411 Tsai, H. F., Chang, Y. C., Li, C. H., Chan, M. H., Chen, C. L., Tsai, W. C., et al. (2021). Type V collagen alpha 1 chain promotes the malignancy of glioblastoma through PPRC1-ESM1 axis activation and extracellular matrix remodeling. Cell Death Discov. 7:313. doi: 10.1038/s41420-021-00661-3 Parkhurst, C. N., Yang, G., Ninan, I., Savas, J. N., Yates, J. R., Lafaille, J. J., et al. (2013). Microglia promote learning- dependent synapse formation through brain-derived neurotrophic factor. Cell 155, 1596–1609. doi: 10.1016/j.cell.2013. 11.030 Verkhratsky, A., and Nedergaard, M. (2018). Physiology of Astroglia. Physiol. Rev. 98, 239–389. Pedroso, J. L., Braga-Neto, P., Felício, A. C., Aquino, C. C. H., do Prado, L. F. B., do Prado, G. F., et al. (2011). Sleep disorders in cerebellar ataxias. Arq. Neuropsiquiatr. 69, 253–257. Wang, X., Allen, M., Li, S., Quicksall, Z. S., Patel, T. A., Carnwath, T. P., et al. (2020). Deciphering cellular transcriptional alterations in Alzheimer’s disease brains. Mol. Neurodegener. 15:38. Pekny, M., Wilhelmsson, U., and Pekna, M. (2014). The dual role of astrocyte activation and reactive gliosis. Neurosci. Lett. 565, 30–38. doi: 10.1016/j.neulet. 2013.12.071 White, J. J., Bosman, L. W. J., Blot, F. G. C., Osório, C., Kuppens, B. W., Krijnen, W. H. J. J., et al. (2021). Region-speciﬁc preservation of Purkinje cell morphology and motor behavior in the ATXN1[82Q] mouse model of spinocerebellar ataxia 1. Brain Pathol. 31:e12946. doi: 10.1111/bpa.12946 Perea, G., Sur, M., and Araque, A. (2014). Neuron-glia networks: Integral gear of brain function. Front. Cell. Neurosci. 8:378. doi: 10.3389/fncel.2014.00378 Wieczorek, L., Majumdar, D., Wills, T. A., Hu, L., Winder, D. G., Webb, D. J., et al. (2012). Absence of Ca 2+-stimulated adenylyl cyclases leads to reduced synaptic plasticity and impaired experience-dependent fear memory. Transl. Psychiatry 2:e126. doi: 10.1038/tp.2012.50 Robeck, T., Skryabin, B. V., Rozhdestvensky, T. S., Skryabin, A. B., and Brosius, J. (2016). References Cerebellar Transcriptome Proﬁles of ATXN1 Transgenic Mice Reveal SCA1 Disease Progression and Protection Pathways. Neuron 89, 1194– 1207. doi: 10.1016/j.neuron.2016.02.011 Miyazaki, T., Yamasaki, M., Hashimoto, K., Kohda, K., Yuzaki, M., Shimamoto, K., et al. (2017). Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells. Proc. Natl. Acad. Sci. U. S. A. 114, 7438–7443. doi: 10.1073/pnas.1617330114 Ingram, M., Wozniak, E. A. L., Duvick, L., Zoghbi, H. Y., Henzler, C., and Orr, H. T. (2016b). Cerebellar Transcriptome Proﬁles of ATXN1 Transgenic Mice Reveal SCA1 Disease Progression and Protection Pathways. Neuron 89, 1194–1207. doi: 10.1016/j.neuron.2016.02.011 Moon, S., and Zhao, Y.-T. (2021). Spatial, temporal and cell-type-speciﬁc expression proﬁles of genes encoding heparan sulfate biosynthesis enzymes and proteoglycan core proteins. Glycobiology 31, 1308–1318. doi: 10.1093/glycob/ cwab054 Jacobi, H., Reetz, K., du Montcel, S. T., Bauer, P., Mariotti, C., Nanetti, L., et al. (2013). Biological and clinical characteristics of individuals at risk for spinocerebellar ataxia types 1, 2, 3, and 6 in the longitudinal RISCA study: Analysis of baseline data. Lancet Neurol. 12, 650–658. doi: 10.1016/S1474-4422(13)70104-2 Moriarty, A., Cook, A., Hunt, H., Adams, M. E., Cipolotti, L., and Giunti, P. (2016). A longitudinal investigation into cognition and disease progression in spinocerebellar ataxia types 1, 2, 3, 6, and 7. Orphanet J. Rare Dis. 11:82. doi: 10.1186/s13023-016-0447 Janeczko, K. (1993). Co-expression of gfap and vimentin in astrocytes proliferating in response to injury in the mouse cerebral hemisphere. A combined autoradiographic and double immunocytochemical study. Int. J. Dev. Neurosci. 11, 139–147. doi: 10.1016/0736-5748(93)90074-n National Research Council (Us) Committee for the Update of the Guide for the Care and Use of Laboratory Animals (2011). Guide for the Care and Use of Laboratory Animals, 8th Edn. Washington, DC: National Academies Press. Kaur, G., and Levy, E. (2012). Cystatin C in Alzheimer’s disease. Front. Mol. Neurosci. 5:79. doi: 10.3389/fnmol.2012.00079 Ng, S. Y., Bogu, G. K., Soh, B. S., and Stanton, L. W. (2013). The long noncoding RNA RMST interacts with SOX2 to regulate neurogenesis. Mol. Cell. 51, 349–359. doi: 10.1016/j.molcel.2013.07.017 Kim, J. H., Lukowicz, A., Qu, W., Johnson, A., and Cvetanovic, M. (2018). Astroglia contribute to the pathogenesis of spinocerebellar ataxia Type 1 (SCA1) Frontiers in Cellular Neuroscience 19 frontiersin.org Borgenheimer et al. 10.3389/fncel.2022.998408 Nicita, F., Tasca, G., Nardella, M., Bellacchio, E., Camponeschi, I., Vasco, G., et al. (2018). Novel Homozygous KCNJ10 Mutation in a Patient with Non- syndromic Early-Onset Cerebellar Ataxia. References Cerebellum 17, 499–503. doi: 10.1007/ s12311-018-0924-7 Servadio, A., Koshy, B., Armstrong, D., Antalﬀy, B., Orr, H. T., and Zoghbi, H. Y. (1995). Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals. Nat. Genet. 10, 94–98. Sheeler, C., Rosa, J., Borgenheimer, E., Mellesmoen, A., and Rainwater, O. (2021). Post-symptomatic Delivery of Brain- Derived Neurotrophic Factor (BDNF) Ameliorates Spinocerebellar Ataxia Type 1 (SCA1) Pathogenesis. Cerebellum 20, 420–429. doi: 10.1007/s12311-020-01226-3 O’Leary, L. A., Davoli, M. A., Belliveau, C., Tanti, A., Ma, J. C., Farmer, W. T., et al. (2020). Characterization of Vimentin- Immunoreactive Astrocytes in the Human Brain. Front. Neuroanat. 14:31. doi: 10.3389/fnana.2020.00031 Ophir, G., Meilin, S., Efrati, M., Chapman, J., Karussis, D., Roses, A., et al. (2003). Human apoE3 but not apoE4 rescues impaired astrocyte activation in apoE null mice. Neurobiol. Dis. 12, 56–64. Sheeler, C., Rosa, J., Ferro, A., Mcadams, B., Borgenheimer, E., and Cvetanovic, M. (2020). Glia in Neurodegeneration?:The Housekeeper, the Defender and the Perpetrator. Int. J. Mol. Sci. 21:9188. doi: 10.3390/ijms21239188 Orr, H. T., and Zoghbi, H. Y. (2007). Trinucleotide Repeat Disorders. Annu. Rev. Neurosci. 30, 575–621. Sochacka, M., Opalinski, L., Szymczyk, J., Zimoch, M. B., Czyrek, A., Krowarsch, D., et al. (2020). FHF1 is a bona ﬁde ﬁbroblast growth factor that activates cellular signaling in FGFR-dependent manner. Cell Commun. Signal. 18:69. doi: 10.1186/ s12964-020-00573-2 Orr, H. T., Chung, M. Y., Banﬁ, S., Kwiatkowski, T. J. Jr., Servadio, A., Beaudet, A. L., et al. (1993). Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nat. Genet. 4, 221–226. Soreq, L., Brain, U. K. C., North American, B. E., Rose, J., Patani, R., and Ule, J. (2017). Major Shifts in Glial Regional Identity Are a Transcriptional Hallmark of Human Brain Aging. Cell Rep. 18, 557–570. doi: 10.1016/j.celrep.2016.12.011 Ousman, S. S., Tomooka, B. H., Van Noort, J. M., Wawrousek, E. F., O’Conner, K., Haﬂer, D. A., et al. (2007). Protective and therapeutic role for αB-crystallin in autoimmune demyelination. Nature 448, 474–479. doi: 10.1038/nature 05935 Tafti, M., and Ghyselinck, N. B. (2007). Functional implication of the vitamin A signaling pathway in the brain. Arch. Neurol. 64, 1706–1711. Parcerisas, A., Ortega-Gascó, A., Pujadas, L., and Soriano, E. (2021). The hidden side of ncam family: Ncam2, a key cytoskeleton organization molecule regulating multiple neural functions. Int. J. Mol. Sci. 22:10021. doi: 10.3390/ijms22181 0021 Tejwani, L., Ravindra, N. References BC1 RNA motifs required for dendritic transport in vivo. Sci. Rep. 6:28300. doi: 10.1038/srep28300 Xin, W., Mironova, Y. A., Shen, H., Marino, R. A. M., Waisman, A., Lamers, W. H., et al. (2019). Oligodendrocytes Support Neuronal Glutamatergic Transmission via Expression of Glutamine Synthetase. Cell Rep. 27, 2262–2271.e5. doi: 10.1016/j.celrep.2019.04.094 Rosa, J., Hamel, K., Sheeler, C., Borgenheimer, E., Soles, A., Ghannoum, F., et al. (2021). Early stage of Spinocerebellar Ataxia Type 1 (SCA1) progression exhibits region- and cell-speciﬁc pathology and is partially ameliorated by Brain Derived Neurotrophic Factor (BDNF). Biorxiv [Preprint]. doi: 10.1101/2021.09.13.460129 Zawi¸slak, A., Jakimowicz, P., McCubrey, J. A., and Rakus, D. (2017). Neuron- derived transthyretin modulates astrocytic glycolysis in hormone-independent manner. Oncotarget 8, 106625–106638. doi: 10.18632/oncotarget.22542 Rothstein, J., Dykes-Hoberg, M., Pardo, C., Bristol, L., Jin, L., Kuncl, R., et al. (1996). Antisense knockout of glutamate transporters reveals a predominant role for astroglial glutamate transport in excitotoxicity and clearance of extracellular glutamate. Neuron 16, 675–686. doi: 10.1016/s0896-6273(00)80086-0 Zhang, Y., Wang, J., Liu, X., and Liu, H. (2020). Exploring the role of RALYL in Alzheimer’s disease reserve by network- based approaches. Alzheimers Res. Ther. 12:165. doi: 10.1186/s13195-020-00733-z Rüb, U., Schöls, L., Paulson, H., Auburger, G., Kermer, P., Jen, J. C., et al. (2013). Clinical features, neurogenetics and neuropathology of the polyglutamine spinocerebellar ataxias type 1, 2, 3, 6 and 7. Prog. Neurobiol. 104, 38–66. doi: 10.1016/j.pneurobio.2013.01.001 Zhong, J., Chuang, S. C., Bianchi, R., Zhao, W., Lee, H., Fenton, A. A., et al. (2009). BC1 regulation of metabotropic glutamate receptor-mediated neuronal excitability. J. Neurosci. 29, 9977–9986. doi: 10.1523/JNEUROSCI.3893-08.2009 Sakers, K., Liu, Y., Llaci, L., Lee, S. M., Vasek, M. J., Rieger, M. A., et al. (2021). Loss of Quaking RNA binding protein disrupts the expression of genes associated with astrocyte maturation in mouse brain. Nat. Commun. 12:1537. doi: 10.1038/s41467-021-21703-5 Zhou, H., Lin, Z., Voges, K., Ju, C., Gao, Z., Bosman, L. W. J., et al. (2014). Cerebellar modules operate at diﬀerent frequencies. Elife. 3:e02536. Zhou, L., Tang, X., Li, X., Bai, Y., Buxbaum, J. N., and Chen, G. (2019). Identiﬁcation of transthyretin as a novel interacting partner for the δ subunit of GABA A receptors. PLoS One 14:e0210094. doi: 10.1371/journal.pone.0210094 Saunders, A., Macosko, E. Z., Wysoker, A., Goldman, M., Krienen, F. M., de Rivera, H., et al. (2018). Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain. Cell 174, 1015–1030.e16. References doi: 10.1016/j.cell.2018.07.028 Zilionis, R., Nainys, J., Veres, A., Savova, V., Zemmour, D., Klein, A. M., et al. (2017). Single-cell barcoding and sequencing using droplet microﬂuidics. Nat. Protoc. 12, 44–73. Sen, A., Nelson, T. J., and Alkon, D. L. (2017). ApoE isoforms diﬀerentially regulates cleavage and secretion of BDNF. Mol. Brain 10:19. doi: 10.1186/s13041- 017-0301-3 Zou, J., Chen, Z., Wei, X., Chen, Z., Fu, Y., Yang, X., et al. (2017). Cystatin C as a potential therapeutic mediator against Parkinson’s disease via VEGF-induced angiogenesis and enhanced neuronal autophagy in neurovascular units. Cell Death Dis. 8:e2854. doi: 10.1038/cddis.2017.240 Serra, H. G., Byam, C. E., Lande, J. D., Tousey, S. K., Zoghbi, H. Y., and Orr, H. T. (2004). Gene proﬁling links SCA1 pathophysiology to glutamate signaling in Purkinje cells of transgenic mice. Hum. Mol. Genet. 13, 2535–2543. doi: 10.1093/ hmg/ddh268 Zu, T., Duvick, L. A., Kaytor, M. D., Berlinger, M. S., Zoghbi, H. Y., Clark, H. B., et al. (2004). Recovery from polyglutamine- induced neurodegeneration in conditional SCA1 transgenic mice. J. Neurosci. 24, 8853–8861. doi: 10.1523/ JNEUROSCI.2978-04.2004 Serra, H. G., Duvick, L., Zu, T., Carlson, K., Stevens, S., Jorgensen, N., et al. (2006). ROR a -Mediated Purkinje Cell Development Determines Disease Severity in Adult SCA1 Mice. Cell 1, 697–708. doi: 10.1016/j.cell.2006.09.036 20 Frontiers in Cellular Neuroscience frontiersin.org
https://openalex.org/W4237003402	https://www.researchsquare.com/article/rs-117672/v1.pdf?c=1631880691000	English	null	Macular Perfusion Analysed by Optical Coherence Tomography Angiography after Uncomplicated Phacoemulsification: Benefits beyond Restoring Vision	Research Square (Research Square)	2,020	cc-by	7,485	Macular Perfusion Analysed by Optical Coherence Tomography Angiography after Uncomplicated Phacoemulsification: Benefits beyond Restoring Vision Ana Križanović Klinička bolnica "Sveti Duh" Mirjana Bjeloš (  dr.mbjelos@gmail.com ) Klinička bolnica "Sveti Duh" Mladen Bušić Klinička bolnica "Sveti Duh" Biljana Kuzmanović Elabjer Klinička bolnica "Sveti Duh" Benedict Rak Klinička bolnica "Sveti Duh" Nenad Vukojević University Hospital Centre Zagreb Ana Križanović Klinička bolnica "Sveti Duh" Mirjana Bjeloš (  dr.mbjelos@gmail.com ) Klinička bolnica "Sveti Duh" Research Article DOI: https://doi.org/10.21203/rs.3.rs-117672/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on February 5th, 2021. See the published version at https://doi.org/10.1186/s12886-021-01837-2. Page 1/18 Page 1/18 Abstract Background: The purpose of the study is to investigate the changes of macular perfusion by OCT-angiography (OCT-A) after uncomplicated phacoemulsification. Background: The purpose of the study is to investigate the changes of macular perfusion by OCT-angiography (OCT-A) after uncomplicated phacoemulsification. Methods: OCT-A was performed before cataract surgery, 1 week, 1 month, and 3 months after surgery recording superficial vascular complex (SVC), nerve fiber layer vascular plexus (NFLVP) superior vascular plexus (SVP), deep vascular complex (DVC), intermediate capillary plexus (ICP) and deep capillary plexus (DCP), as well as large choroidal blood vessels and choriocapillaris (CC) were recorded. Explant area (EA), vessels area (VA), vessels percentage area (VPA), total number of junctions (TNJ), junctions density (JD), total vessels length (TVL), average vessels length (AVL), total number of end points (TNEP), mean E lacunarity (MEL), and foveal avascular zone (FAZ) area throughout all layers were analysed. Results: Significant changes of vascular parameters in 55 eyes of 55 patients mostly reached plateau one week after surgery and remained stable up to three months after surgery, occurring in all retinal layers but not in choroid and choriocapillaris. Changes of FAZ area ensued across all retinal layers within one month after surgery. The greatest increase in VPA (22.79%), TVL (16.71%), AVL (166.71%) and JD (29.49%) was in SVC causing the largest FAZ surface decrease (-25.54%) in SVC likewise. On the contrary, the greatest change of MEL (-53.41%) appeared in DVC. Conclusions: This is the first OCT-A study demonstrating that perfusion alterations observed in macula after phacoemulsification are response to functional hyperaemia prompted by augmented visual stimulation. Phacoemulsification in elderly population proved as advantageous feature in addition to restoring visual acuity. Background The exact impact of phacoemulsification, one of the most common surgical procedures in the world [1], on macular perfusion is unknown [2, 3]. Changes in arterial blood pressure, position of blood vessels, venous return of blood and CO2 levels all influence eye perfusion during cataract surgery [4]. Within retina, one of the tissues with the highest metabolic requirements in the body [5, 6], three layers of the retinal blood vessels exist: superficial, deep, and intermediate layer [6]. Superficial vascular complex (SVC), formed of nerve fiber layer vascular plexus (NFLVP) and superficial vascular plexus (SVP), is located in the nerve fiber layer (NFL) and ganglion cell layer (GCL) respectively [5]. Deep vascular complex (DVC), formed of intermediate capillary plexus (ICP) and deep capillary plexus (DCP), is located deeper in the inner nuclear (INL) and outer plexiform layer (OPL) [5]. Inner plexiform layer (IPL) is thus supported by two capillary networks: SVP and ICP. The superficial layer contains arterioles, venules, and capillaries, while the deep layer consists of capillary-sized blood vessels [5]. The aim of this study was to investigate the changes of macular perfusion analyzed by optical coherence tomography angiography (OCT-A) after uncomplicated phacoemulsification in normal ageing subjects. If such changes do exist, it is necessary to define whether they are favorable or adverse and thus to provide an evidence based recommendation for the indication of cataract surgery if patients can reach further benefits beyond improvement in visual acuity. Methods All patients underwent history taking, determination of best corrected visual acuity (BCVA), auto-refractokeratometry, endothelial biomicroscopy, optical biometry, arterial pressure measurement, IOP measurement by Goldmann tonometry, optical coherence tomography (OCT) of the macula, fundus examination in mydriasis and OCT-A imaging. BCVA was determined using the ETDRS table and recorded in the logMAR unit [8]. Corneal endothelial cells were analyzed by specular biomicroscope (CEM-530, Nidek, Gamagori, Japan). The AL was determined using optical biometer (IOLMaster 700®, Zeiss, Oberkochen, Germany). Arterial pressure was measured with a digital pressure gauge (M6 Comfort, Omron, Kyoto, Japan) and IOP by Goldmann applanation tonometry. Mean arterial pressure (MAP) and ocular perfusion pressure (OPP) were calculated using MAP = (SBP + 2 x DBP) / 3 and OPP = 2 x (MAP - IOP) / 3 formulas respectively. Examination of the anterior segment of the eye was performed using a slit lamp. Tropicamide 1% (Mydriacyl®, Alcon Laboratories Inc., Geneva, Switzerland) was dripped into each eye of the patient three times at intervals of 15 minutes to maximize mydriasis and cycloplegia. OCT (FAST mode recording 20 x 20° of the central macula using 512 A-scans x 25 sections with 240 µm distance between sections) and autofluorescence (multi-color, infrared, blue and green) were recorded using HRA+OCT Spectralis® (Heidelberg Engineering, Heidelberg, Germany). Each patient underwent objective refraction measuring using auto-refractokeratometer (Righton Speedy-K Autorefractor Keratometer, Right Group, Tokyo, Japan), imaging on Pentacam® HR device (OCULUS Optikgeräte GmbH, Wetzlar, Germany) to calculate PNS and fundus examination with an indirect ophthalmoscope. OCT-A was performed on HRA+OCT Spectralis® device recording 10 x 10˚ of the central macula, accordingly 2.9 x 2.9 mm, with 512 A-scans x 512 sections, 6 µm distance between sections and resolution of 5.7 µm/pixel. Automated segmentation performed with integrated Spectralis software of superficial layer (NFLVP and SVP forming SVC) and deep layer (DCP and ICP forming DVC) of retinal vessels, CC and large choroidal blood vessels is considered reliable in case of unremarkable retinal layer pathology [9]. Recording was done using high resolution mode, reported for low measurement variability [10, 11]. In case of possible misleading artefacts, the measurement was repeated. Images with quality index Q ≥ 30, as computed by integrated software, were further analyzed and carefully reviewed by two graders for accurate segmentation of the vascular layers and presence of artefacts prior to final analysis [9]. Patients The ain of the study was to investigate the changes of macular perfusion by OCT-A after uncomplicated phacoemulsification. The study was conducted at the Department of Ophthalmology, University Hospital "Sveti Duh" Zagreb on one group of patients scheduled to have microincision phacoemulsification under topical anesthesia. All Page 2/18 patients were operated by the same surgeon (BKE) and monitored and analyzed by one examiner (AK). The analyzed parameters refer to only one eye. The inclusion criteria for the study were: 1. uncomplicated senile cataract; 2. cataract Pentacam® Nucleus Staging (PNS) assessed as 1, 2 or 3 by Oculus Pentacam® nucleus grading system; 3. Quality index (Q) of OCT-A images ≥ 30; 4. axial length (AL) 20-25 mm measured by optical biometry; 5. intraocular pressure (IOP) measured by Goldmann tonometry 10- 21 mmHg; 6. systolic blood pressure (SBP) ≥ 90 and ≤ 140 mmHg, and diastolic blood pressure (DBP) ≥ 60 and ≤ 90 mmHg. The exclusion criteria for the study were: corneal diseases, pseudoexfoliation syndrome, cataracts other than uncomplicated senile, glaucoma, age-related macular degeneration, signs of hypertensive retinopathy [7], degenerative myopia, diabetes, intraoperative and/or postoperative complications, poor image quality. Results 59 patients met the inclusion criteria, yet 55 eyes of 55 patients were included in final analysis. One patient developed postoperative uveitis and was excluded from further analysis. Two patients missed the follow-up appointments. One patient developed pseudophakic macular edema (PME) one month after surgery based on OCT criteria [14], with no signs of PME three months after surgery. General characteristics of the participants, and surgery parameters are presented in the Additional file 1: Table S1. Methods Patients were evaluated before and 7 days, 1 month, and 3 months after surgery. Images were exported to Angiotool 0.6 software for quantitative analysis [12]. A vessel is defined after segmentation as a segment between two branching points or a branching point and an end point [12]. Analyzed vascular parameters include: explant area (EA): the analyzed area; vessels area (VA): area of the segmented vessels; vessels percentage area (VPA): percentage of area containing vessels inside the explant area (VA/EA); total Page 3/18 Page 3/18 number of junctions (TNJ): total number of vessels junctions in the image; junctions’ density (JD): number of vessel junctions per unit area (branch points/unit area); total vessels length (TVL): sum of Euclidean distances between the pixels of all the vessels in the image; average vessels length (AVL): mean length of all the vessels in the image; total number of end points (TNEP): number of open-ended segments; mean lacunarity (ML): mean lacunarity over all size boxes. number of junctions (TNJ): total number of vessels junctions in the image; junctions’ density (JD): number of vessel junctions per unit area (branch points/unit area); total vessels length (TVL): sum of Euclidean distances between the pixels of all the vessels in the image; average vessels length (AVL): mean length of all the vessels in the image; total number of end points (TNEP): number of open-ended segments; mean lacunarity (ML): mean lacunarity over all size boxes. Foldable intraocular lens (Zeiss CT Lucia 611PY or AMO Tecnis PCB00) were implanted. Total cumulative dissipated energy (CDE) and total ultrasound time (PHACO time) were automatically recorded using the Centurion® Vision System (Alcon Inc., Fort Worth, USA) [13]. Postoperatively dexamethasone 0.1% drops (Maxidex®, Alcon Laboratories Inc., Geneva, Switzerland) were prescribed q.i.d. for 7 days followed by b.i.d. for 7 days. General parameters IOP before surgery was significantly higher than one week, one month and three months after surgery. IOP one week after surgery was significantly higher than one month and three months after surgery. SBP before surgery was significantly higher than one month and three months after surgery. BCVA significantly improved one week after surgery, with further significant improvement when comparing one week and three months after surgery. DBP, MAP and OPP did not display any change (Additional file 1: Table S2). Statistical analysis Statistical analysis was performed using MedCalc statistical software (MedCalc Software Ltd, Ostend, Belgium). Data are presented by median and interquartile ranges. A comparison of pre-operative values with values obtained one week, one month, and three months after surgery was made using non-parametric Friedman ANOVA test. Inter-layer comparison of vascular parameters three months after surgery was made using Student's t test. The significance level was set to P < 0.05. Quality index Quality of OCT-A images before and after phacoemulsification demonstrated no significant changes (Table 1). Page 4/18 Page 4/18 Page 4/18 Table 1 Quality index of OCT-A images before and after phacoemulsification OCT-A image Before 1 week after 1 month after 3 months after P Quality index 34 (31-37.5) 34 (32-36.8) 35 (32.25-37.75) 35 (33-38) 0.170 OCT-A optical coherence tomography angiography, Q quality index. This table shows median and interquartile ranges for quality index (25th and 75th percentiles). Friedman ANOVA test, the significance level was set to P < 0.05. Vascular parameters No difference was found in EA. An increase in parameters: VA, VPA, TNJ, JD, TVL and AVL was found in all layers except in CC and choroid. A decrease in parameter TNEP was found in all layers except in choroid and NFLVP. ML decreased in all layers but choroid. The most pronounced changes were found in NFLVP and SVC. Average vessels length has undergone the greatest change in all layers except in NFLVP. For most vascular parameters a statistically significant difference was observed between values obtained before surgery and values obtained one week, one month, and three months after surgery within which there was no statistically significant difference (Additional file 1: Table S3; Table S4). ber layer vascular plexus, superficial vascular plexus and superficial vascular complex Nerve fiber layer vascular plexus, superficial vascular plexus and superficial vascular complex In NFLVP layer, VA, VPA, TNJ, JD, TVL and AVL one week after surgery were lower than one month and three months after surgery, within which there was no significant difference (Table 2). For TNEP no alterations were detected (Table 2). In NFLVP layer, VA, VPA, TNJ, JD, TVL and AVL one week after surgery were lower than one month and three months after surgery, within which there was no significant difference (Table 2). For TNEP no alterations were detected (Table 2). In NFLVP layer, VA, VPA, TNJ, JD, TVL and AVL one week after surgery were lower than one month and three months after surgery, within which there was no significant difference (Table 2). For TNEP no alterations were detected (Table 2). LVP nerve fiber layer vascular plexus, EA explant area, VA vessels area, VPA vessels percentage area, TNJ total num e lower one week compared to one and three months after surgery, within which there was no significant difference In SVP and SVC an increase in VA, VPA, TNJ, JD and TVL with decrease in TNEP and ML was found one week postoperatively (Table 3; Table 4). In addition, TNEP was higher one week after surgery than three months after surgery (Table 3; Table 4). In SVP layer, TVL one week after surgery was lower than three months after surgery (Table 3). In SVC layer TNJ, JD, TVL and AVL values were lower one week compared to three months after surgery (Table 4). sity, TVL total vessels length, AVL average vessels length, TNEP total number of end points, ML mean lacunarity. , significant difference (bold values) was found for values with P ˂ 0.05. Observed values were lower one week compared to one and three months after surgery, within which there was n Friedman ANOVA test, significant difference (bold values) was found for values with P ˂ 0.05. Vascular parameters Page 5/18 Table 2 Statistical analysis of changes in vascular parameters in nerve fiber layer vascular plexus NFLVP Before 1 week after 1 month after 3 months after P Bias EA (mm2) 8.3791 (8.3755- 8.3804) 8.3796 (8.3770- 8.3805) 8.3794 (8.3768- 8.3807) 8.3794 (8.3759- 8.3803) 0.740 0.05% VA (mm2) 1.7746 (1.3408- 2.4250) 2.3389 (1.7994- 2.7072) 2.4731 (2.0283- 3.0603) 2.4584 (1.9830- 2.9862) <0.001 31.72%* VPA (%) 21.1911 (16.0203- 28.9405) 27.9786 (21.4717- 32.3195) 29.5066 (24.2454- 36.5190) 29.3376 (23.6625- 35.6347) <0.001* 31.67%* TNJ 456 (300-725) 692 (457-871) 781 (583-1000) 729 (558-996) <0.001* 44.48%* JD (junctions/mm2) 54.5141 (35.8141- 86.5340) 82.5760 (54.5604- 103.8807) 93.3164 (69.6196- 119.4013) 86.9921 (66.5249- 118.8970) <0.001* 44.43%* TVL (mm) 66.7446 (51.4339- 93.6245) 87.9231 (70.0598- 102.8570) 94.7294 (76.9631- 114.5119) 92.3583 (78.0480- 112.1177) <0.001* 29.22%* AVL (mm) 0.1761 (0.1287- 0.2544) 0.2275 (0.1656- 0.3089) 0.2604 (0.1823- 0.3953) 0.2691 (0.1864- 0.3845) <0.001* 24.72%* TNEP 954 (864-1031) 984 (874-1061) 966 (855-1059) 959 (850-1124) 0.426 4.39% ML 0.2865 (0.2111- 0.4280) 0.2288 (0.1935- 0.3086) 0.2311 (0.1543- 0.2945) 0.2151 (0.1603- 0.2506) <0.001 -31.15% NFLVP nerve fiber layer vascular plexus, EA explant area, VA vessels area, VPA vessels percentage area, TNJ total number of junctions, JD junctions In SVP and SVC an increase in VA, VPA, TNJ, JD and TVL with decrease in TNEP and ML was found one week postoperatively (Table 3; Table 4). In addition, TNEP was higher one week after surgery than three months after surgery (Table 3; Table 4). In SVP layer, TVL one week after surgery was lower than three months after surgery (Table 3). In SVC layer TNJ, JD, TVL and AVL values were lower one week compared to three months after surgery (Table 4). postoperatively (Table 3; Table 4). In addition, TNEP was higher one week after surgery than three months after surgery (Table 3; Table 4). In SVP layer, TVL one week after surgery was lower than three months after surgery (Table 3). In SVC layer TNJ, JD, TVL and AVL values were lower one week compared to three months after surgery (Table 4). Vascular parameters Page 6/18 Page 6/18 Table 3 Statistical analysis of changes in vascular parameters in superficial vascular plexus SVP Before 1 week after 1 month after 3 months after P Bias EA (mm2) 8.3792 (8.3777- 8.3806) 8.3794 (8.3780- 8.3806) 8.3801 (8.3793- 8.3807) 8.3801 (8.3790- 8.3809) 0.066 0.01% VA (mm2) 4.4132 (3.8255- 4.9965) 4.9826 (4.6831- 5.2155) 5.0491 (4.7088- 5.3848) 5.0094 (4.7490- 5.4082) <0.001 15.00% VPA (%) 52.6630 (45.6480- 59.7126) 59.4532 (55.8881- 62.2341) 60.2560 (56.1915- 64.2580) 59.7772 (56.6644- 64.6154) <0.001 14.93% TNJ 1242 (1086- 1377) 1398 (1337- 1467) 1421 (1345- 1495) 1433 (1364- 1507) <0.001 16.58% JD (junctions/mm2) 148.2088 (129.6466- 164.2696) 166.8051 (159.6096- 175.0851) 169.5579 (160.4704- 178.4466) 170.9742 (162.8021- 179.8361) <0.001 16.50% TVL (mm) 135.6119 (126.7246- 143.7911) 145.2219 (140.3096- 149.2926) 147.1460 (142.4993- 150.2033) 146.6076 (141.8954- 151.0425) <0.001* 9.56%* AVL (mm) 3.3768 (1.5705- 5.5304) 6.7711 (4.8339- 9.0807) 7.7264 (4.1957- 10.0277) 7.5436 (4.5400- 10.4743) <0.001 116.90% TNEP 327 (190-447) 194 (162-235) 188 (145-243) 182 (136-220) <0.001** -38.54%† ML 0.04859 (0.03868- 0.06534) 0.03816 (0.03027- 0.04640) 0.03720 (0.02838- 0.04485) 0.03701 (0.02614- 0.04696) <0.001 -35.99% SVP superficial vascular plexus, EA explant area, VA vessels area, VPA vessels percentage area, TNJ total number of junctions, JD junctions density, TVL total vessels length, AVL average vessels length, TNEP total number of end points, ML mean lacunarity.This table shows median and interquartile ranges for each parameter (25th and 75th percentile). P values and percentages of change are presented one week after phacoemulsification. Friedman ANOVA test, significant difference (bold values) was found for values with P ˂ 0.05. Observed value was lower one week compared to three months after surgery. Observed value was higher one week compared to three months after surgery. Vascular parameters Page 7/18 Page 7/18 Table 4 Statistical analysis of changes in vascular parameters in superficial vascular complex SVC Before 1 week after 1 month after 3 months after P Bias EA (mm2) 8.3796 (8.3770- 8.3806) 8.3795 (8.3778- 8.3806) 8.3801 (8.3794- 8.3807) 8.3799 (8.3787- 8.3807) 0.444 0.01% VA (mm2) 3.5392 (2.9247- 4.4603) 4.4781 (3.9746- 4.7150) 4.5563 (4.0255- 4.9281) 4.6398 (4.2046- 5.0134) <0.001 22.82% VPA (%) 42.2612 (34.9094- 53.2560) 53.4469 (47.4008- 56.2615) 54.3727 (48.0558- 58.8102) 55.3807 (50.2028- 59.8245) <0.001 22.79% TNJ 1025 (794- 1345) 1320 (1187- 1442) 1360 (1215- 1482) 1393 (1259- 1501) <0.001 29.51%* JD (junctions/mm2) 122.3430 (94.7414- 160.8583) 157.5173 (141.6555- 172.0434) 162.3467 (145.0291- 176.7771) 166.2187 (150.3590- 179.1949) <0.001* 29.49%* TVL (mm) 121.1787 (104.0833- 141.6036) 140.6202 (133.4555- 146.7111) 142.2354 (134.4940- 148.7362) 144.0417 (136.0600- 149.4173) <0.001* 16.71%* AVL (mm) 1.1413 (0.6458- 4.2501) 4.5943 (2.3966- 6.4788) 4.5737 (2.4302- 7.2035) 5.5527 (3.0329- 9.1357) <0.001* 166.71%* TNEP 477 (282-621) 262 (212-402) 234 (196-379) 228 (190-339) <0.001** -39.56%† ML 0.06767 (0.05250- 0.1128) 0.04874 (0.03855- 0.05780) 0.04805 (0.03578- 0.06185) 0.04450 (0.03148- 0.05804) <0.001 -44.15% SVC superficial vascular complex, EA explant area, VA vessels area, VPA vessels percentage area, TNJ total number of junctions, JD junctions density, TVL total vessels length, AVL average vessels length, TNEP total number of end points, ML mean lacunarity. Intermediate capillary plexus, deep capillary plexus and deep vascular complex In ICP and DVC an increase in VA, VPA, TNJ, JD, TVL and AVL along with decrease in TNEP and ML was found one week postoperatively. No difference between one week, one month and three months after surgery was observed for any parameter. In addition, the largest significant change was observed for AVL (75.78% and 113.40% respectively) (Table 5; Table 6). Page 8/18 Table 5 Statistical analysis of changes in vascular parameters in intermediate capillary plexus ICP Before 1 week after 1 month after 3 months after P Bias EA (mm2) 8.3800 (8.3774- 8.3806) 8.3797 (8.3779- 8.3808) 8.3801 (8.3788- 8.3809) 8.3800 (8.3786- 8.3808) 0.359 0.01% VA (mm2) 4.4108 (3.7545- 4.8631) 4.8974 (4.6741- 5.1877) 4.9264 (4.5317- 5.1928) 5.0351 (4.7020- 5.3053) <0.001 16.06% VPA (%) 52.6315 (44.8284- 58.0264) 58.4357 (55.8038- 62.1985) 58.7958 (54.0681- 61.9713) 60.0813 (56.1073- 63.3031) <0.001 16.02% TNJ 1446 (1197- 1576) 1593 (1504- 1675) 1603 (1452- 1682) 1619 (1506- 1670) <0.001 16.46% JD (junctions/mm2) 172.6665 (142.9233- 188.0191) 190.1043 (179.5759- 200.7896) 191.3712 (173.2411- 200.7275) 193.1983 (179.7861- 202.8178) <0.001 16.42% TVL (mm) 145.8206 (129.0303- 153.5934) 154.3064 (150.4366- 158.9588) 155.3216 (146.2016- 158.3196) 155.9789 (148.7249- 160.2269) <0.001 10.80% AVL (mm) 3.1665 (1.1356- 5.2864) 6.2908 (4.1408- 8.3364) 6.2008 (3.8223- 8.0361) 6.2737 (3.8795- 8.4574) <0.001 75.78% TNEP 366 (236-550) 233 (191-296) 236 (202-313) 231 (190-304) <0.001 -36.58% MEL 0.03303 (0.02445- 0.05412) 0.02367 (0.02006- 0.02980) 0.02469 (0.01956- 0.03311) 0.02373 (0.01769- 0.03048) <0.001 -50.61% ICP intermediate capillary plexus, EA explant area, VA vessels area, VPA vessels percentage area, TNJ total number of junctions, JD junctions density, TVL total vessels length, AVL average vessels length, TNEP total number of end points, ML mean lacunarity. This table shows median and interquartile ranges for each parameter (25th and 75th percentile). P values and percentages of change are presented one week after phacoemulsification. Friedman ANOVA test, significant difference (bold values) was found for values with P ˂ 0.05. week after phacoemulsification. Friedman ANOVA test, significant difference (bold values) was found for values with P ˂ 0.05. Page 9/18 In DCP layer an increase in TNJ, JD, TVL and AVL and decrease in TNEP and ML was found postoperatively one week, one month and three months after surgery with no significant difference between them. VA and VPA before surgery were lower than one week, one month and three months after surgery. VA and VPA one week after surgery were lower than three months after surgery. In DCP, the largest statistically significant change in preoperative versus postoperative value was observed for AVL (103.51%) (Table 7) Page 9/18 Table 6 Statistical analysis of changes in vascular parameters in deep vascular complex DVC Before 1 week after 1 month after 3 months after P Bias EA (mm2) 8.3798 (8.3767- 8.3807) 8.3799 (8.3788- 8.3808) 8.3804 (8.3791- 8.3810) 8.3799 (8.3786- 8.3808) 0.128 0.02% VA (mm2) 4.6295 (3.7813- 5.1470) 5.2785 (5.0204- 5.5748) 5.3497 (4.9653- 5.6558) 5.3920 (4.9867- 5.7371) <0.001 18.17% VPA (%) 55.2483 (45.1905- 61.4192) 63.0098 (59.9148- 66.5252) 63.8886 (59.2817- 67.4735) 64.3579 (59.5031- 68.4626) <0.001 18.10% TNJ 1601 (1270- 1665) 1716 (1670- 1771) 1729 (1647- 1772) 1722 (1634- 1780) <0.001 15.41% JD (junctions/mm2) 191.0277 (151.6214- 198.5983) 204.7507 (199.3202- 211.4425) 206.7926 (196.6997- 211.4956) 205.4722 (194.6548- 212.3658) <0.001 15.34% TVL (mm) 152.8660 (130.5779- 158.5100) 160.3862 (156.7585- 163.6309) 161.0969 (156.1049- 163.2658) 160.5890 (155.0713- 163.9713) <0.001 10.74% AVL (mm) 3.8988 (0.9151- 10.1915) 11.0566 (6.7885- 23.2487) 13.4610 (7.3215- 20.4125) 12.1796 (7.0645- 20.5614) <0.001 113.40% TNEP 333 (177-525) 163 (117-221) 155 (115-225) 140 (112-213) <0.001 -49.04% ML 0.03602 (0.02654- 0.07429) 0.02452 (0.01976- 0.03203) 0.02530 (0.01902- 0.03206) 0.02564 (0.01882- 0.03265) <0.001 -53.41% DVC deep vascular complex, EA explant area, VA vessels area, VPA vessels percentage area, TNJ total number of junctions, JD junctions density, TVL total vessels length, AVL average vessels length, TNEP total number of end points, ML mean lacunarity. This table shows median and interquartile ranges for each parameter (25th and 75th percentile). P values and percentages of change are presented one week after phacoemulsification. Friedman ANOVA test, significant difference (bold values) was found for values with P ˂ 0.05. In DCP layer an increase in TNJ, JD, TVL and AVL and decrease in TNEP and ML was found postoperatively one week, one month and three months after surgery with no significant difference between them. VA and VPA before surgery were lower than one week, one month and three months after surgery. VA and VPA one week after surgery were lower than three months after surgery. In DCP, the largest statistically significant change in preoperative versus postoperative value was observed for AVL (103.51%) (Table 7) In DCP layer an increase in TNJ, JD, TVL and AVL and decrease in TNEP and ML was found postoperatively one week, one month and three months after surgery with no significant difference between them. VA and VPA before surgery were lower than one week, one month and three months after surgery. VA and VPA one week after surgery were lower than three months after surgery. Page 9/18 In DCP, the largest statistically significant change in preoperative versus postoperative value was observed for AVL (103.51%) (Table 7) Page 10/18 Table 7 Statistical analysis of changes in vascular parameters in deep capillary plexus DCP Before 1 week after 1 month after 3 months after P Bias EA (mm2) 8.3792 (8.3766- 8.3803) 8.3802 (8.3790- 8.3808) 8.3802 (8.3789- 8.3807) 8.3801 (8.3790- 8.3810) 0.357 0.01% VA (mm2) 4.2319 (3.4769- 4.8241) 4.9539 (4.4996- 5.2425) 4.9380 (4.5294- 5.3485) 4.9121 (4.5870- 5.4160) <0.001* 17.65%* VPA (%) 50.5055 (41.6312- 57.5592) 59.1286 (53.7127- 62.5724) 58.9327 (54.1117- 63.8239) 58.6169 (54.7285- 64.6397) <0.001* 17.59%* TNJ 1375 (1078- 1546) 1573 (1442- 1646) 1572 (1446- 1650) 1572 (1440- 1654) <0.001 16.77% JD (junctions/mm2) 164.1052 (128.8104- 184.4084) 187.7513 (172.1331- 196.4872) 187.7643 (172.532- 196.884) 187.5586 (171.8801- 197.3193) <0.001 16.71% TVL (mm) 138.1963 (120.0253- 150.4007) 151.3618 (144.6869- 156.4053) 152.0403 (145.4315- 156.7864) 151.0493 (145.3439- 156.7136) <0.001 11.00% AVL (mm) 1.9423 (0.7977- 4.6726) 5.8517 (3.7833- 8.4856) 5.3625 (3.2110- 8.1661) 5.2044 (3.3965- 8.1440) <0.001 103.51% TNEP 411 (237-597) 239 (180-328) 219 (171-311) 225 (167-298) <0.001 -37.06% ML 0.05597 (0.04235- 0.08264) 0.04124 (0.0304- 0.05118) 0.03980 (0.03073- 0.04955) 0.03709 (0.03059- 0.05586) <0.001 -44.00% DCP deep capillary plexus, EA explant area, VA vessels area, VPA vessels percentage area, TNJ total number of junctions, JD junctions density, TVL total vessels length, AVL average vessels length, TNEP total number of end points, ML mean lacunarity. This table shows median and interquartile ranges for each parameter (25th and 75th percentile). P values and percentages of change are presented one week after phacoemulsification. Friedman ANOVA test, significant difference (bold values) was found for values with P ˂ 0.05. *Observed values were lower one week than three months after surgery. Retina Observing the whole retina, an increase in VA, VPA, TNJ, JD, TVL and AVL with decrease in TNEP and ML was found one week postoperatively. No difference between one week, one month and three months after surgery was observed for any parameter (Table 8). Page 11/18 Table 8 Statistical analysis of changes in vascular parameters in retina RETINA Before 1 week after 1 month after 3 months after P Bias EA (mm2) 8.3794 (8.3782- 8.3804) 8.3796 (8.3781- 8.3807) 8.3798 (8.3789- 8.3805) 8.3800 (8.3794- 8.3808) 0.567 0.00% VA (mm2) 4.4237 (3.8317- 4.7725) 4.8044 (4.4337- 5.0568) 4.7583 (4.4779- 5.1489) 4.7814 (4.4562- 5.0985) <0.001 8.86% VPA (%) 52.8002 (45.7295- 56.9454) 57.3259 (52.9224- 60.3462) 56.7723 (53.4809- 61.4520) 57.0595 (53.1767- 60.8384) <0.001 8.86% TNJ 1544 (1302- 1648) 1690 (1512- 1750) 1638 (1547- 1758) 1672 (1594- 1747) <0.001 10.72% JD (junctions/mm2) 184.2823 (155.3695- 196.6816) 201.6892 (180.4112- 209.4030) 195.4505 (184.6068- 209.7366) 199.4898 (190.1586- 208.4696) <0.001 10.71% TVL (mm) 150.0282 (135.6528- 156.1119) 158.3173 (149.9265- 161.9888) 157.3335 (151.8571- 162.0794) 158.2087 (153.6966- 162.5753) <0.001 6.19% AVL (mm) 2.4171 (1.0409- 4.7464) 5.4592 (3.5082- 7.8666) 4.8714 (3.4563- 7.4278) 5.8118 (3.0205- 7.7420) <0.001 87.65 % TNEP 419 (269-587) 255 (215-373) 270 (209-356) 263 (196-369) <0.001 -30.99% ML 0.03724 (0.02724- 0.05949) 0.03017 (0.02426- 0.03485) 0.03040 (0.02414- 0.03666) 0.02909 (0.02460- 0.03849) <0.001 -30.26% EA explant area, VA vessels area, VPA vessels percentage area, TNJ total number of junctions, JD junctions density, TVL total vessels length, AVL average vessels length, TNEP total number of end points, ML mean lacunarity.This table shows median and interquartile ranges for each parameter (25th and 75th percentile). P values and percentages of change are presented one week after phacoemulsification. Friedman ANOVA test, significant difference (bold values) was found for values with P ˂ 0.05. Choroid No statistically significant changes in vascular parameters were observed (Additional file 1: Table S6). Choriocapillaris In choriocapillaris TNEP was higher before surgery than one week, one month and three months after surgery, with no significant difference between them. ML before surgery was higher than one month and three months after surgery (Additional file 1: Table S5). Inter-layer differences Morphometric differences between the vascular layers are presented online in Additional file 1: Tables S7-S10. Page 12/18 Major results A significant increase in VA, VPA, TNJ, JD, TVL and AVL was found, followed by the decrease in TNEP and ML manifesting rise in blood supply of the central macula after phacoemulsification (Additional file 1: Table S3; Table S4). Most changes in vascular parameters were evident one week after surgery and remained stable up to three months after surgery. These changes affected all retinal layers but not CC and choroid (Additional file 1: Table S5; Table S6). Observed patterns of alterations between the retina and choroid were somewhat expected as these two layers have different hemodynamic properties. Functional hyperemia Inflammatory response after uncomplicated phacoemulsification is imputed to be the cause of increased macular thickness, reaching its maximum between one week and one month after surgery and returning to baseline after 2-6 months [2, 15, 16]. However, none of these studies actually measured the local release of inflammatory metabolites. On the contrary, the prevailing view modelled from our study results' was that increase in macular hemodynamics could not be the result of postoperative inflammation as the changes persisted three months after the surgery, when inflammatory response should have been over [2, 15, 17-19], nor decrease in IOP due to OPP consistency (Additional file 1: Table S2) [3]. Moreover, all patients underwent uncomplicated phacoemulsification with favorable, low PHACO time and CDE (Additional file 1: Table S1), while factors contributing to neural damage and blood-brain barrier breakdown were excluded prior to enrolment. Thus, the third mechanism, functional hyperemia, disclosed that blood flow in retinal vasculature significantly varied due to increased intensity of light stimulus after cataract removal, in stark contrast to choroidal circulation [5]. Namely, cataract blocks up to 40% of light at different wavelengths [20]. Increase in metabolism which accompanies neuronal activity lowers O2 and glucose levels and leads to production of vasoactive metabolites [21]. Products of neuronal activity adenosine, lactate and arachnoidic acid cause vasodilatation and functional hyperemia to compensate for energy consumption and increase in ganglion cell activity due to light stimulation restoring O2 and glucose levels [5, 22, 23]. Likewise, larger blood flow is induced after a period of dark adaptation [23], what in our case, hypothetically, we could consider cataract to be as cataracts gradually weaken the intensity of light stimuli. Discussion This study monitored vascular parameters of retinal blood vessels across NFLVP, SVP, SVC, ICP, DCP, DVC, as well as CC and large and medium choroidal blood vessels revealing that uncomplicated phacoemulsification significantly improved macular hemodynamics. We consider these changes to be favorable and beneficial. Page 12/18 Page 12/18 SVC vs DVC The greatest increase in VA, VPA, TNJ, JD, TVL and AVL was found in SVC (Additional file 1: Table S4). Due to functional hyperemia, AVL increased more than TVL suggesting coiling of blood vessels (Additional file 1: Table S4). On the contrary, the greatest change of ML as an index for vascular structural non-uniformity, [12] appeared in DVC (Additional file 1: Table S4). These differences suggest different metabolic demands of retinal layers supplied by SVC and DVC. Page 13/18 Page 13/18 Page 13/18 NFLVP, a layer of long capillaries with small number of anastomosis located only in the posterior pole [6, 24], is presented with unique configuration (Additional file 1: Table S7) manifesting the highest number of end points and the lowest number of junctions (Table 2; Additional file 1: Table S7; Table S9). NFLVP, a layer of long capillaries with small number of anastomosis located only in the posterior pole [6, 24], is presented with unique configuration (Additional file 1: Table S7) manifesting the highest number of end points and the lowest number of junctions (Table 2; Additional file 1: Table S7; Table S9). Three months after surgery VA, VPA, TNJ, JD, TVL and AVL were significantly higher in DVC compared to SVC, while TNEP and ML were significantly higher in SVC (Additional file 1: Table S10). On the contrary, while comparing SVP and DCP (Additional file 1: Table S8), greater VA and VPA for SVP network were found likewise [10], but DCP showed higher TNJ, JD and TVL. Aforesaid attributes could correlate to the larger diameter of the SVP vasculature opposed to DVP being solely capillary meshwork [25]. Morphometry of ICP and DCP did not demonstrate significant differences (Additional file 1: Table S8). Until this date, only two OCT-A studies analyzed retinal blood vessels after uncomplicated phacoemulsification [2, 3]. Performed on a relatively small subject samples (N = 9 and N = 32), they used different OCT-A devices while Q and EA comparison before and after cataract surgery was not performed [2, 3]. Zhao et al observed parafoveal and perifoveal blood vessels density increase one week after phacoemulsification lasting up to three months after surgery [3]. Furthermore, cataract surgery was followed by a decrease in the foveal avascular zone surface, and an increase in full and inner retinal thickness, occurring one week after surgery, and still increasing up to three months after the surgery [3]. Outer retinal thickness remained almost unchanged [3]. The vascular pattern responses observed here could give the anatomical background for the aforementioned results. Although all retinal layers demonstrated increase in perfusion, SVC underwent greater change than DVC (Additional file 1: Table S4). Inner plexiform layer contains both superficial and deep blood vessels [6]. Thus, the functional hyperemia observed could cause an increase in full and inner retinal thickness [3]. Page 13/18 On the contrary, we hypothesize that functional hyperemia identified in DCP cannot elicit significant changes of outer retinal thickness as DCP is intersected between INL and OPL [6], justifying Zhao’s et al results [3]. Choroid CC presented with the greatest VA, VPA, TNJ, JD, TVL and AVL, and the lowest TNEP and ML (Additional file 1: Tables S5; Table S9) confirming its structure as a continuous capillary meshwork with a high number of anastomosis [25]. Opposed to the retina, we found no significant changes in the choroid (Additional file 1: Table S6) and CC (Additional file 1: Table S5), except for TNEP and ML in CC. Light stimulation has a little effect on choroidal circulation, insensitive to pO2 fluctuations [5]. We thus concluded that physiological requirements outlined with low PHACO time and CDE did not reach the threshold to induce outer blood-retinal barrier breakdown and inflammatory response. Further, significant decrease of TNEP and ML in CC (Additional file 1: Table S5) could result from increased demand in heat dissipation through opening of anastomoses corroborating with published studies [2, 16, 26]. Some authors hypothesized increase in subfoveal thickness to be a consequence of local choroidal inflammatory response but found no reasoning for these changes, while correlation to CDE was not reported [18, 25, 27]. Ageing Ageing causes altered hemodynamics and hypoperfusion mostly in neural tissues with high metabolic demand [28]. Decline in metabolic activity under physiological ageing was further supported by our study correspondingly, as retinal macular perfusion significantly increased after uncomplicated phacoemulsification and augmented visual stimulation. Conclusions This is the first OCT-A study that has clearly demonstrated persistent increase in macular perfusion most likely due to functional hyperemia prompted by augmented visual stimulation. In this study, phacoemulsification in elderly population proved as an advantageous feature in addition to restoring visual acuity. This beneficial event could facilitate the decision-making process with regard to earlier timing for cataract removal in healthy aging patients. Further studies with a longer follow-up period are warranted to validate our results and reveal temporal trends. Thus, to conclude if functional hyperemia is a long-term condition. Image quality Page 14/18 Page 14/18 Page 14/18 One might ask if these changes in macular perfusion after surgery were the result of better image quality after cataract removal. Thus, we counterbalanced this potential bias. OCT-A may overcome early stage cataract in contrast to clinically significant ones [29]. As follows, this study included only patients with mild to moderate opacities, graded objectively (Table S1). Secondly and more important, OCT-A image quality (Q) quantified by the software integrated in HRA+OCT Spectralis® before and after surgery was statistically the same (Table 1). Consequently, perfusion changes demonstrated here were unlikely the result of the improvement of the ocular optics after cataract removal. Furthermore, different time frames of perfusion alterations were demonstrated (Tables 2-8; Additional file 1: Table S5). In addition, the latest software version of Spectralis® uses the Position Artefact Removal tool, eliminating blood movement artefacts and enabling even more distinct analysis of deeper layers [9]. With TruTrack Active Eye Tracking technology high-quality retinal imaging even with eye movements is allowed [9]. There are few limitations to this analysis. First, we reported the values for only 55 subjects of Caucasian descent for whom we did not measure retinal metabolic activity. Further studies using OCT-A are needed to establish a normative database of observed vascular parameters for other demographic variables. Second, the analysis of retinal and choroidal pathology was beyond the scope of this report. List Of Abbreviations AL: axial length; AVL: average vessels length; BCVA: best corrected visual acuity; CC: choriocapillaris; CDE: cumulative dissipated energy; DBP: diastolic blood pressure; DCP: deep capillary plexus; DVC: deep vascular complex; EA: explant area; GCL: ganglion cell layer; ICP: intermediate capillary plexus; INL: inner nuclear; IOP: intraocular pressure; IPL: inner plexiform layer; JD: junctions density; MAP: mean arterial pressure; ML: mean lacunarity; NFL: nerve fiber layer; NFLVP: nerve fiber layer vascular plexus; OCT: optical coherence tomography; OCT-A: optical coherence tomography angiography OPL: outer plexiform layer; OPP: ocular perfusion pressure; PHACO time: total ultrasound time; PME: pseudophakic macular oedema; PNS: Pentacam® Nucleus Staging; Q: images quality index; SBP: systolic blood pressure; SVC: superficial vascular complex; SVP: superior vascular plexus; TNEP: total number of end points; TNJ: total number of junctions; TVL: total vessels length; VA: vessels area; VPA: vessels percentage area Ethics approval and consent to participate The study protocol was approved by the Institutional Review Board of University Hospital „Sveti Duh“, Zagreb, Croatia (IRB no. 01-4212/2) and was conducted in accordance with the tenets of the World Medical Association Declaration of Helsinki. Written informed consent to participate was obtained from all patients before inclusion in the study. Consent for publication Not applicable. Availability of data and material The datasets generated and/or analysed during the current study are not publicly available due to extensive and large- scale datasheets but are available from the corresponding author on reasonable request. Funding This research did not receive any funding. Authors’ contributions Conception and design of the study: AK, MBj, MB, NV. Analysis and interpretation: AK, MBj, MB, BKE, BR, NV. Writing of the article: AK, MBj; Critical revision of the article: AK, MBj, MB, BKE, BR, NV. Data collection: AK, BKE. Provision of patients. AK. Statistical expertise: AK, MBj, BR. Literature search: AK, MBj, MB, BKE, BR. Administrative support: AK, MB, BKE, BR. All authors read and approved the final manuscript. Acknowledgements Not applicable. Competing interests The authors declare that they have no competing interests. References 1. Micieli JA, Arshinoff SA. Cataract surgery. 2011;183:1621. 1. Micieli JA, Arshinoff SA. Cataract surgery. 2011;183:1621. 1. Micieli JA, Arshinoff SA. Cataract surgery. 2011;183:1621. 2. Pilotto E, Leonardi F, Stefanon G, Longhin E, Torresin T, Deganello D, et al. Early retinal and choroidal OCT and OCT angiography signs of inflammation after uncomplicated cataract surgery. Br J Ophthalmol. 2019;103:1001-7. 2. Pilotto E, Leonardi F, Stefanon G, Longhin E, Torresin T, Deganello D, et al. Early retinal and choroidal OCT and OCT angiography signs of inflammation after uncomplicated cataract surgery. Br J Ophthalmol. 2019;103:1001-7. 2. Pilotto E, Leonardi F, Stefanon G, Longhin E, Torresin T, Deganello D, et al. Early retinal and choroidal OCT and OCT angiography signs of inflammation after uncomplicated cataract surgery. Br J Ophthalmol. 2019;103:1001-7. 3. Zhao Z, Wen W, Jiang C, Lu Y. Changes in macular vasculature after uncomplicated phacoemulsification surgery: Optical coherence tomography angiography J Cataract Refract Surg. 2018;44:453-8. 3. Zhao Z, Wen W, Jiang C, Lu Y. Changes in macular vasculature after uncomplicated phacoemulsification surgery: Optical coherence tomography angiography J Cataract Refract Surg. 2018;44:453-8. 4. Rich WJ, James ML. Cataract surgery and vascular changes in the eye. Acta Ophthalmol Suppl. 1989;191:39-42. 4. Rich WJ, James ML. Cataract surgery and vascular changes in the eye. Acta Ophthalmol Suppl. 1989;191:39-42. 4. Rich WJ, James ML. Cataract surgery and vascular changes in the eye. Acta Ophthalmol Suppl. 1989;191:39-42. 5. Kur J, Newman EA, Chan-Ling T. Cellular and physiological mechanisms underlying blood flow regulation in the retina , g y g y p pp ; 5. Kur J, Newman EA, Chan-Ling T. Cellular and physiological mechanisms underlying blood flow regulation in the retina and choroid in health and disease. Prog Retin Eye 2012;31:377-406. 5. Kur J, Newman EA, Chan-Ling T. Cellular and physiological mechanisms underlying blood flow regulation in the retina and choroid in health and disease. Prog Retin Eye 2012;31:377-406. 6. Coscas F, Sellam A, Glacet-Bernard A, Jung C, Goudot M, Miere A, et al. Normative Data for Vascular Density in Superficial and Deep Capillary Plexuses of Healthy Adults Assessed by Optical Coherence Tomography Angiography. Invest Ophthalmol Vis Sci. 2016;57:211-23. 6. Coscas F, Sellam A, Glacet-Bernard A, Jung C, Goudot M, Miere A, et al. Normative Data for Vascular Density in Superficial and Deep Capillary Plexuses of Healthy Adults Assessed by Optical Coherence Tomography Angiography. Invest Ophthalmol Vis Sci. 2016;57:211-23. A, Raiji V, Garcia Gonzalez JM. References Review of hypertensive retinopathy. Dis Mon. 2017;63:63 8. Kniestedt C, Stamper RL. Visual acuity and its measurement. Ophthalmol Clin North Am. 2003;16:155–70. 8. Kniestedt C, Stamper RL. Visual acuity and its measurement. Ophthalmol Clin North Am. 2003;16:155–70. 9. Spectralis Product Family. User Manual Softvare version 6.8. Heidelberg: Heidelberg Engineering GmbH; 2017. 9. Spectralis Product Family. User Manual Softvare version 6.8. Heidelberg: Heidelberg Engineering GmbH; 2017. 9. Spectralis Product Family. User Manual Softvare version 6.8. Heidelberg: Heidelberg Engineering GmbH; 2017. Page 16/18 10. Hosari S, Hohberger B, Theelke L, Sari H, Lucio M, Mardin CY. OCT Angiography: Measurement of Retinal Macular Microvasculature with Spectralis II OCT Angiography - Reliability and Reproducibility. Ophthalmologica. 2020;243:75- 84. 11. Lupidi M, Coscas F, Cagini C, Fiore T, Spaccini E, Fruttini D, et al. Automated Quantitative Analysis of Retinal Microvasculature in Normal Eyes on Optical Coherence Tomography Angiography. Am J Ophthalmol. 2016;169:9-23. 12. Zudaire E, Gambardella L, Kurcz C, Vermeren S. A computational tool for quantitative analysis of vascular networks. PLoS One. 2011;6:e27385. 13. Oh LJ, Nguyen C, Wong E, Wang SSY. Prospective study of Centurion® versus Infiniti® phacoemulsification systems: surgical and visual outcomes. Int J Ophthalmol. 2017;10:1698-702. 14. Kim SJ, Belair ML, Bressler NM, Dunn JP, Thorne JE, Kedhar SR, et al. A method of reporting macular edema after cataract surgery using optical coherence tomography. Retina. 2008;28:870-6. 15. Gharbiya M, Cruciani F, Cuozzo G, Parisi F, Russo P, Abdolrahimzadeh S. Macular thickness changes evaluated with spectral domain optical coherence tomography after uncomplicated phacoemulsification. Eye (Lond). 2013;27:605– 11. 16. Falcão MS, Gonçalves NM, Freitas-Costa P, Beato JB, Rocha-Sousa A, Carneiro A, et al. Choroidal and macular thickness changes induced by cataract surgery. Clin Ophthalmol. 2014;8:55–60. 17. Neatrour K, McAlpine A, Owens TB, Trivedi RH, Poole Perry LJ. Evaluation of the etiology of persistent iritis after cataract surgery. Ophthalmic Inflamm Infect. 2019;9:4. 18. Ibrahim AM, Elgouhary SM, Nassar MK, El Batanony AH. Changes in Choroidal Thickness after Cataract Surgery. Semin Ophthalmol. 2018;33:664-70. 19. Alio JL, Bodaghi B, Tassignon MJ. Guidelines for managing post-cataract surgery inflammation: Can we reach a consensus? Ophthalmology Times Europe. 2008. 19. Alio JL, Bodaghi B, Tassignon MJ. Guidelines for managing post-cataract surgery inflammation: Can we reach a consensus? Ophthalmology Times Europe. 2008. https://www.ophthalmologytimes.com/sites/default/files/legacy/mm/OphthalmologyTimesEurope/PDF/Guidelines- for-managing-post-cataract-surgery-inflammation.pdf. Accessed 25 Sep 2020. https://www.ophthalmologytimes.com/sites/default/files/legacy/mm/OphthalmologyTimesEurope/PDF/Guidelines- for-managing-post-cataract-surgery-inflammation.pdf. Accessed 25 Sep 2020. 20. Artigas JM, Felipe A, Navea A, Fandiño A, Artigas C. References Spectral transmission of the human crystalline lens in adult and elderly persons: color and total transmission of visible light. Invest Ophthalmol Vis Sci. 2012;53:4076-84. 21. Roy CS, Sherrington CS. On the Regulation of the Blood-supply of the Brain. J Physio 22. Attwell D, Buchan A, Charpak S, Lauritzen M, Macvicar BA, Newman EA. Glial and neuronal control of brain blood flow. 2010;468:232–43. 23. Noonan JE, Lamoureux EL, Sarossy M. Neuronal activity-dependent regulation of retinal blood flow. Clin Exp Ophthalmol. 2015;43:673-82. 24. Chan G, Balaratnasingam C, Yu PK, Morgan WH, McAllister IL, Cringle SJ, et al. Quantitative morphometry of perifoveal capillary networks in the human retina. Invest Ophthalmol Vis Sci. 2012;53:5502-14. 25. Zeng S, Liang C,He Y, Zhao Q, Dai S, Cheng F, et al. Changes of Subfoveal Choroidal Thickness after Cataract Surgery: A Meta-Analysis. J Ophthalmol. 2018;2018:2501325. 26. Brito PN, Rosas VM, Coentrão LM, Carneiro ÂV, Rocha-Sousa A, Brandão E, et al. Evaluation of visual acuity, macular status, and subfoveal choroidal thickness changes after cataract surgery in eyes with diabetic retinopathy. Retina. 2015;35(2):294-302. 27. Chen H, Wu Z, Chen Y, He M, Wang J. Short-term changes of choroidal vascular structures after phacoemulsification surgery. BMC Ophthalmol. 2018;18:81. 28. Lin Y, Jiang H, Liu Y, Rosa Gameiro G, Gregori G, Dong C, et al. Age-Related Alterations in Retinal Tissue Perfusion and Volumetric Vessel Density. Invest Ophthalmol Vis 2019;60:685-93. Page 17/18 Page 17/18 29. Yu S, Frueh BE, Steinmair D, Ebneter A, Wolf S, Zinkernagel MS, et al. Cataract significantly influences quantitative measurements on swept-source optical coherence tomography angiography imaging. PLoS One. 2018;13(10):e0204501. 29. Yu S, Frueh BE, Steinmair D, Ebneter A, Wolf S, Zinkernagel MS, et al. Cataract significantly influences quantitative measurements on swept-source optical coherence tomography angiography imaging. PLoS One. 2018;13(10):e0204501. 29. Yu S, Frueh BE, Steinmair D, Ebneter A, Wolf S, Zinkernagel MS, et al. Cataract significantly influences quantitative measurements on swept-source optical coherence tomography angiography imaging. PLoS One. 2018;13(10):e0204501. Additionalfile1.pdf Additionalfile1.pdf Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionalfile1.pdf Additionalfile1.pdf Additionalfile1.pdf Page 18/18 Page 18/18
https://openalex.org/W2767195211	https://www.frontiersin.org/articles/10.3389/fphys.2017.00868/pdf?isPublishedV2=False	English	null	A “Wearable” Test for Maximum Aerobic Power: Real-Time Analysis of a 60-m Sprint Performance and Heart Rate Off-Kinetics	Frontiers in physiology	2,017	cc-by	7,370	ORIGINAL RESEARCH published: 01 November 2017 doi: 10.3389/fphys.2017.00868 Edited by: Luca Paolo Ardigò, University of Verona, Italy Edited by: Luca Paolo Ardigò, University of Verona, Italy Reviewed by: Takeshi Otsuki, Ryutsu Keizai University, Japan Sergej Ostojic, University of Novi Sad, Serbia Capelli Carlo, Norwegian School of Sport Sciences Oslo, Norway Reviewed by: Takeshi Otsuki, Ryutsu Keizai University, Japan Sergej Ostojic, University of Novi Sad, Serbia Capelli Carlo, Norwegian School of Sport Sciences Oslo, Norway Reviewed by: Takeshi Otsuki, Ryutsu Keizai University, Japan Sergej Ostojic, University of Novi Sad, Serbia Capelli Carlo, Norwegian School of Sport Sciences Oslo, Norway Correspondence: Alberto E. Minetti alberto.minetti@unimi.it Specialty section: This article was submitted to Exercise Physiology, a section of the journal Frontiers in Physiology Received: 21 August 2017 Accepted: 17 October 2017 Published: 01 November 2017 A “Wearable” Test for Maximum Aerobic Power: Real-Time Analysis of a 60-m Sprint Performance and Heart Rate Off-Kinetics Jorge L. Storniolo, Gaspare Pavei and Alberto E. Minetti Jorge L. Storniolo, Gaspare Pavei and Alberto E. Minetti* Laboratory of Locomotion Physiomechanics, Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy Maximum aerobic power ( ˙VO2peak) as an indicator of body ﬁtness is today a very well-known concept not just for athletes but also for the layman. Unfortunately, the accurate measurement of that variable has remained a complex and exhaustive laboratory procedure, which makes it inaccessible to many active people. In this paper we propose a quick estimate of it, mainly based on the heart rate off-kinetics immediately after an all-out 60-m sprint run. The design of this test took into account the recent availability of wrist wearable, heart band free, multi-sensor smart devices, which could also inertially detect the different phases of the sprint and check the distance run. 25 subjects undertook the 60-m test outdoor and a ˙VO2peak test on the laboratory treadmill. Running average speed, HR excursion during the sprint and the time constant (τ) of HR exponential decay in the off-kinetics were fed into a multiple regression, with measured ˙VO2peak as the dependent variable. Statistics revealed that within the investigated range (25–55 ml O2/(kg min)), despite a tendency to overestimate low values and underestimate high values, the three predictors conﬁdently estimate individual ˙VO2peak (R2 = 0.65, p < 0.001). The same analysis has been performed on a 5-s averaged time course of the same measured HR off-kinetics, as these are the most time resolved data for HR provided by many modern smart watches. Results indicate that despite of the substantial reduction in sample size, predicted ˙VO2peak still explain 59% of the variability of the measured ˙VO2peak. Keywords: maximum aerobic power, heart rate, off kinetics, sprint running, smart devices Experimental Protocol Submaximal metabolic eﬀort such as walking, running, hiking, swimming at moderate speed has been classically included in the activity monitor function of “smart” portable/wearable devices. The estimate of burned calories is obtained from short term average HR, average speed and from accelerometry-based recognition of locomotion type (Chowdhury et al., 2017). Subjects performed two diﬀerent tests in diﬀerent days separated by a minimum of 48 h: a 60-m maximal sprint accomplished on an outdoor athletic track and an incremental exercise test for the determination of ˙VO2peak in the laboratory. Participants were instructed to arrive at the experimental session in a rested and fully hydrated state and to avoid strenuous exercise in the 24 h preceding each testing session. In addition, they were told to avoid alcohol (24 h) and caﬀeine (6 h) intake before the exercise test. Diﬀerently, no estimate of ˙VO2peak from smart devices has been implemented so far, to the authors’ knowledge. Potential reasons for this is, as mentioned, the infancy of wearable sensor technology that strives to compete with professional analogs. Our challenge in the present investigation was to design a simple test exploiting sensors already incorporated in smart watches/bracelets. The ﬁrst session consisted of a 60-m maximal sprint trial preceded by a short warm-up (5 min with jogging and stretching) and 10 min of resting period (5 min in a seated and 5 min in a standing position). Heart rate (HR) was recorded beat-by- beat throughout all phases of the sprint test (rest, running and 5 min of recovery) by a heart rate monitor with transmitter belt (Polar S410, Kempele, Finland). All tests were performed at the same time of day (10–11 a.m.) to limit the inﬂuences of circadian rhythm on muscle performance and heart rate response/variability. 60-m sprint duration was recorded by using a manual stopwatch and the average running speed (vtest, m.s−1) was obtained. Subjects were encouraged to accomplish their best performance. The idea originated from transport engineering: race car engines increase and decrease rpm (revolutions per minute, a “sound” particularly apparent when gear is disengaged) much faster than in a normal car. In the biological realm we face a similar phenomenon: athletes display a faster ˙VO2 increase (at the start of a heavy exercise) and decrease (during the recovery) than sedentary subjects (for a review see Jones and Poole, 2005; Rossiter, 2011). Citation: In the last few decades we assisted to a growing interest toward personally keeping one’s health in a better shape, a condition that would enrich the entire life and likely prevents early deterioration of many body functions. This passes, among others, through the development and maintenance of a maximum oxygen consumption ( ˙VO2peak) higher than for a sedentary. However, portable professional metabographs are out of reach for most of the athletes, not to mention the laymen, who represents the vast majority of the potential audience in the need to periodically check the aerobic ﬁtness level. Storniolo JL, Pavei G and Minetti AE (2017) A “Wearable” Test for Maximum Aerobic Power: Real-Time Analysis of a 60-m Sprint Performance and Heart Rate Off-Kinetics. Front. Physiol. 8:868. doi: 10.3389/fphys.2017.00868 November 2017 \| Volume 8 \| Article 868 Frontiers in Physiology \| www.frontiersin.org ˙VO2peak Test for Smart Wearables Storniolo et al. MATERIALS AND METHODS At the same time, the progress in terms of portable technology (tablets, “smart” phones, bracelets/bands and watches) makes us move equipped with a redundancy of sensors. In addition to the ubiquitary camera, most of the devices bring GPSs, accelerometers, gyroscopes, magnetometers, proximity sensors and, most recently, infrared emitter/detector LED systems to measure heart rate (HR) in real-time. Although not all of them provide data accurately and precisely enough to compete with the analogous laboratory equipment (Chowdhury et al., 2017), their improvement is just a matter of time and scenarios for new and diﬀerent biomedical tests could be certainly hypothesized to be implemented in the near future. Subjects Twenty-ﬁve subjects (7 women and 18 men, 25.0 ± 5.0 year, 1.77 ± 0.08 m height, 71.4 ± 8.6 kg body mass; mean ± SD) took part in the study; they were physically active subjects involved either in recreational activity or in amateur sport activity with a maximum of four sessions per week. The study was approved by the Ethics Committee of the University of Milan (031511), and participants, after becoming aware of the potential risks involved in the experimental sessions, gave written informed consent. Frontiers in Physiology \| www.frontiersin.org Experimental Protocol HR is a fundamental determinant of ˙VO2 on- and oﬀ-kinetics, since its time course closely mimics the changes in gas exchange (Hickson et al., 1978; Hagberg et al., 1980; Norris and Petersen, 1998). Thus, similarly to engine rpm, HR kinetics is expected to be faster the higher the metabolic power of human engine (Darr et al., 1988; Sugawara et al., 2001; Otsuki et al., 2007; Ostojic et al., 2010, 2011; Watson et al., 2017). This applies to other important kinetics, such as the enzymatic chain (Timmons et al., 1998), within the whole metabolic/mechanical “turn on/oﬀ” process of muscular exercise. Peak aerobic power ( ˙VO2peak) was determined with an incremental running test performed on a treadmill (Ergo LG, Woodway). After 10 min of standing resting period, the protocol began with subjects running at 9 km.h−1 for 4 min, then the belt speed was increased by 1 km.h−1 every minute until volitional exhaustion. Pulmonary ventilation ( ˙VE, BTPS), O2 consumption ( ˙VO2), and CO2 output ( ˙VCO2), both STPD, were determined breath by breath by a portable metabograph (K4b2, Cosmed, Italy). ˙VO2peak values were taken as the highest 30 s average ˙VO2 value attained before the subject’s volitional exhaustion. Respiratory exchange ratio (RER) was calculated as the ratio of ˙VCO2 to ˙VO2. At rest and at various times (5, 7, and 9 min) during recovery, 0.6 µL of capillary blood was obtained from a preheated earlobe for the determination of blood lactate concentration ([La]b) (Lactate Plus, Nova Biomedical). A very short maximal sprint (60-m) was adopted in order to design a quick test that could be performed in a non-specialized environment: only rubber soles and a short straight path, in addition to the smart watch, would be necessary. We decided to use only the HR oﬀ-kinetics because even more professional HR sensors (i.e., the thoracic belt) have troubles to detect just the heart signal when many other muscles in the body are intensively activated, as during maximal propulsion. Heart Rate Kinetics A paired t-test did not show signiﬁcant diﬀerence (p = 0.97) between measured ˙VO2peak and the predicted value (b˙VO2peak) and the SEE was 5.28 ml.kg−1.min−1. Figure 4 shows the Bland- Altman plot ˙VO2diﬀ(= b˙VO2peak −˙VO2peak) vs. mean ˙VO2peak, both for a beat-to-beat analysis and for a 5 s average of HR oﬀ-kinetics (see below). Statistics Data are presented as mean ± standard deviation (SD). Multiple linear regression analysis was adopted to explain the variance of the individual ˙VO2peak, based on independent variables vtest, voﬀ and 1HR. Linear regressions were used to analyse correlations between variables and residuals of predicted (b˙VO2peak) vs. measured ˙VO2peak. The Standard Error of the Estimate (SEE) was calculated to measure the accuracy of the prediction and to compare it to other published predictors of ˙VO2peak. Statistical signiﬁcance was granted at p ≤0.05. Statistical analysis was performed by using SPSS v20 (IBM, USA). The idea behind this investigation has been to ﬁnd a simple and short test that could reasonably predict individual ˙VO2peak, based on signals from sensors that constitute the current “equipment” inside mobile/smart devices (phones or watches). The idea behind this investigation has been to ﬁnd a simple and short test that could reasonably predict individual ˙VO2peak, based on signals from sensors that constitute the current “equipment” inside mobile/smart devices (phones or watches). The software algorithm has been designed as to use the most meaningful part of the post-sprint HR time course: it was noted that signal is often still increasing or almost constant before starting the decay toward the resting value (Figures 1, 5). Thus, in order to better quantify HR oﬀ-kinetics, a routine trimmed the data and fed the statistical procedure (exponential regression Least Squares Method) with just-decay values. We did not analyse the on-kinetics because, as expected, during the sprint, HR values are scattered (see the data between the ﬁrst two vertical lines in Figures 1, 5) presumably due to both the interferences of the contracting thoracic muscles and of the belt vibrations on the ECG signal. However, as an indicator of the on-kinetics the overall HR variation from rest baseline to the beginning of the oﬀ-kinetics (1HR) was included in the model. Heart Rate Kinetics Aim of the study was to propose a simple methodology and algorithm predicting individual aerobic ﬁtness, and check its adherence to experimentally measured ˙VO2peak values. This test could be implemented in many smart wearable devices and would certainly beneﬁt from the inevitable improvement in sensor technology. Heart rate oﬀ-kinetics (HR decrease after 60-m sprint) was modeled according to a mono-exponential function of time by using a Least Squares Method (minimizing the sum of squared vertical distances between experimental points and the exponential curve): November 2017 \| Volume 8 \| Article 868 2 ˙VO2peak Test for Smart Wearables Storniolo et al. HR (t) = HRbaseline + Ampl · e(−t/τoﬀ) HR (t) = HRbaseline + Ampl · e(−t/τoﬀ) (1) HR (t) = HRbaseline + Ampl · e(−t/τoﬀ) (Figures 2A,B), whereas 1HR was not related to ˙VO2peak (r = −0.18, p = 0.39) (Figure 2C). where HRbaseline is the average of HR (bpm) during the last 60 s of the recovery period; Ampl is the asymptotic amplitude for the exponential term (maximal HR values −HRbaseline, bpm); τoﬀis the time constant (s) of the exponential, i.e., the time from the end of the sprint to reach 27% of HR maximum excursion [which corresponds to HR = HRbaseline + Ampl (1–63%)]. The velocity of HR decays after the sprint (voﬀ, s−1) was inferred as the reciprocal of τoﬀ. Also, the heart rate range from the sprint start to the beginning of the oﬀ-kinetics phase (1HR) was calculated (Figure 1). All data have been analyzed with purposely written LabView programs (release 13, National Instruments). Multiple regression analysis ( ˙VO2peak = 7.46 · vtest + 261.4 · voﬀ−0.19 · 1HR) showed that a linear combination of vtest, voﬀand HR from the sprint test explained 65% of the ˙VO2peak variance (R2 = 0.65, p < 0.001) (Figure 3). oﬀ ) test, voﬀand HR from the sprint test explained 65% of the ˙VO2peak variance (R2 = 0.65, p < 0.001) (Figure 3). A paired t-test did not show signiﬁcant diﬀerence (p = 0.97) between measured ˙VO2peak and the predicted value (b˙VO2peak) and the SEE was 5.28 ml.kg−1.min−1. Figure 4 shows the Bland- Altman plot ˙VO2diﬀ(= b˙VO2peak −˙VO2peak) vs. mean ˙VO2peak, both for a beat-to-beat analysis and for a 5 s average of HR oﬀ-kinetics (see below). RESULTS ˙VO2peak range was 29.1–56.6 ml.kg−1.min−1 (mean ± SD, 42.5 ± 8.7 ml.kg−1.min−1). Peak net blood lactate concentration after the incremental test was 8.5 ± 1.3 mM. All groups attained maximal HR values corresponding to 95% of the age predicted maximum and RER values > 1.1. Thus, taking into account also [La]b peak values, it can be assumed that subjects reached the maximum exercise capacity. The Multiple Regression has been designed to correlate a measure of metabolic power (= metabolic work/time, ˙VO2peak) to three predictors: as two of them originally have units with time at the denominator (vtest and 1HR), we decided to transform τoﬀinto voﬀ= 1 τoﬀ, to increase the “linearity” of their statistical eﬀect. The vtest and voﬀ were signiﬁcantly related to ˙VO2peak (r = 0.74, p < 0.001; r = 0.43, p = 0.03, respectively) FIGURE 1 \| Example of a representative heart rate time course from rest to recovery of the maximal sprint test. The thick vertical line coincides with the start of the sprint, the dashed vertical line with the end of the sprint, the thin vertical line represents the start decay of HR, while the dotted line expresses the τoff and the gray continuous curve represents the mono-exponential best ﬁt. Despite of the short duration and the simplicity of the test, its reliability in predicting the “real” ˙VO2peak values has been witnessed by the signiﬁcant correlation of Multiple Regression and by an acceptable standard error of the estimate (also in relation to previous literature, see Figure 6 and below). Among the predictors, when individually compared to ˙VO2peak, only 1HR does not signiﬁcantly correlate (albeit negatively) with ˙VO2peak (see Figure 2C). This can be ascribed to two contrasting eﬀects: (1) 1HR should be higher in highly ﬁt subjects due to their faster on-kinetics, and (2) highly ﬁt subjects (as shown by a signiﬁcant correlation in Figure 2A) run the same 60-m in a shorter time, thus not allowing HR to reach a high value. However, the regression made just by vtest and voﬀas variates explains a smaller portion of the experimental ˙VO2peak variance (55%), witnessing the value of 1HR in the multiple regression model. FIGURE 1 \| Example of a representative heart rate time course from rest to recovery of the maximal sprint test. RESULTS Solid line (average bias = −0.04 ml.kg−1.min−1); dashed line indicates 95% limits of agreement. The trend line equation expresses y = 0.28x −11.9, with r = 0.40 (p = 0.04) with conﬁdence intervals (95%). Gray circles: predicted values based on 5 s average of HR time course (see section Discussion). FIGURE 3 \| Relation between measured ˙VO2peak and predicted b˙VO2peak estimated from multiple linear regression b˙VO2peak = 7.46·vtest + 261.4·voff − 0.19·1HR. Trend line (thick black line) expresses the linear regression between the variables (b˙VO2peak = 0.62 ˙VO2peak + 15.9; r = 0.80). The thin black lines are the conﬁdence interval (95%) of the trend line, while the dashed gray line is the identity line. FIGURE 3 \| Relation between measured ˙VO2peak and predicted b˙VO2peak estimated from multiple linear regression b˙VO2peak = 7.46·vtest + 261.4·voff − 0.19·1HR. Trend line (thick black line) expresses the linear regression between the variables (b˙VO2peak = 0.62 ˙VO2peak + 15.9; r = 0.80). The thin black lines are the conﬁdence interval (95%) of the trend line, while the dashed gray line is the identity line. have been shown to inﬂuence the speed/tau of HR oﬀ-kinetics due to diﬀerent energetic contribution and released metabolites during exercise and recovery (Pierpont et al., 2000; Buchheit et al., 2007; Borresen and Lambert, 2008; Al Haddad et al., 2009; Nakamura et al., 2009; do Nascimento Salvador et al., 2016). Such heterogeneity in exercise related factors and diﬀerent indexes used for deﬁning the oﬀ-kinetics make a comparison of decays among diﬀerent studies quite troublesome. line with previous literature (Darr et al., 1988; Yamamoto et al., 1991; Sugawara et al., 2001; Carnethon et al., 2005; Giallauria et al., 2005; Ostojic et al., 2011; de Mendoca et al., 2017). HR oﬀ-kinetics is characterized by a coordinated interaction of parasympathetic re-activation and sympathetic withdrawal (Perini et al., 1989; Imai et al., 1994; Dewland et al., 2007; Borresen and Lambert, 2008; Maeder et al., 2009; Daanen et al., 2012) and it seems that in trained subjects the adaptations in the eﬀerent parasympathetic pathway could accelerate the vagus- mediated heart rate recovery (Imai et al., 1994; Dewland et al., 2007). On the other hand Hagberg et al. (1979) found that this faster recovery was not related to a more rapid recovery of the sympathetic response to exercise. RESULTS The thick vertical line coincides with the start of the sprint, the dashed vertical line with the end of the sprint, the thin vertical line represents the start decay of HR, while the dotted line expresses the τoff and the gray continuous curve represents the mono-exponential best ﬁt. FIGURE 1 \| Example of a representative heart rate time course from rest to recovery of the maximal sprint test. The thick vertical line coincides with the start of the sprint, the dashed vertical line with the end of the sprint, the thin vertical line represents the start decay of HR, while the dotted line expresses the τoff and the gray continuous curve represents the mono-exponential best ﬁt. The HR oﬀ-kinetics (voﬀFigure 2B) is positively related to ˙VO2peak: ﬁtter subjects showed a faster decay; this result is in November 2017 \| Volume 8 \| Article 868 Frontiers in Physiology \| www.frontiersin.org Frontiers in Physiology \| www.frontiersin.org 3 ˙VO2peak Test for Smart Wearables Storniolo et al. FIGURE 2 \| Relation between ˙VO2peak and vtest (A), voff (B), and 1HR. (C) are illustrated. FIGURE 3 \| Relation between measured ˙VO2peak and predicted b˙VO2peak estimated from multiple linear regression b˙VO2peak = 7.46·vtest + 261.4·voff − 0.19·1HR. Trend line (thick black line) expresses the linear regression between the variables (b˙VO2peak = 0.62 ˙VO2peak + 15.9; r = 0.80). The thin black lines are the conﬁdence interval (95%) of the trend line, while the dashed gray line is the identity line. FIGURE 4 \| Bland-Altman plot of ˙VO2peak difference (measured −predicted values) vs. mean ˙VO2peak (black circles). Solid line (average bias = −0.04 ml.kg−1.min−1); dashed line indicates 95% limits of agreement. The trend line equation expresses y = 0.28x −11.9, with r = 0.40 (p = 0.04) with conﬁdence intervals (95%). Gray circles: predicted values based on 5 s average of HR time course (see section Discussion). have been shown to inﬂuence the speed/tau of HR oﬀ-kinetics due to diﬀerent energetic contribution and released metabolites during exercise and recovery (Pierpont et al., 2000; Buchheit et al., 2007; Borresen and Lambert, 2008; Al Haddad et al., 2009; Nakamura et al., 2009; do Nascimento Salvador et al., 2016). Such heterogeneity in exercise related factors and diﬀerent indexes FIGURE 2 \| Relation between ˙VO2peak and vtest (A), voff (B), and 1HR. (C) are illustrated. FIGURE 2 \| Relation between ˙VO2peak and vtest (A), voff (B), and 1HR. (C) are illustrated. RESULTS FIGURE 2 \| Relation between ˙VO2peak and vtest (A), voff (B), and 1HR. (C) are illustrated. FIGURE 3 \| Relation between measured ˙VO2peak and predicted b˙VO2peak estimated from multiple linear regression b˙VO2peak = 7.46·vtest + 261.4·voff − 0.19·1HR. Trend line (thick black line) expresses the linear regression between the variables (b˙VO2peak = 0.62 ˙VO2peak + 15.9; r = 0.80). The thin black lines are the conﬁdence interval (95%) of the trend line, while the dashed gray line is the identity line. FIGURE 4 \| Bland-Altman plot of ˙VO2peak difference (measu values) vs. mean ˙VO2peak (black circles). Solid line (average b ml.kg−1.min−1); dashed line indicates 95% limits of agreeme equation expresses y = 0.28x −11.9, with r = 0.40 (p = 0.0 conﬁdence intervals (95%). Gray circles: predicted values bas average of HR time course (see section Discussion). have been shown to inﬂuence the speed/tau of H due to diﬀerent energetic contribution and releas during exercise and recovery (Pierpont et al., 2 et al., 2007; Borresen and Lambert, 2008; Al Hadd Nakamura et al., 2009; do Nascimento Salvador et heterogeneity in exercise related factors and di FIGURE 3 \| Relation between measured ˙VO2peak and predicted b˙VO2peak estimated from multiple linear regression b˙VO2peak = 7.46·vtest + 261.4·voff − 0.19·1HR. Trend line (thick black line) expresses the linear regression between the variables (b˙VO2peak = 0.62 ˙VO2peak + 15.9; r = 0.80). The thin black lines are the conﬁdence interval (95%) of the trend line, while the dashed gray line is the identity line. FIGURE 4 \| Bland-Altman plot of ˙VO2peak difference (measured −predicted values) vs. mean ˙VO2peak (black circles). Solid line (average bias = −0.04 ml.kg−1.min−1); dashed line indicates 95% limits of agreement. The trend line equation expresses y = 0.28x −11.9, with r = 0.40 (p = 0.04) with conﬁdence intervals (95%). Gray circles: predicted values based on 5 s average of HR time course (see section Discussion). FIGURE 4 \| Bland-Altman plot of ˙VO2peak difference (measured −predicted values) vs. mean ˙VO2peak (black circles). Solid line (average bias = −0.04 ml.kg−1.min−1); dashed line indicates 95% limits of agreement. The trend line equation expresses y = 0.28x −11.9, with r = 0.40 (p = 0.04) with conﬁdence intervals (95%). Gray circles: predicted values based on 5 s average of HR time course (see section Discussion). FIGURE 4 \| Bland-Altman plot of ˙VO2peak difference (measured −predicted values) vs. mean ˙VO2peak (black circles). RESULTS Exercise intensity and type line with previous literature (Darr et al., 1988; Yamamoto et al., 1991; Sugawara et al., 2001; Carnethon et al., 2005; Giallauria et al., 2005; Ostojic et al., 2011; de Mendoca et al., 2017). HR oﬀ-kinetics is characterized by a coordinated interaction of parasympathetic re-activation and sympathetic withdrawal (Perini et al., 1989; Imai et al., 1994; Dewland et al., 2007; Borresen and Lambert, 2008; Maeder et al., 2009; Daanen et al., 2012) and it seems that in trained subjects the adaptations in the eﬀerent parasympathetic pathway could accelerate the vagus- mediated heart rate recovery (Imai et al., 1994; Dewland et al., 2007). On the other hand Hagberg et al. (1979) found that this faster recovery was not related to a more rapid recovery of the sympathetic response to exercise. Exercise intensity and type In the present study, subjects performed a shorter eﬀort than those present in literature, reached a maximal HR of 150 ± 20 bpm, which is the 79% of the maximal HR of the incremental test, in about 20 s. At the beginning of the exercise, the onset of HR is characterized by the fast vagal withdraw and the slower sympathetic activation. Even if our exercise duration was very short it seems that both mechanisms would have been activated in order to reach (and then recover from) the 79% of the maximal HR. As shown in Figures 3, 4, the Multiple Regression overestimates and underestimates measured values at low and high ˙VO2peak, respectively. The predictive equation was November 2017 \| Volume 8 \| Article 868 Frontiers in Physiology \| www.frontiersin.org 4 Storniolo et al. ˙VO2peak Test for Smart Wearables FIGURE 5 \| Heart rate recording of each participant (n = 25) during the sprint test and their respective markers: thick black vertical line as start of the sprint, dashed vertical line representing end of the sprint, thin vertical black line denotes the start decay of HR, and dotted line expresses the τoff. FIGURE 5 \| Heart rate recording of each participant (n = 25) during the sprint test and their respective markers: thick black vertical line as start of the sprint, dashed vertical line representing end of the sprint, thin vertical black line denotes the start decay of HR, and dotted line expresses the τoff. RESULTS Apple Watch (Apple Inc., California, USA), which has been mentioned for a very high reliability in processing physiological data during physical exercise (Chowdhury et al., 2017; Wang et al., 2017), does not output beat-to-beat intervals. Blood oxygenation pulsations (photo-plethysmography) are detected by photodetectors measuring the bounced back infrared light emitted by LED diodes located between wrist and watch. The ﬂuctuations in blood color absorption due to the local volume changes are measured resulting in HR data. A few studies have emphasized the accuracy of these wrist-worn devices (Spierer et al., 2015; Wallen et al., 2016; Chowdhury et al., 2017; Wang et al., 2017) during rest and exercise. Nevertheless, no system at present seems to be conﬁdent enough to deliver single beat interval/frequency, probably due to motion induced artifacts, a problem that could be solved by sensor redundancy and/or signal processing enhancing signal-to-noise ratio. veriﬁed with a random sampling approach. From the whole sample (n = 25), 12 subjects were randomly extracted and used as a new control group for the predictions of the multiple regression that was performed on the other 13 subjects. This process was performed 35 times (taking care of avoiding duplicated group composition). We obtained 35 new predictive equations and average discrepancies between the measured and predicted ˙VO2peak. The mean SEE was 6.31 ± 0.82, similar to 5.28 obtained from processing the whole sample. Although we used lab-quality sensor technology, the implementation of the proposed test on consumer wearables could manage the whole experimental protocol locally: continuous beat-by-beat HR would be used both to monitor/warn the subjects on the most appropriate time at which to start the 60-m sprint (i.e., when a rest steady state is reached) and to collect the recovery phase. GPS and 3-axis accelerometers could provide where and when, respectively, the 60-m sprint started and ended, from which the overall distance traveled can be checked and the average speed calculated. Current technology conﬁnes the time resolution of most of those devices (Parak et al., 2015) to about 5 s, within which an average heart frequency is computed. Although our investigation is particularly meant for next, beat-by-beat sensors, we tested the predictive ability of the proposed algorithm when HR data was provided at 0.2 Hz (as in the actual versions). Frontiers in Physiology \| www.frontiersin.org RESULTS This was achieved by manipulating the recorded single-beat sequences as to obtain The use of a “traditional” thoracic belt sensor (Polar S410, Kempele, Finland) has been driven by the need of the most accurate, beat-by-beat HR sensor. There is no such a capability, so far, in most consumer wrist-wearable devices. Even November 2017 \| Volume 8 \| Article 868 5 Storniolo et al. ˙VO2peak Test for Smart Wearables FIGURE 6 \| Accuracy of the ˙VO2peak prediction, as SEE (ml.kg−1.min−1), is presented in relation to average protocol duration (t, min) clustered in exercise (red positive bars) and rest (blue negative bars) time. Present data is shown as thick lines. SEE of other predictive equations on submaximal protocols are shown for comparison with their bibliographic reference. A more detailed discussion about the quoted investigations can be found in Sartor et al. (2013) review. FIGURE 6 \| Accuracy of the ˙VO2peak prediction, as SEE (ml.kg−1.min−1), is presented in relation to average protocol duration (t, min) clustered in exercise (red positive bars) and rest (blue negative bars) time. Present data is shown as thick lines. SEE of other predictive equations on submaximal protocols are shown for comparison with their bibliographic reference. A more detailed discussion about the quoted investigations can be found in Sartor et al. (2013) review. inter-subject repeatability could enhance the power of the predictive equation. an average value every 5 s. In Figure 4 gray points reﬂect the approximation involved in using 5 s average HR data, which resulted quite similar to single-beat regressions (R2 = 59%). Results from the current investigation encourage to develop new simple methods to infer individual physiologic variables by exploiting the current and next portable technology. Watches and bracelets are the perfect candidates, with respect to smart phones, because of their small size and the increased computational power capable to sample and process the data on-board, with no immediate need of external connection. The proposed, indirect ˙VO2peak test is certainly not meant to replace the usual direct metabolic measurements and protocols done in a research or clinical laboratory setting, where a much higher accuracy is required in the assessment of subject/athlete’s aerobic ﬁtness. By using a portable smart device with multiple sensors and the suggested algorithm, a handy ˙VO2peak estimation is at reach for individuals who could later decide whether or not to deepen the awareness of their health status. CONCLUSION A simple and short test (60-m sprint run) could reasonably predict individual ˙VO2peak based on the heart rate oﬀ-kinetics immediately after the sprint. This test can be easily managed by all individuals with the new wrist wearable, heart band free, multi-sensor smart devices and the proposed algorithm. The proposed test leaves space for improvement: (a) HR kinetics are known (Astrand et al., 1986) to be aﬀected by a number of conditions (age, body and ambient temperature, over-training, altitude, fatigue, hydration, etc.) here not taken into account, (b) oﬀ-kinetics only have been considered, but new processing techniques (e.g., Salehizadeh et al., 2016) could allow to include HR on-kinetics to better infer ˙VO2peak, and (c) new reﬁned modeling approaches (e.g., Zakynthinaki, 2015) could help to incorporate in the algorithm and detect slightly diﬀerences in the delayed oﬀ-kinetics start that could better estimate the ﬁtness level, (d) a greater sample size with AUTHOR CONTRIBUTIONS AM conceived the study, JS and GP collected data, JS, GP, and AM analyzed data, wrote the manuscript and revised the ﬁnal version. RESULTS However, when compared with many other ˙VO2peak predicting submaximal protocols done on similar subjects, based on diﬀerent physical activities and with much longer exercise duration (thus more distressful conditions, quoted in Figure 6), the SEE was quite similar: 5.28 for the 60-m sprint vs. 4.63 ± 1.58 (average) of the literature. Frontiers in Physiology \| www.frontiersin.org REFERENCES Physiol. 254, 340–343. de Mendoca, G. V., Teodosio, C., and Bruno, P. M. (2017). Sexual dimorphism in heart rate recovery from peak exercise. Eur. J. Appl. Physiol. 117, 1373–1381. doi: 10.1007/s00421-017-3627-8 Ostojic, S. M., Stojanovic, M. D., and Calleja-Gonzalez, J. (2011). Ultra short- term heart rate recovery after maximal exercise: relations to aerobic power in Ostojic, S. M., Stojanovic, M. D., and Calleja-Gonzalez, J. (2011). Ultra short- term heart rate recovery after maximal exercise: relations to aerobic power in sportsmen. Chin. J. Physiol. 54, 105–110. doi: 10.4077/CJP.2011.AMM018 y p sportsmen. Chin. J. Physiol. 54, 105–110. doi: 10.4077/CJP.2011.AMM018 Dewland, T. A., Androne, A. S., Lee, F. A., Lampert, R. J., and Katz, S. D. (2007). Eﬀect of acetylcholinesterase inhibition with pyridostigmine on cardiac parasympathetic function in sedentary adults and trained athletes. Am. J. Physiol. Heart Circ. Physiol. 293, 86–92 doi: 10.1152/ajpheart.01339.2006 Otsuki, T., Maeda, S., Iemitsu, M., Saito, Y., Tanimura, Y., Sugawara, J., et al. (2007). Postexercise heart rate recovery accelerates in strength-trained athletes. Med. Sci. Sports Exerc. 39, 365–370. doi: 10.1249/01.mss.0000241647. 13220.4c Parak, J., Tarniceriu, A., Renevey, P., Bertschi, M., Delgado-Gonzalo, R., and Korhonen, I. (2015). Evaluation of the beat-to-beat detection accuracy of PulseOn wearable optical heart rate monitor. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2015, 8099–8102. doi: 10.1109/EMBC.2015.7320273 do Nascimento Salvador, P. C., de Aguiar, R. A., Teixeira, A. S., Souza, K. M., de Lucas, R. D., Denadai, B. S., et al. (2016). Are the oxygen uptake and heart rate oﬀ-kinetics inﬂuenced by the intensity of prior exercise? Respir Physiol Neurobiol. 230, 60–67. doi: 10.1016/j.resp.2016.05.007 Perini, R., Orizio, C., Comande, A., Castellano, M., Beschi, M., and Veicsteinas, A. (1989). Plasma norepinephrine and heart rate dynamics during recovery from submaximal exercise in man. Eur. J. Appl. Physiol. Occup. Physiol. 58, 879–883. doi: 10.1007/BF02332222 Fox, E. L. (1973). A simple, accurate technique for predicting maximal aerobic power. J. Appl. Physiol. 35, 914–916. George, J. D., Vehrs, P. R., Allsen, P. E., Fellingham, G. W., and Fisher, A. G. (1993). VO2max estimation from a submaximal 1-mile track jog for ﬁt college-age individuals. Med. Sci. Sports Exerc. 25, 401–406. doi: 10.1249/00005768-199303000-00017 Pierpont, G. L., Stolpman, D. R., and Gornick, C. C. (2000). Heart rate recovery post-exercise as an index of parasympathetic activity. J. Auton. Nerv. Syst. 80, 169–174. doi: 10.1016/S0165-1838(00)00090-4 Giallauria, F., Del Forno, D., Pilerci, F., De Lorenzo, A., Manakos, A., Lucci, R., et al. (2005). REFERENCES Hickson, R. C., Bomze, H. A., and Holloszy, J. O. (1978). Faster adjustments of O2 uptake to the energy requirement of exercise in the trained state. J. Appl. Physiol. Respir. Environ. Exerc. Physiol. 44, 877–881. Akalan, C., Robergs, R. A., and Kravitz, L. (2008). Prediction of VO2max from an individualized submaximal cycle ergometer protocol. J. Exerc. Physiol. Online. 11, 1–17. Imai, K., Sato, H., Hori, M., Kusuoka, H., Ozaki, H., Yokoyama, H., et al. (1994). Vagally mediated heart rate recovery after exercise is accelerated in athletes but blunted in patients with chronic heart failure. J. Am. Coll Cardiol. 24, 1529–1535. doi: 10.1016/0735-1097(94)90150-3 Al Haddad, H., Laursen, P. B., Ahmaidi, S., and Buchheit, M. (2009). Nocturnal heart rate variability following supramaximal intermittent exercise. Int. J. Sports Physiol. Perform. 4, 435–447. doi: 10.1123/ijspp.4.4.435 Jetté, M., Campbell, J., Mongeon, J., and Routhier, R. (1976). The Canadian home ﬁtness test as a predictor for aerobic capacity. Can. Med. Assoc. J. 114, 680–682. Astrand, P. O., Rodahl, K., Dahl, H. A., and Stromme, S. B. (1986). Textbook of Work Physiology - Physiological Bases of Exercise. Champaign: Human Kinetics. Jones, A. M., and Poole, D. C. (2005). Oxygen Uptake Kinetics in Sport, Exercise and Medicine. Abingdon: Routledge, Taylor & Francis Books. Beekley, M. D., Brechue, W. F., de Hoyos, D. V., Garzarella, L., Werber-Zion, G., and Pollock, M. (2004). Cross-validation of the YMCA submaximal cycle ergometer test to predict VO2max. Res. Q. Exerc. Sport. 75, 337–342. doi: 10.1080/02701367.2004.10609165 Kline, G. M., Porcari, J. P., Hintermeister, R., Freedson, P. S., Ward, A., McCarron, R. F., et al. (1987). Estimation of VO2max from a one-mile track walk, gender, age, and body weight. Med. Sci. Sports Exerc. 19, 253–259. doi: 10.1249/00005768-198706000-00012 Bonen, A., Heyward, V. H., Cureton, K. J., Boileau, R. A., and Massey, B. H. (1979). Prediction of maximal oxygen uptake in boys, ages 7–15 years. Med Sci Sports. 11, 24–29. Larsen, G. E., George, J. D., Alexander, J. L., Fellingham, G. W., Aldana, S. G., and Parcell, A. C. (2002). Prediction of maximum oxygen consumption from walking, jogging, or running. Res. Q. Exerc. Sport. 73, 66–72. doi: 10.1080/02701367.2002.10608993 Borresen, J., and Lambert, M. I. (2008). Autonomic control of heart rate during and after exercise: measurements and implications for monitoring training status. Sports Med. 38, 633–646. doi: 10.2165/00007256-200838080-00002 Maeder, M. T., Ammann, P., Rickli, H., and Brunner-La Rocca, H. P. (2009). REFERENCES Impact of the exercise mode on heart rate recovery after maximal exercise. Eur. J. Appl. Physiol. 105, 247–255. doi: 10.1007/s00421-008-0896-2 Buchheit, M., Laursen, P. B., and Ahmaidi, S. (2007). Parasympathetic reactivation after repeated sprint exercise. Am. J. Physiol. Heart Circ. Physiol. 293, 133–141. doi: 10.1152/ajpheart.00062.2007 Magrani, P., and Pompeu, F. A. (2010). Equations for predicting aerobic power VO2 of young Brazilian adults. Arq. Bras. Cardiol. 94, 763–770. doi: 10.1590/S0066-782X2010005000054 Buono, M. J., Borin, T. L., Sjoholm, N. T., and Hodgdon, J. A. (1996). Validity and reliability of a timed 5 km cycle ergometer ride to predict maximum oxygen uptake. Physiol. Meas. 17, 313–317. doi: 10.1088/0967-3334/17/4/009 Mier, C. M., and Gibson, A. L. (2004). Evaluation of a treadmill test for predicting the aerobic capacity of ﬁreﬁghters. Occup. Med. 54, 373–378. doi: 10.1093/occmed/kqh008 Carnethon, M. R., Jacobs, D. R., Sidney, S., Sternfeld, B., Gidding, S. S., Shoushtari, C., et al. (2005). A longitudinal study of physical activity and heart rate recovery: CARDIA, 1987–1993. Med. Sci. Sports Exerc. 37, 606–612. doi: 10.1249/01.MSS.0000158190.56061.32 Nakagaichi, M., Lee, M. S., and Tanaka, K. (2001). Accuracy of two simple methods for the assessment of health-related physical ﬁtness. Percept. Mot. Skills. 92, 37–49. doi: 10.2466/pms.2001.92.1.37 Chowdhury, E. A., Western, M. J., Nightingale, T. E., Peacock, O. J., and Thompson, D. (2017). Assessment of laboratory and daily energy expenditure estimates from consumer multi-sensor physical activity monitors. PLoS ONE 12:e0171720. doi: 10.1371/journal.pone.0171720 Nakamura, F. Y., Soares-Caldeira, L. F., Laursen, P. B., Polito, M. D., Leme, L. C., and Buchheit, M. (2009). Cardiac autonomic responses to repeated shuttle sprints. Int. J. Sports Med. 30, 808–813. doi: 10.1055/s-0029-1234055 Norris, S. R., and Petersen, S. R. (1998). Eﬀects of endurance training on transient oxygen uptake responses in cyclists. J. Sports Sci. 16, 733–738. doi: 10.1080/026404198366362 Daanen, H. A., Lamberts, R. P., Kallen, V. L., Jin, A., and Van Meeteren, N. L. (2012). A systematic review on heart-rate recovery to monitor changes in training status in athletes. Int. J. Sports Physiol. Perform. 7, 251–260. doi: 10.1123/ijspp.7.3.251 Ostojic, S. M., Markovic, G., Calleja-Gonzalez, J., Jakvoljevic, D. G., Vucetic, V., and Stojanovic, M. D. (2010). Ultra short-term heart rate recovery after maximal exercise in continuous versus intermittent endurance athletes. Eur. J. Appl. Physiol. 108, 1055–1059. doi: 10.1007/s00421-009-1313-1 Darr, K. C., Bassett, D. R., Morgan, B. J., and Thomas, D. P. (1988). Eﬀects of age and training status on heart rate recovery after peak exercise. Am. J. ACKNOWLEDGMENTS JS acknowledges the ﬁnancial support from National Council for Scientiﬁc and Technological Development-CNPq/Brazil. November 2017 \| Volume 8 \| Article 868 6 ˙VO2peak Test for Smart Wearables Storniolo et al. REFERENCES Improvement of heart rate recovery after exercise training in older people. J. Am. Geriatr Soc. 53, 2037–2038. doi: 10.1111/j.1532-5415.2005.00479_4.x Pober, D. M., Freedson, P. S., Kline, G. M., McInnis, K. J., and Rippe, J. M. (2002). Development and validation of a one-mile treadmill walk test to predict peak oxygen uptake in healthy adults ages 40 to 79 years. Can. J. Appl. Physiol. 27, 575–589. doi: 10.1139/h02-033 Hagberg, J. M., Hickson, R. C., Eshani, A. A., and Holloszy, J. O. (1980). Faster adjustments to and recovery from submaximal exercise in the trained state. J. Appl. Physiol. 48, 218–224. Rossiter, H. B. (2011). Exercise: kinetic considerations for gas Exchange. Compr Physiol. 1, 203–244. doi: 10.1002/cphy.c090010 Salehizadeh, S. M. A., Dao, D., Bolkhovsky, J., Cho, C., Mendelson, Y., and Chon, K. H. (2016). A novel time-varying spectral ﬁltering algorithm for reconstruction of motion artifact corrupted heart rate signals during intense Hagberg, J. M., Hickson, R. C., McLane, J. A., Ehsani, A. A., and Winder, W. W. (1979). Disappearance of norepinephrine from the circulation following strenuous exercise. J. Appl. Physiol. 47, 1311–1314. November 2017 \| Volume 8 \| Article 868 Frontiers in Physiology \| www.frontiersin.org 7 ˙VO2peak Test for Smart Wearables Storniolo et al. Timmons, J. A., Gustafsson, T., Sundberg, C. J., Jansson, E., Hultman, E., and Kaijser, L. (1998). Substrate availability limits human skeletal muscle oxidative ATP generation at the onset of ischemic exercise. J. Clin. Invest. 101, 79–85. doi: 10.1172/JCI1146 physical activities using a wearable photoplethysmogram sensor. Sensors 16:10. doi: 10.3390/s16010010 Santo, A. S., and Golding, L. A. (2003). Predicting maximum oxygen uptake from a modiﬁed 3-minute step test. Res. Q. Exerc. Sport. 74, 110–115. doi: 10.1080/02701367.2003.10609070 Vehrs, P. R., George, J. D., Fellingham, G. W., Plowman, S. A., and Dustman- Allen, K. (2007). Submaximal treadmill exercise test to predict VO2max in ﬁt adults. Meas. Phys. Educ. Exerc. Sci. 11, 61–72 doi: 10.1080/109136707012 94047 Sartor, F., Vernillo, G., Morree, H. M., Bonomi, A. G., La Torre, A., Kubis, H.- P., et al. (2013). Estimation of the maximal oxygen uptake via submaximal exercise testing in sports, clinical, and home settings. Sports Med. 43, 865–873 doi: 10.1007/s40279-013-0068-3 Wallen, M. P., Gomersall, S. R., Keating, S. E., Wisloﬀ, U., and Coombes, J. S. (2016). Accuracy of heart rate watches: implications for weight management. PLoS ONE. 11:e0154420. doi: 10.1371/journal.pone.0154420 Siconolﬁ, S. F, Garber, C. E, Lasater, T. M., and Carleton, R. A. (1985). REFERENCES A simple, valid step test for estimating maximal oxygen uptake in epidemiologic studies. Am. J. Epidemiol. 121, 382–390. doi: 10.1093/oxfordjournals.aje.a114010 Wang, R., Gordon, B. G., Desai, M., Phelan, D., Gillinov, L., Houghtaling, P., et al. (2017). Accuracy of wrist-worn heart rate monitors. JAMA Cardiol. 2, 104–106. doi: 10.1001/jamacardio.2016.3340 Spierer, D. K., Rosen, Z., Litman, L. L., and Fujii, K. (2015). Validation of photoplethysmography as a method to detect heart rate during rest and exercise. J. Med. Eng. 39, 264–271. doi: 10.3109/03091902.2015.1047536 Watson, A., Brickson, S., Prawda, B. S., and Sanﬁlippo, J. L. (2017). Short-term heart rate recovery is related to aerobic ﬁtness in elite intermittent sport athletes. J. Strength Cond Res. 31, 1055–1061. doi: 10.1519/JSC.0000000000001567 g Sugawara, J., Murakami, H., Maeda, S., Kuno, S., and Matsuda, M. (2001). Change in post-exercise vagal reactivation with exercise training and detraining in young men. Eur. J. Appl. Physiol. 85, 259–263. doi: 10.1007/s004210100443 Weyand, P. G., Kelly, M., Blackadar, T., Darley, J. C., Oliver, S. R., Ohlenbusch, N. E., et al. (2001). Ambulatory estimates of maximal aerobic power from foot- ground contact times and heart rates in running humans. J. Appl. Physiol. 91, 451–458. Swain, D. P., Parrott, J. A., Bennett, A. R., Branch, J. D., and Dowling, E. A. (2004). Validation of a new method for estimating VO2max based on VO2 reserve. Med. Sci. Sports Exerc. 36, 1421–1426. doi: 10.1249/01.MSS.0000135774.28494.19 Swain, D. P., and Wright, R. L. (1997). Prediction of VO2peak from submaximal cycle ergometry using 50 versus 80 rpm. Med. Sci. Sports Exerc. 29, 268–272. doi: 10.1097/00005768-199702000-00016 Yamamoto, Y., Hughson, R. L., and Peterson, J. C. (1991). Autonomic control of heart rate during exercise studied by heart rate variability spectral analysis. J. Appl. Physiol. 71, 1136–1142. Swank, A. M., Serapiglia, L., Funk, D., Adams, K. J., Durham, M, and Berning, J. M. (2001). Development of a branching submaximal treadmill test for predicting VO2max. J. Strength Cond. Res. 15, 302–308. doi: 10.1519/00124278-200108000-00007 Zakynthinaki, M. S. (2015). Modeling heart rate kinetics. PLoS ONE 10:e0118263. doi: 10.1371/journal.pone.0118263 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Sykes, K., and Roberts, A. (2004). The Chester step test - a simple yet eﬀective tool for the prediction of aerobic capacity. Physiotherapy 90, 183–188. doi: 10.1016/j.physio.2004.03.008 Teh, K. C., and Aziz, A. R. (2000). Frontiers in Physiology \| www.frontiersin.org November 2017 \| Volume 8 \| Article 868 REFERENCES A stair-climb test of cardiorespiratory ﬁtness for Singapore. Singapore Med. J. 41, 588–594. Copyright © 2017 Storniolo, Pavei and Minetti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Tierney, M. T., Lenar, D., Stanforth, P.R., Craig, J. N., and Farrar, R. P. (2010). Prediction of aerobic capacity in ﬁreﬁghters using submaximal treadmill and stairmill protocols. J. Strength Cond. Res. 24, 757–764. doi: 10.1519/JSC.0b013e3181c7c282 November 2017 \| Volume 8 \| Article 868 Frontiers in Physiology \| www.frontiersin.org 8
https://openalex.org/W2041352679	https://europepmc.org/articles/pmc3297151?pdf=render	English	null	Nonlinear Dynamics and Chaos of Microcantilever-Based TM-AFMs with Squeeze Film Damping Effects	Sensors	2,009	cc-by	11,657	Sensors 2009, 9, 3854-3874; doi:10.3390/s90503854 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors Article Nonlinear Dynamics and Chaos of Microcantilever-Based TM-AFMs with Squeeze Film Damping Effects Wen-Ming Zhang , Guang Meng, Jian-Bin Zhou and Jie-Yu Chen State Key Laboratory of Mechanical System and Vibration, School of Mechanical Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China; E-Mails: gmeng@sjtu.edu.cn (G.M.); giantbean@sjtu.edu.cn (J.-B.Z.); jerrysmiling@hotmail.com (J.-Y.C.) Author to whom correspondence should be addressed; E-Mail: wenmingz@sjtu.edu.cn; Tel. +86-21-34206006; Fax: +86-21-34206831-206 Received: 27 March 2009; in revised form: 23 April 2009 / Accepted: 13 May 2009 / Published: 20 May 2009 OPEN ACCESS Sensors 2009, 9, 3854-3874; doi:10.3390/s90503854 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS Keywords: TM-AFM; microcantilever; squeeze film damping; Lennard-Jones (LJ) potential Nonlinear Dynamics and Chaos of Microcantilever-Based TM-AFMs with Squeeze Film Damping Effects Wen-Ming Zhang , Guang Meng, Jian-Bin Zhou and Jie-Yu Chen Wen-Ming Zhang , Guang Meng, Jian-Bin Zhou and Jie-Yu Chen State Key Laboratory of Mechanical System and Vibration, School of Mechanical Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China; E-Mails: gmeng@sjtu.edu.cn (G.M.); giantbean@sjtu.edu.cn (J.-B.Z.); jerrysmiling@hotmail.com (J.-Y.C.) * Author to whom correspondence should be addressed; E-Mail: wenmingz@sjtu.edu.cn; Tel. +86-21-34206006; Fax: +86-21-34206831-206 * Author to whom correspondence should be addressed; E-Mail: wenmingz@sjtu.edu.cn; Tel. +86-21-34206006; Fax: +86-21-34206831-206 Received: 27 March 2009; in revised form: 23 April 2009 / Accepted: 13 May 2009 / Published: 20 May 2009 Received: 27 March 2009; in revised form: 23 April 2009 / Accepted: 13 May 2009 / Published: 20 May 2009 Abstract: In Atomic force microscope (AFM) examination of a vibrating microcantilever, the nonlinear tip-sample interaction would greatly influence the dynamics of the cantilever. In this paper, the nonlinear dynamics and chaos of a tip-sample dynamic system being run in the tapping mode (TM) were investigated by considering the effects of hydrodynamic loading and squeeze film damping. The microcantilever was modeled as a spring-mass- damping system and the interaction between the tip and the sample was described by the Lennard-Jones (LJ) potential. The fundamental frequency and quality factor were calculated from the transient oscillations of the microcantilever vibrating in air. Numerical simulations were carried out to study the coupled nonlinear dynamic system using the bifurcation diagram, Poincaré maps, largest Lyapunov exponent, phase portraits and time histories. Results indicated the occurrence of periodic and chaotic motions and provided a comprehensive understanding of the hydrodynamic loading of microcantilevers. It was demonstrated that the coupled dynamic system will experience complex nonlinear oscillation as the system parameters change and the effect of squeeze film damping is not negligible on the micro-scale. Keywords: TM-AFM; microcantilever; squeeze film damping; Lennard-Jones (LJ) potential (LJ) Sensors 2009, 9 Sensors 2009, 9 Sensors 2009, 9 3855 1. Introduction Atomic force microscopy (AFM) has been developed to a nearly ubiquitous tool for studying physics, chemistry, biology, medicine and engineering at the nano-scale [1-5]. AFM could significantly impact many fabrication and manufacturing processes due to its advantages such as 3D topography of nano-fabrication and metrology for MEMS [2]. As a typical dynamic mode, the tapping mode (TM) is widely used in the operation of AFM where the cantilever is driven at a fixed frequency close or equal to the fundamental resonance frequency of vertical bending [4,6]; a schematic of the TM-AFM setup is shown in Figure 1. Moreover, the vibration amplitude of the cantilever is much bigger than the equilibrium separation between the tip and the sample. The TM-AFM has attracted extensive attention due to its ability to deal with compliant materials as well as to overcome adhesion forces. Figure 1. Schematic of the typical TM-AFM setup. Figure 1. Schematic of the typical TM-AFM setup. x y z The inherently and highly nonlinear tip-sample interaction will give rise to complex dynamics of the cantilever in TM-AFM [7]. Nonlinearity is essential in understanding the dynamics of cantilevers as there are many nonlinear forces in TM-AFM, such as the attractive van der Waals forces, the short- range repulsive interactions, contact nonlinearities and capillary forces et al. [8-10]. Ashhab et al. [11] concluded that chaos in AFM depend on the damping, excitation and tip-sample distance, and suggested that a state feedback control can be used to eliminate the possibility of chaotic behavior. Using nonlinear analysis methods and numerical simulations, Basso et al. [12] found that the chaotic behavior may occur via a cascade of period doubling bifurcations. In their studies [11,12], Melnikov theory was used to predict the existence of chaos in AFM. The nonlinear dynamics to frequency sweeps in TM-AFM was simulated using the van der Waals forces and the Derjaguin-Muller-Toporov (DMT) and Johnson-Kendall-Roberts (JKR) contact models [6,13]. Lee et al. [6] carried out numerical analysis using modern continuation tools for computational nonlinear dynamics and bifurcation problems where the tip-surface interaction was represented by the van der Waals and DMT contact forces. Nonlinear hysteresis and jumps in the dynamic response were examined as the tip approaches x y z The inherently and highly nonlinear tip-sample interaction will give rise to complex dynamics of the cantilever in TM-AFM [7]. 1. Introduction Nonlinearity is essential in understanding the dynamics of cantilevers as there are many nonlinear forces in TM-AFM, such as the attractive van der Waals forces, the short- range repulsive interactions, contact nonlinearities and capillary forces et al. [8-10]. Ashhab et al. [11] concluded that chaos in AFM depend on the damping, excitation and tip-sample distance, and suggested that a state feedback control can be used to eliminate the possibility of chaotic behavior. Using nonlinear analysis methods and numerical simulations, Basso et al. [12] found that the chaotic behavior may occur via a cascade of period doubling bifurcations. In their studies [11,12], Melnikov theory was used to predict the existence of chaos in AFM. The nonlinear dynamics to frequency sweeps in TM-AFM was simulated using the van der Waals forces and the Derjaguin-Muller-Toporov (DMT) and Johnson-Kendall-Roberts (JKR) contact models [6,13]. Lee et al. [6] carried out numerical analysis using modern continuation tools for computational nonlinear dynamics and bifurcation problems where the tip-surface interaction was represented by the van der Waals and DMT contact forces. Nonlinear hysteresis and jumps in the dynamic response were examined as the tip approaches The inherently and highly nonlinear tip-sample interaction will give rise to complex dynamics of the cantilever in TM-AFM [7]. Nonlinearity is essential in understanding the dynamics of cantilevers as there are many nonlinear forces in TM-AFM, such as the attractive van der Waals forces, the short- range repulsive interactions, contact nonlinearities and capillary forces et al. [8-10]. Ashhab et al. [11] concluded that chaos in AFM depend on the damping, excitation and tip-sample distance, and suggested that a state feedback control can be used to eliminate the possibility of chaotic behavior. Using nonlinear analysis methods and numerical simulations, Basso et al. [12] found that the chaotic behavior may occur via a cascade of period doubling bifurcations. In their studies [11,12], Melnikov theory was used to predict the existence of chaos in AFM. The nonlinear dynamics to frequency sweeps in TM-AFM was simulated using the van der Waals forces and the Derjaguin-Muller-Toporov (DMT) and Johnson-Kendall-Roberts (JKR) contact models [6,13]. Lee et al. [6] carried out numerical analysis using modern continuation tools for computational nonlinear dynamics and bifurcation problems where the tip-surface interaction was represented by the van der Waals and DMT contact forces. 1. Introduction Nonlinear hysteresis and jumps in the dynamic response were examined as the tip approaches Sensors 2009, 9 3856 Sensors 2009, 9 to or retracts from the sample at a fixed excitation frequency [14]. Zitzler et al. [15] considered the influence of hysteretic capillary forces in TM-AFM and studied the effect of the relative humidity on the amplitude and phase of the cantilever oscillation. By using the forward-time simulation and numerical continuation techniques [15], Hashemi et al. [10] investigated the nonlinear dynamics of a TM-AFM with tip-surface interactions, which include attractive, repulsive, and capillary forces. Hersam [3] conducted experiments to verify the importance of nonlinear dynamics in TM-AFM measurements. Rutzel et al. [9] used the Lennard-Jones (LJ) potential to model the tip-surface interactions and carried out a comprehensive investigation to the nonlinear dynamics and stability of the TM-AFM, and the results showed that considering the LJ interaction potential in modeling the dynamics of AFM could improve the qualitative prediction of the real system response. Nonlinear dynamics and chaos of the cantilever for the TM-AFM remain a challenging and critical issue. After experimental observations, Couturier et al. [16] mentioned that the motion of the cantilever could become chaotic under instability conditions. When the cantilever is driven close to the surface of the sample, the squeeze film between the cantilever and the sample surface contributes significantly to the damping and gives rise to the complicated nonlinear behavior [17,18]. When studying the frequency response of AFM cantilevers in liquid media contained in a commercial fluid cell, Motamedi and Wood-Adams [19] found that such systems could exhibit complicated dynamics. However, a rational connection of the tip-sample-interaction and the nonlinear dynamics analysis of the cantilever where the coupled effects of squeeze film damping and hydrodynamic loading are considered has not been presented and addressed satisfactorily. To order to make the TM-AFM achieve good performance, it is necessary to identify and so as to eliminate the possible chaotic motion of the cantilever of the AFM. In this paper, the cantilever is modeled as a single spring-mass-damper system and a nonlinear dynamic model is developed to study the cantilever-sample interaction by using the LJ potential including the long-range attractive forces and short-range repulsive forces. A comprehensive investigation of the nonlinear dynamics and chaos of the TM-AFM is carried out. The rest of the paper is organized as follows. 1. Introduction Section 2 describes the mathematic model of the cantilever vibrating in air considering the effects of hydrodynamic loading and squeeze film damping and the physical model of the cantilever-sample interaction is established in Section 3 using the LJ potential. Numerical results and discussions of the quality factor and resonant frequency of the frequency response, and nonlinear chaos and bifurcation of the dynamic TM-AFM are presented in Section 4. Finally, we end the paper with our conclusions in Section 5 . Sensors 2009, 9 3857 It has been found that the wavelength of vibration greatly exceeds the dominant length scale in the flow [20,21]. The nominal width B is the dominant length scale in the gas flow and the appropriate Reynolds number Re can be expressed as [21]: 2 4 e B R    (2) 2 4 e B R    (2) where  is a characteristic radial vibration frequency and Re is a normalized Reynolds number which indicates the importance of viscous forces relative to inertial forces in the gas fluid. It can be found that the gas could be considered to be non-viscous in the limit as Re → ∞, and the non-viscous gas model is applicable for practical cases where Re >> 1 [21]. However, if ω is close to the resonant frequency of the cantilever in vacuum, a reduction in dimensions of the beam will result in a reduction in Re. where  is a characteristic radial vibration frequency and Re is a normalized Reynolds number which indicates the importance of viscous forces relative to inertial forces in the gas fluid. It can be found that the gas could be considered to be non-viscous in the limit as Re → ∞, and the non-viscous gas model is applicable for practical cases where Re >> 1 [21]. However, if ω is close to the resonant frequency of the cantilever in vacuum, a reduction in dimensions of the beam will result in a reduction in Re. The surrounding viscous fluid medium plays an important role on the dynamics of the cantilever in AFM, Sader et al. [21,22] studied the effect of viscous fluid medium on the AFM cantilever and found that the shift in resonant frequency of the cantilever from vacuum to fluid (gas or liquid) was strongly dependent on both the density and viscosity of the fluid. 2.1. Hydrodynamic Loading Effect The gas flow around the cantilever is assumed to be incompressible and the Navier-Stokes equation is given by [20]:   2 p t        v v v v (1) (1) where , v , p and μ are the density, velocity, pressure and viscosity of the gas, respectively. where , v , p and μ are the density, velocity, pressure and viscosity of the gas, respectively. 2.2. Squeeze Film Damping Effect When the cantilever is driven close to the sample, the squeeze film between the cantilever and the surface of the sample causes significant damping except for the fluid dissipation at the edges and above the cantilever. For the damping problems encountered at micro-scale [24], the squeeze film damping can be expressed by the nonlinear Reynolds equation:   3 3 12 h h p h p t                                  (9) (9) Under the basic assumptions including the gas in the gap has been regarded as a continuum and the gas undergoes an isothermal process, the squeeze film damping can be modeled as: Under the basic assumptions including the gas in the gap has been regarded as a continuum and the gas undergoes an isothermal process, the squeeze film damping can be modeled as: 2 2 2 2 2 12 a eff a a a p h p p ξ p μ p p t h t p ξ ζ                                            (10) (10) where the pressure p is small compared to the ambient pressure pa ( 5 1.013 10 ap Pa   ), μeff is the effective gas viscosity, and h is the gap between the cantilever and the surface of the sample. At micro- scale, when the ratio between the mean free path of the air λ and the film thickness h, i.e., Knudsen number Kn = λ/h, is not small, the no-slip boundary condition at the interface may be inadequate. Considering the effect of slip flow, the effective viscosity μeff can be expressed as [25]: 1.159 1 9.638 eff n μ μ K  (11) (11) where μ is the viscosity coefficient at 1 atm. where μ is the viscosity coefficient at 1 atm. For the normal motion of parallel plates, h and μeff are not functions of the position. Equation (10) can be simplified as: For the normal motion of parallel plates, h and μeff are not functions of the position. Sensors 2009, 9 The quality factor for the bending modes can be written as [20]: The quality factor for the bending modes can be written as [20]: The quality factor for the bending modes can be written as [20]: 2 4 4 1 ( ) ( ) ( ) ( ) r gas r gas gas i gas i gas B Q                (5) (5) For small amplitude of the normal vibration of the cantilever, the local interaction stiffness in the vertical direction kn and damping coefficient Cn is given by [23]: For small amplitude of the normal vibration of the cantilever, the local interaction stiffness in the vertical direction kn and damping coefficient Cn is given by [23]: 0 cos 1 n n L n A k k A          (6) (6) Sensors 2009, 9 Sensors 2009, 9 3858 Sensors 2009, 9 For a rectangular cantilever beam [21], when the quality factor of the fundamental mode of the cantilever in gas exceeds 1, which is typically satisfied when the cantilever is placed in air, the relationship between the vacuum resonant frequency ωvac and the resonant frequency in gas ωgas can be written as [20]: 1/ 2 1/ 2 1 ( ) 1 ( ) 4 4 gas r gas r gas vac c B H                            (3) (3) where ρc and H are the density and thickness of the cantilever, respectively, and the natural scaling parameter is c B H    , which is defined as the ratio of the added mass of the gas to the mass of the cantilever. To study the effect of hydrodynamic loading on the dynamics of the cantilever, the hydrodynamic functions Γ(ω), which represents the real and imaginary pressure of the surrounding on the cantilever in two dimensions, is given by [20,21]: r i j  (4) r i j  (4) (4) where 2 1 r e a a R   and 1 2 i e e b b R R   , and a1 = 1.0553, a2 = 3.7997, b1 = 3.8018, and b2 = 2.7364 are selected from [20]. selected from [20]. selected from [20]. And: 0 sin L n n n k A C A    (7) (7) where An0 is the free amplitude in the normal direction, An is the measured amplitude, φ is the measured phase of the cantilever, ω is the drive frequency, and kL is the normal bending stiffness and 3 3 4 L EBH k L  , in which E, B, H and L are the elastic modulus, width, thickness and length of the cantilever, respectively. When the cantilever in AFM operates far below the resonance, the phase angle is close to zero, then kn and Cn become:  1 n L k k    , 0 n C  (8) (8) where 0 / n n A A  . Film Damping Effect 2.2. Squeeze Film Damping Effect Sensors 2009, 9 Sensors 2009, 9 3859 Then, the damping pressure can be obtained by direct integration with the boundary conditions, i.e.: Then, the damping pressure can be obtained by direct integration with the boundary conditions, i.e.: 2 2 3 6 ( , ) 2 eff B dh p t dt h                     (13) (13) The damping force on the cantilever is: The damping force on the cantilever is: The damping force on the cantilever is: The damping force on the cantilever is: The damping force on the cantilever is: 3 /2 /2 3 ( , ) eff B s B B L dh F p t Ld dt h         (14) (14) and the coefficient of damping force is given by: and the coefficient of damping force is given by: and the coefficient of damping force is given by: 3 3 eff s B L C h   (15) (15) 2.2. Squeeze Film Damping Effect Equation (10) can be simplified as: 3 ( ) 12 eff h p p ph p p μ ξ ξ ζ ζ t                               (12) (12) 3. The Physical Model The LJ force can be defined as the sum of the attractive and repulsive forces and is expressed as [9]:     0 1 2 8 2 0 0 0 ( , ) ( ) 180 6 LJ LJ U x z A R A R F x z z x z x         (17) (17) During the AFM operates in the TM, a low-dimensional model reduction can provide an accurate description of the cantilever dynamics. As shown in Figure 2, the cantilever is driven by the harmonic driving force, the tip-sample interaction force FLJ (LJ force) and the force due to squeeze film damping Fs. The governing equation of the motion of the cantilever can be determined by: During the AFM operates in the TM, a low-dimensional model reduction can provide an accurate description of the cantilever dynamics. As shown in Figure 2, the cantilever is driven by the harmonic driving force, the tip-sample interaction force FLJ (LJ force) and the force due to squeeze film damping Fs. The governing equation of the motion of the cantilever can be determined by:   3 0 0 0 ( , ) , , cos c LJ s mx cx kx k x F x z F x x z f ωt          (18) (18) where x is the instantaneous displacement of the cantilever tip measured from the equilibrium tip position in the absence of external forces with positive values toward the sample surface, and x and x are the instantaneous velocity and acceleration of the cantilever tip,. m, k and c are the equivalent mass, spring stiffness and damping coefficients of the cantilever in air, respectively, and L n k k k   and 1 / n c m Q C    , in which 1 / L k m  is the first-order mode frequency, and kc is the nonlinear cubic stiffness. f0 and ω are the amplitude and angular frequency of the harmonic driving force. 3. The Physical Model The AFM is composed of an elastic cantilever and the achievable sensitivity and resolution of AFM depend largely on the geometry of the cantilever [11,26]. Considering only the first vibration mode, the cantilever can be modeled as a simplified spring-mass-damping system, as shown in Figure 2. The tip is modeled as a sphere of radius R, and the cantilever-sample distance is characterized by z0, which is the distance between the equilibrium position of the cantilever and the sample when only the gravity acts on it. The cantilever position is given by x measured from the equilibrium position. Figure 2. Schematic of the lumped spring-mass-damping model for the TM-AFM cantilever vibrating near a sample surface. k ck c sc R 0z x m ck The LJ potential models the dispersive van der Waals forces as well as the short-range repulsive exchange interactions between two molecules [9]. The interaction between a cantilever tip and sample surface can be modeled as the interaction between a sphere and a flat surface. The tip-sample interaction is modeled by the LJ potential given by [9,11]:     1 2 0 7 0 0 ( , ) 6 1260 LJ A R A R U x z z x z x     (16) (16) Sensors 2009, 9 3860 where A1 and A2 are the Hamaker constants for the attractive and repulsive potentials, respectively. The Hamaker constants are defined as A1 = π2ρ1ρ2c1 and A2 = π2ρ1ρ2c2, in which ρ1 and ρ2 are the densities of the two interaction components, and c1 and c2 are the interaction constants, respectively. When the cantilever is driven close to the sample, there are three different kinds of forces including the spring force, the van deer Waals attractive force which is proportional to the inverse square power of the distance between the cantilever tip and the sample, and the repulsive force which is proportional to the inverse eighth power of the distance between the tip and the sample. 3. The Physical Model q s ( )( ) s [ ], 2 ( ) Introducing dimensionless variables: Introducing dimensionless variables: 1 τ ω t  ， s x y Z  ， 1 s x y Z     ， 4 27 d  ， 0 s z Z  ， 2 c s k Z k  ， 1 sc m    ， 3 3 1 eff s B L m Z     ， 0 0 s f F kZ  ， 1    ， 1/ 6 1 2 A A        ， s Z   ， 1 Q  (20) (20) Then, the LJ force FLJ and the squeeze film damping force Fs can be rewritten as:     6 2 8 30 LJ d d F y y        (21)     6 2 8 30 LJ d d F y y        (21) And: Sensors 2009, 9 3861   3 1 sF χ y α y       3 1 sF χ y α y     (22) (22) erefore, the dynamic equation of the system can be given by: Therefore, the dynamic equation of the system can be given by: Therefore, the dynamic equation of the system can be given by:       6 3 2 8 3 Σ 1 ΓcosΩ 30 d d y ζy y βy ε τ η y α y α y α y                      (23) (23) where, Γ = F0/ε, /   , /    , and ε is a small perturbation. where, Γ = F0/ε, /   , /    , and ε is a small perturbation. 3. The Physical Model The damping force   0 , , sF x x z  due to the squeeze film on the cantilever can be obtained from equation (14) and is given by:     3 0 3 0 , , eff s s μ B L F x x z c x x x z       (19) (19) In order to facilitate the investigation of the qualitative behaviors of the dynamic system, the equilibrium distance variable Zs is defined and 1/ 3 (3/ 2)(2 ) s Z D  [11], in which 2 /(6 ) D A R k  . In order to facilitate the investigation of the qualitative behaviors of the dynamic system, the equilibrium distance variable Zs is defined and 1/ 3 (3/ 2)(2 ) s Z D  [11], in which 2 /(6 ) D A R k  . 4. Results and Discussion This section aims at numerically investigating the characteristics and nonlinear dynamics of a TM- AFM cantilever-sample system driven by the harmonic excitation. The general properties of the cantilever and interaction properties with the respective sample are referred to [9], are listed in Table 1. The 4th order Runge-Kutta method is used to integrate the set of Equation (23). A small integration step (2π/200) has to be chosen to ensure a stable solution and to avoid the numerical divergence at the points where derivatives of FLJ and Fs are discontinuous. The effects of system parameters on the dynamic behavior of the cantilever vibrating system are investigated by using the bifurcation diagram, Poincaré maps, largest Lyapunov exponent, phase portraits, time histories and amplitude spectrum. Table 1. Parameters of the silicon tip tapping and silicon sample used in the numerical simulations. Description Value Length 449 μm Width 46 μm Thickness 1.7 μm Tip radius 150 nm Material density 2,330 kg/m3 Young’s modulus 176 GPa Bending stiffness 0.11 N·m-1 First-order resonant frequency 11.804 kHz Quality factor 100 Hamaker constant (Repulsive) 1.3596 × 10-70 J·m6 Hamaker constant (Attractive) 1.865 × 10-19 J 4.1. Effect of External Forcing Term Γ 4.1. Effect of External Forcing Term Γ The external forcing term is one of the most important parameters affecting the dynamic characteristics of the TM-AFM tip-sample system. Figure 3 and Figure 4 show the bifurcation diagram and largest Lyapunov exponent map of the dynamic system where the amplitude of the external excitation is the control parameter and the small perturbation ε = 0.1 is added. 3862 Sensors 2009, 9 Figure 3. Bifurcation diagram of the amplitude of the external forcing term Γ. Figure 3. Bifurcation diagram of the amplitude of the external forcing term Γ. Figure 4. Largest Lyapunov exponent map of the amplitude of the external forcing term Γ. Figure 4. Largest Lyapunov exponent map of the amplitude of the external forcing term Γ. Figure 4. Largest Lyapunov exponent map of the amplitude of the external forcing term Γ. It can be seen from Figure 3 that the system responses contain periodic and chaotic motions alternately at the interval of 0 < Γ< 80. When Γ = 6.8, the vibration amplitude of the cantilever is small, the motion is synchronous with period-eight (P-8), and eight points are correspondingly displayed in the Poincaré map, as shown in Figure 5(a). With the increase of the amplitude of the forcing term , the motion becomes synchronous with period-two (P-2) at Γ = 33.0, as illustrated in Figure 5(b). Moreover, the period-1 motion with only one isolated point in Poincaré map and one circle in phase portrait can be observed at Γ = 65.0 in Figure 5(c), and the corresponding largest Lyapunov exponent becomes negative, according to Figure 4. At Γ = 75.2, the chaos is shown in Figure 5(d), the strange attractor has a fractal structure in Poincaré map, and it can be found in Figure 4 that the corresponding largest Lyapunov exponent is positive. Therefore, as the amplitude of the It can be seen from Figure 3 that the system responses contain periodic and chaotic motions alternately at the interval of 0 < Γ< 80. When Γ = 6.8, the vibration amplitude of the cantilever is small, the motion is synchronous with period-eight (P-8), and eight points are correspondingly displayed in the Poincaré map, as shown in Figure 5(a). With the increase of the amplitude of the forcing term , the motion becomes synchronous with period-two (P-2) at Γ = 33.0, as illustrated in Figure 5(b). 4.1. Effect of External Forcing Term Γ Moreover, the period-1 motion with only one isolated point in Poincaré map and one circle in phase portrait can be observed at Γ = 65.0 in Figure 5(c), and the corresponding largest Lyapunov exponent becomes negative, according to Figure 4. At Γ = 75.2, the chaos is shown in Figure 5(d), the strange attractor has a fractal structure in Poincaré map, and it can be found in Figure 4 that the corresponding largest Lyapunov exponent is positive. Therefore, as the amplitude of the It can be seen from Figure 3 that the system responses contain periodic and chaotic motions alternately at the interval of 0 < Γ< 80. When Γ = 6.8, the vibration amplitude of the cantilever is small, the motion is synchronous with period-eight (P-8), and eight points are correspondingly displayed in the Poincaré map, as shown in Figure 5(a). With the increase of the amplitude of the forcing term , the motion becomes synchronous with period-two (P-2) at Γ = 33.0, as illustrated in Figure 5(b). Moreover, the period-1 motion with only one isolated point in Poincaré map and one circle in phase portrait can be observed at Γ = 65.0 in Figure 5(c), and the corresponding largest Lyapunov exponent becomes negative, according to Figure 4. At Γ = 75.2, the chaos is shown in Figure 5(d), the strange attractor has a fractal structure in Poincaré map, and it can be found in Figure 4 that the corresponding largest Lyapunov exponent is positive. Therefore, as the amplitude of the 3863 Sensors 2009, 9 Sensors 2009, 9 forcing term increases, the changes of the system responses are very complex, with alternative periodic and chaotic motions. Figure 5. The Poincaré maps, phase portraits, amplitude spectrums and time histories of different Γ. (a) 6.8  (b) 33.0  (c) 65.0  (d) 75.2  4.2. Effect of Squeeze Film Damping η (d) 75.2  (a) 6.8  (b) 33.0  (c) 65.0  4.2. Effect of Squeeze Film Damping η At the micro-scale, the squeeze film damping coefficient and ratio are the key parameters for the dynamic responses of the micro-devices. The larger the squeeze film damping is, the higher the noise level results. The ratio of the fundamental resonant frequency in gas to that in vacuum ωgas/ωvac is numerically calculated from Equation (3). 4.1. Effect of External Forcing Term Γ Figure 6 gives the ratio of resonant frequencies in vacuum and gas ωgas/ωvac as a function of the Reynolds number Re at different natural scaling parameter . It could be found that ωgas/ωvac is increasing with the increase of Reynolds number Re and ωgas/ωvac decreases with the increase of the scaling parameter П. Figure 7 gives the quality factor Qgas as a function of both natural scaling parameters, Re and П, which can be obtained directly from Equation (5). It is indicated that the quality factor Qgas increases with the increase of the Reynolds number Re and the decrease of the natural scaling parameter П. However, when the natural scaling parameter П Sensors 2009, 9 3864 Sensors 2009, 9 tends to be a larger value, i. e., П = 10, the quality factor Qgas has small change. For example, when П changes from 10 to 100, the quality factor Qgas has very small change, as shown in Figure 7. Figure 6. The relationship between the vacuum resonant frequency ωvac and the resonant frequency in gas ωgas (ωgas/ωvac) as a function of the Reynolds number Re at different natural scaling parameter П. Figure 6. The relationship between the vacuum resonant frequency ωvac and the resonant frequency in gas ωgas (ωgas/ωvac) as a function of the Reynolds number Re at different natural scaling parameter П. Figure 7. The quality factor Qgas as a function of the Reynolds number Re for the fundamental mode at different natural scaling parameter П. Figure 7. The quality factor Qgas as a function of the Reynolds number Re for the fundamental mode at different natural scaling parameter П. Microstructures undergoing motion transverse to a fixed plate exhibit damping effects that should be considered in the dynamics simulation. The heat transfer analogy is applied to obtain the damping and stiffness coefficients of the microstructures with PLANE 55 thermal elements in ANSYS 11.0 [28]. The effective damping and stiffness coefficients are determined by the finite element thermal analogy approach and theoretic analyses at different operation frequencies and the results are listed in Table 2. It can be seen that the damping coefficient decreases tardily with the increase of the operation frequency and the stiffness coefficient increases fleetly at the same time with slip and without slip. Microstructures undergoing motion transverse to a fixed plate exhibit damping effects that should be considered in the dynamics simulation. 4.1. Effect of External Forcing Term Γ The heat transfer analogy is applied to obtain the damping and stiffness coefficients of the microstructures with PLANE 55 thermal elements in ANSYS 11.0 [28]. The effective damping and stiffness coefficients are determined by the finite element thermal analogy approach and theoretic analyses at different operation frequencies and the results are listed in Table 2. It can be seen that the damping coefficient decreases tardily with the increase of the operation frequency and the stiffness coefficient increases fleetly at the same time with slip and without slip. Sensors 2009, 9 3865 Sensors 2009, 9 3866 Figure 9 displays the bifurcation diagrams of squeeze film damping ratio η in the range of 0.05 < η < 0.1 for the coupling nonlinear dynamic system, from which it can be that the system response changes between periodic and chaotic motions alternately. Figure 10 shows the Poincaré maps and phase plane portraits of different squeeze film damping ratio η on the responses of the coupling system. The system response starts synchronous motion with period-4 at η = 0.0525 (shown in Figure 10a), and then becomes synchronous motion with period-1 at η = 0.0635(shown in Figure 10b), and then leaves synchronous motion with period-1 and enters chaotic motion at η = 0.07, which can be seen in Figure 10c. The strange attractor has a fractal structure and the corresponding largest Lyapunov exponent is positive. As indicated in Figure 10d, with the increase of squeeze film damping ratio, the system response becomes synchronous motion with period-1 from chaotic motion. Therefore, the effect of squeeze film damping on the system response cannot be neglected for structures at the micro-scale. Figure 10. The Poincaré maps and phase portraits of different squeeze film damping ratios η. Figure 10. The Poincaré maps and phase portraits of different squeeze film damping ratios η. (a) η = 0.0525 (b) η = 0.0635 (c) η = 0.07 (d) η = 0.0835 4.3. Effect of material property parameter Σ At micro-scale, the material properties of the AFM tip and sample play an important role on the surface force between them and, as a result, the dynamic response of the tip-sample system displays very rich nonlinear characteristics. Figure 11 is the bifurcation diagram of the material property parameter Σ for the coupling nonlinear dynamic system with different cubic stiffness ratios and squeeze film damping ratios. The system parameters are taken as follows: equilibrium parameter α = 1.2, excitation frequency ratio Ω = 1, integration step numbers N = 200 and the bifurcation step ΔΣ 0.005  . Fi 11 h h bif i di f h i l i h f (a) η = 0.0525 (b) η = 0.0635 (c) η = 0.07 (d) η = 0.0835 4.3. Effect of material property parameter Σ (c) η = 0.07 (d) η = 0.0835 (a) η = 0.0525 (b) η = 0.0635 (c) η = 0.07 (a) η = 0.0525 (b) η = 0.0635 4.3. Sensors 2009, 9 Meanwhile, the damping and stiffness coefficients with slip effect are smaller than those without slip. Figure 8 shows the pressure distribution of the damping component with slip at the resonant frequency. The pressure distribution is approximately parabola in the directions of length and width, and the peak appears at the center of the film. Meanwhile, the damping and stiffness coefficients with slip effect are smaller than those without slip. Figure 8 shows the pressure distribution of the damping component with slip at the resonant frequency. The pressure distribution is approximately parabola in the directions of length and width, and the peak appears at the center of the film. Table 2. Effective damping and stiffness coefficients of the squeeze film at different frequencies. Table 2. Effective damping and stiffness coefficients of the squeeze film at different frequencies. Frequency f (Hz) Damping coefficient Stiffness coefficient PLANE55 Analytic(slip ) [27] PLANE55 Analytic(slip ) [27] No slip Slip No slip Slip 1 1.5199e – 4 1.3499e – 4 1.2529e – 4 9.2051e – 10 7.2610e – 10 6.2304e – 10 1 000 1.5199e – 4 1.3499e – 4 1.2529e – 4 9.2051e – 4 7.2610e – 4 6.2304e – 4 11 804 1.5197e – 4 1.3497e – 4 1.2528e – 4 0.1282 0.1012 0.0868 50 000 1.5163e – 4 1.3473e – 4 1.2509e – 4 2.296 1.8118 1.555 100 000 1.5056e – 4 1.3398e – 4 1.2449e – 4 9.117 7.206 6.190 Figure 8. Pressure distributions of the cantilever at the resonant frequency. Figure 8. Pressure distributions of the cantilever at the resonant frequency. Figure 9. Bifurcation diagram of the squeeze film damping ratio . Figure 9. Bifurcation diagram of the squeeze film damping ratio . Figure 9. Bifurcation diagram of the squeeze film damping ratio . Sensors 2009, 9 Effect of material property parameter Σ At micro-scale, the material properties of the AFM tip and sample play an important role on the surface force between them and, as a result, the dynamic response of the tip-sample system displays very rich nonlinear characteristics. Figure 11 is the bifurcation diagram of the material property parameter Σ for the coupling nonlinear dynamic system with different cubic stiffness ratios and squeeze film damping ratios. The system parameters are taken as follows: equilibrium parameter α = 1.2, excitation frequency ratio Ω = 1, integration step numbers N = 200 and the bifurcation step ΔΣ 0.005  . Figure 11a shows the bifurcation diagram of the material property parameter in the range of 0.3 < Σ < 0.42 with the cubic stiffness ratio β = 0.3. The response of the coupled nonlinear system undergoes a complete process from chaotic motion through period-2, period-1, period-4 and period-8 motions to steady-state motion with period-1 by the forms of period-doubling and anti period-doubling Sensors 2009, 9 Sensors 2009, 9 3867 bifurcations. At the interval of 0.42 < Σ < 0.47, the system response alters between chaotic motion with long time and periodic motion with short time. When Σ increases to Σ > 0.47, the system response becomes synchronous motion with period-1. With the change of the cubic stiffness ratio of the cantilever tip from β = 0.3 to β = 0.5, the response of the coupled system undergoes the process of chaotic and periodic motions alternatively. It comes into period-2 motion from chaotic motion with anti period-doubling bifurcation, and then enters period-1 motion in a large range of material property parameter (Σ > 0.39), as illustrated in Figure 11b. In addition, it is found that the chaotic motion disappears at higher Σ with the increase of cubic stiffness ratio (from β = 0.3 to β = 0.5). Therefore, with the changes of the measured samples in experimental tests, the values of Σ vary accordingly, and as a result the response of the coupled system displays various nonlinear dynamic behaviors. Figure 11. Bifurcation diagram of th 1 cm e material property parameter Σ at different cubic stiffness ratios: (a) β = 0.3; (b) β = 0.5. Sensors 2009, 9 Figure 12 is the bifurcation diagram of the cubic stiffness ratio β on the response of TM-AFM tip- sample system at the interval of 0.3 < β < 0.6 for various combinations of squeeze film damping ratios, material and equilibrium parameters, and the bifurcation step Δ 0.01 β  . It can be observed from Figure 12(a) that the response of the coupled system has a complete process from chaotic motion through periodic motion and chaotic motion to period-1 motion. At the interval of 0.3 < β < 0.43, the system response enters periodic motion from chaotic motion, then it becomes chaotic motion again, and finally it comes into steady-state motion with period-1 in the range of 0.43 < β < 0.6. With the increase of squeeze film damping η (η = 0.14), the system response changes noticeably and it mainly contains the periodic components, such as period-1, period-3 and period-6 motions, as illustrated in Figure 12(c). As the equilibrium parameter α increases, the chaotic components of the system response decrease, while the periodic components increase and contain period-2, period-4 and period-8 motions with the case of α = 1.6, as shown in Figure 12(d). Figure 12 is the bifurcation diagram of the cubic stiffness ratio β on the response of TM-AFM tip- sample system at the interval of 0.3 < β < 0.6 for various combinations of squeeze film damping ratios, material and equilibrium parameters, and the bifurcation step Δ 0.01 β  . It can be observed from Figure 12(a) that the response of the coupled system has a complete process from chaotic motion through periodic motion and chaotic motion to period-1 motion. At the interval of 0.3 < β < 0.43, the system response enters periodic motion from chaotic motion, then it becomes chaotic motion again, and finally it comes into steady-state motion with period-1 in the range of 0.43 < β < 0.6. With the increase of squeeze film damping η (η = 0.14), the system response changes noticeably and it mainly contains the periodic components, such as period-1, period-3 and period-6 motions, as illustrated in Figure 12(c). As the equilibrium parameter α increases, the chaotic components of the system response decrease, while the periodic components increase and contain period-2, period-4 and period-8 motions with the case of α = 1.6, as shown in Figure 12(d). Sensors 2009, 9 Figure 12 is the bifurcation diagram of the cubic stiffness ratio β on the response of TM-AFM tip- sample system at the interval of 0.3 < β < 0.6 for various combinations of squeeze film damping ratios, material and equilibrium parameters, and the bifurcation step Δ 0.01 β  . It can be observed from Figure 12(a) that the response of the coupled system has a complete process from chaotic motion through periodic motion and chaotic motion to period-1 motion. At the interval of 0.3 < β < 0.43, the system response enters periodic motion from chaotic motion, then it becomes chaotic motion again, and finally it comes into steady-state motion with period-1 in the range of 0.43 < β < 0.6. With the increase of squeeze film damping η (η = 0.14), the system response changes noticeably and it mainly contains the periodic components, such as period-1, period-3 and period-6 motions, as illustrated in Figure 12(c). As the equilibrium parameter α increases, the chaotic components of the system response decrease, while the periodic components increase and contain period-2, period-4 and period-8 motions with the case of α = 1.6, as shown in Figure 12(d). The Poincaré maps and phase portraits for different cubic stiffness ratios are displayed in Figure 13. When the cubic stiffness ratio is small, i.e., β = 0.35, the exhibited motion is period-6 motion, six points and circles can be seen in the Poincaré map and phase portrait respectively from Figure 13(a). As the cubic stiffness ratio becomes larger, the closed circle is decomposed and the points in the Poincaré map gradually scatter. At β = 0.42, the system response comes into chaotic motion, and then the points of the attractor are decomposed again and finally converge to one point. When β = 0.48, a synchronous motion with period-1 can be observed. Then the system response comes into period-4 motion. It is indicated that the components of chaotic motions become wider with the increase of the Sensors 2009, 9 Sensors 2009, 9 3868 squeeze film damping. Material properties of the tip and sample and equilibrium coefficient ratio play very important role in the nonlinear dynamics of the coupled system. Figure 12. Sensors 2009, 9 Bifurcation diagram of the cubic stiffness ratio β at different combinations of squeeze film damping ratios, material parameters and equilibrium parameters: (a) 0.08  , 0.3  , 1.2  ; (b) 0.08  , 0.5  , 1.2  ; (c) 0.14  , 0.5  , 1.2  ; (d) 0.14  , 0.5  , 1.6  . Figure 12. Bifurcation diagram of the cubic stiffness ratio β at different combinations of squeeze film damping ratios, material parameters and equilibrium parameters: (a) 0.08  , 0.3  , 1.2  ; (b) 0.08  , 0.5  , 1.2  ; (c) 0.14  , 0.5  , 1.2  ; (d) 0.14  , 0.5  , 1.6  . Figure 13. The Poincaré maps and phase portraits of different cubic stiffness ratios β. Figure 13. The Poincaré maps and phase portraits of different cubic stiffness ratios β (a) β = 0.35 (b) β = 0.42 (c) β = 0.48 (d) β = 0.55 (c) β = 0.48 (d) β = 0.55 Sensors 2009, 9 Sensors 2009, 9 3869 Sensors 2009, 9 3870 To explain the dynamic responses of the system clearly, Figure 16 shows the local bifurcation diagram and Poincaré maps of the dynamic system at the interval of 1.6 < α < 2. It can be found that the system responses exhibit the alternation of periodic and chaotic motions. The system response comes into steady-state synchronous motion with period-1 from chaotic motion, and enters period-2 motion from period-1 motion as the equilibrium parameter α increases, and then becomes chaotic motion with period-doubling bifurcation. Moreover, at 1.7 < α < 1.9, the system response changes between period-1 and period-2 motions alternately. When α > 1.9, the system response comes into steady-state synchronous motion with period-1. These phenomena indicate that the dynamic responses of the coupled system are very complex. The cantilever tip can undergo a period-doubling cascade to possible chaos about the original equilibrium. It is demonstrated that, away from the surface, the net force on the tip is always in the downward direction and causes the tip to accelerate the sample until it passes the key point, where the repulsive force plus the spring force becomes larger than the van der Waals force, and then the tip is forced away from the sample. Figure 16. Local bifurcation and Poincaré maps of equilibrium parameter . Figure 16. Local bifurcation and Poincaré maps of equilibrium parameter . Figure 17 shows the bifurcation diagram of the equilibrium parameter  on the response of TM- AFM tip-sample system at the interval of 1.0 1.8    , the squeeze film damping ratio is taken as η = 0.008 and the bifurcation step is Δ 0.01 α  . It can be seen from Figure 17(a) that the response of the coupled dynamic system comes into period-2 motion from period-1 motion and then enters chaotic motion with period-doubling bifurcation. With the increase of , the system responses enters period-2 motion from chaotic motion, and it subsequently becomes period-1 motion again when 0.3  and Γ = 2. As illustrated in Figure 17(b), the response of the coupled system has a complete process from chaotic motion through periodic motion to chaotic motion with the forms of period-doubling bifurcation and anti period-doubling bifurcations at the interval of 1 1.8    when 0.3  and Γ = 4. Sensors 2009, 9 With the increases of the material parameter coefficients, as shown in Figure 17(c) and (d), the system response changes noticeably and it mainly contains the periodic components, such as period-1, period- 2, period-3 and period-6 motions. Meanwhile, the chaotic components of the system response decrease and, on the contrary, the periodic components increase. In addition, the chaotic components of the Figure 17 shows the bifurcation diagram of the equilibrium parameter  on the response of TM- AFM tip-sample system at the interval of 1.0 1.8    , the squeeze film damping ratio is taken as η = 0.008 and the bifurcation step is Δ 0.01 α  . It can be seen from Figure 17(a) that the response of the coupled dynamic system comes into period-2 motion from period-1 motion and then enters chaotic motion with period-doubling bifurcation. With the increase of , the system responses enters period-2 motion from chaotic motion, and it subsequently becomes period-1 motion again when 0.3  and Γ = 2. As illustrated in Figure 17(b), the response of the coupled system has a complete process from chaotic motion through periodic motion to chaotic motion with the forms of period-doubling bifurcation and anti period-doubling bifurcations at the interval of 1 1.8    when 0.3  and Γ = 4. With the increases of the material parameter coefficients, as shown in Figure 17(c) and (d), the system response changes noticeably and it mainly contains the periodic components, such as period-1, period- 2, period-3 and period-6 motions. Meanwhile, the chaotic components of the system response decrease and, on the contrary, the periodic components increase. In addition, the chaotic components of the Figure 17 shows the bifurcation diagram of the equilibrium parameter  on the response of TM- AFM tip-sample system at the interval of 1.0 1.8    , the squeeze film damping ratio is taken as η = 0.008 and the bifurcation step is Δ 0.01 α  . It can be seen from Figure 17(a) that the response of the coupled dynamic system comes into period-2 motion from period-1 motion and then enters chaotic motion with period-doubling bifurcation. With the increase of , the system responses enters period-2 motion from chaotic motion, and it subsequently becomes period-1 motion again when 0.3  and Γ = 2. 4.4. Effect of Equilibrium Parameter α 4.4. Effect of Equilibrium Parameter α Equilibrium coefficient ratio α is one of the important parameters for determining the equilibrium position of the cantilever tip in AFM and it becomes the key factor to reflect the dynamic responses of the tip-sample model. The dynamic behavior of the coupled system depends on the value of α. Figures 14 and 15 give the bifurcation diagram and largest Lyapunov exponent map of the dynamic system with the control parameter of the equilibrium parameter α at Ω = 1. It can be seen from Figure 14 that the dynamic responses are very complicated, and the components contain periodic and chaotic motions at the interval of 1< α < 2. The corresponding largest Lyapunov exponents are alternately positive and negative, as shown in Figure 15. Figure 14. Bifurcation diagram of the equilibrium parameter α. Figure 14. Bifurcation diagram of the equilibrium parameter α. Figure 15. Largest Lyapunov exponent map of the equilibrium parameter α. Figure 14. Bifurcation diagram of the equilibrium parameter α. Figure 15. Largest Lyapunov exponent map of the equilibrium parameter α. Figure 15. Largest Lyapunov exponent map of the equilibrium paramet Figure 15. Largest Lyapunov exponent map of the equilibrium parameter α. Figure 15. Largest Lyapunov exponent map of the equilibrium parameter α. Sensors 2009, 9 As illustrated in Figure 17(b), the response of the coupled system has a complete process from chaotic motion through periodic motion to chaotic motion with the forms of period-doubling bifurcation and anti period-doubling bifurcations at the interval of 1 1.8    when 0.3  and Γ = 4. With the increases of the material parameter coefficients, as shown in Figure 17(c) and (d), the system response changes noticeably and it mainly contains the periodic components, such as period-1, period- 2, period-3 and period-6 motions. Meanwhile, the chaotic components of the system response decrease and, on the contrary, the periodic components increase. In addition, the chaotic components of the Sensors 2009, 9 Sensors 2009, 9 3871 system response shift to the smaller equilibrium parameter. It demonstrates that the components of chaotic motions become wider as the amplitude of external forcing term increases, and the material properties of the tip and sample and equilibrium coefficient ratio affect the nonlinear dynamics of the coupled system. Figure 17. Bifurcation diagram of the equilibrium parameter α at different combined material parameters and external forcing terms: (a) 0.3  , 2  ; (b) 0.3  , 4  ; (c) 0.5  , 2  ; (d) 0.5  , 4  . Figure 17. Bifurcation diagram of the equilibrium parameter α at different combined material parameters and external forcing terms: (a) 0.3  , 2  ; (b) 0.3  , 4  ; (c) 0.5  , 2  ; (d) 0.5  , 4  . (c) 0.5  , 2  ; (d) 0.5  , 4  . To illustrate the various motions, Figure 18 shows the nonlinear characteristics of the coupled ystem with the plots of the Poincaré maps and phase portraits for different equilibrium parameters  at different conditions. The motion of the coupled system changes between periodic and chaotic motions alternately. At α = 1.2, the motion with period-1 represented by a point in the Poincaré maps and characterized by a close curve in phase portraits is shown in Figure 18(a). As illustrated in Figure 18(b), the system response comes into period-6 motion at α = 1.42 from synchronous motion with period-1 at α = 1.2, as displayed in Figure 18 (a), then leaves period-6 motion and enters chaotic motion at α = 1.6, which can be seen from Figure 18(c). Sensors 2009, 9 The strange attractor has a fractal structure and the corresponding largest Lyapunov exponent is positive. With the increase of the equilibrium parameter coefficient, as shown in Figure 18(d), the system response becomes periodic motion from chaotic motion again, and one can find the period-3 motion marked by three isolated points in Poincaré map and three circles in phase portrait at 1.74  . It is indicated that the components of chaotic To illustrate the various motions, Figure 18 shows the nonlinear characteristics of the coupled system with the plots of the Poincaré maps and phase portraits for different equilibrium parameters  To illustrate the various motions, Figure 18 shows the nonlinear characteristics of the coupled system with the plots of the Poincaré maps and phase portraits for different equilibrium parameters  at different conditions. The motion of the coupled system changes between periodic and chaotic motions alternately. At α = 1.2, the motion with period-1 represented by a point in the Poincaré maps and characterized by a close curve in phase portraits is shown in Figure 18(a). As illustrated in Figure 18(b), the system response comes into period-6 motion at α = 1.42 from synchronous motion with period-1 at α = 1.2, as displayed in Figure 18 (a), then leaves period-6 motion and enters chaotic motion at α = 1.6, which can be seen from Figure 18(c). The strange attractor has a fractal structure and the corresponding largest Lyapunov exponent is positive. With the increase of the equilibrium parameter coefficient, as shown in Figure 18(d), the system response becomes periodic motion from chaotic motion again, and one can find the period-3 motion marked by three isolated points in Poincaré map and three circles in phase portrait at 1.74  . It is indicated that the components of chaotic To illustrate the various motions, Figure 18 shows the nonlinear characteristics of the coupled system with the plots of the Poincaré maps and phase portraits for different equilibrium parameters  at different conditions. The motion of the coupled system changes between periodic and chaotic motions alternately. At α = 1.2, the motion with period-1 represented by a point in the Poincaré maps and characterized by a close curve in phase portraits is shown in Figure 18(a). Sensors 2009, 9 As illustrated in Figure 18(b), the system response comes into period-6 motion at α = 1.42 from synchronous motion with period-1 at α = 1.2, as displayed in Figure 18 (a), then leaves period-6 motion and enters chaotic motion at α = 1.6, which can be seen from Figure 18(c). The strange attractor has a fractal structure and the corresponding largest Lyapunov exponent is positive. With the increase of the equilibrium parameter coefficient, as shown in Figure 18(d), the system response becomes periodic motion from chaotic motion again, and one can find the period-3 motion marked by three isolated points in Poincaré map and three circles in phase portrait at 1.74  . It is indicated that the components of chaotic Sensors 2009, 9 Sensors 2009, 9 3872 motions of the coupled system increases obviously with the increase of the amplitude of the force term. In general, the effect of equilibrium parameter on the system response should be considered for the design of the TM-AFM. Figure 18. The Poincaré maps and phase portraits of different equilibrium parameters . (a) 1.2  (b) 1.42  (c) 1.6  (d) 1.74  5. Conclusions The cantilever in tapping mode Atomic force microscope (TM-AFM) is one of crucial components and so it is very important to carry out a thorough dynamic analysis for the cantilever to further enhance the performance of the TM-AFM. In this paper, the cantilever-sample interaction and a harmonically forcing term in the TM-AFM have been considered. Numerical simulations have been used to investigate the nonlinear behaviors between the tip and the sample. The chaotic behavior appears to be generated via various system parameters including the amplitude of the external forcing term, equilibrium parameter, squeeze film damping and material property. The dynamic system responses display very rich nonlinear dynamic characteristics under the effects of these parameters and show an alternate changing process among periodic motion, quasi-periodic motion and chaotic motion. The component of the chaotic motion in the system response increases with the increase of the amplitude of the external excitation, but will be weaken by the squeeze film damping. In addition, the increase of the equilibrium parameter coefficient would strengthen the component of the chaotic motion but weaken the component of the periodic motion increases in the system response. It is indicated that the external excitation and squeeze film damping are important for the design of dynamic TM-AFM. 5. Conclusions The cantilever in tapping mode Atomic force microscope (TM-AFM) is one of crucial components and so it is very important to carry out a thorough dynamic analysis for the cantilever to further enhance the performance of the TM-AFM. In this paper, the cantilever-sample interaction and a harmonically forcing term in the TM-AFM have been considered. Numerical simulations have been used to investigate the nonlinear behaviors between the tip and the sample. The chaotic behavior appears to be generated via various system parameters including the amplitude of the external forcing term, equilibrium parameter, squeeze film damping and material property. The dynamic system responses display very rich nonlinear dynamic characteristics under the effects of these parameters and show an alternate changing process among periodic motion, quasi-periodic motion and chaotic motion. The component of the chaotic motion in the system response increases with the increase of the amplitude of the external excitation, but will be weaken by the squeeze film damping. In addition, the i f h ilib i ffi i ld h h f h h i The cantilever in tapping mode Atomic force microscope (TM-AFM) is one of crucial components and so it is very important to carry out a thorough dynamic analysis for the cantilever to further enhance the performance of the TM-AFM. In this paper, the cantilever-sample interaction and a harmonically forcing term in the TM-AFM have been considered. Numerical simulations have been used to investigate the nonlinear behaviors between the tip and the sample. The chaotic behavior appears to be generated via various system parameters including the amplitude of the external forcing term, equilibrium parameter, squeeze film damping and material property. The dynamic system responses display very rich nonlinear dynamic characteristics under the effects of these parameters and show an alternate changing process among periodic motion, quasi-periodic motion and chaotic motion. The component of the chaotic motion in the system response increases with the increase of the amplitude of the external excitation, but will be weaken by the squeeze film damping. In addition, the increase of the equilibrium parameter coefficient would strengthen the component of the chaotic motion but weaken the component of the periodic motion increases in the system response. It is indicated that the external excitation and squeeze film damping are important for the design of dynamic TM-AFM. 5. Conclusions It is of significance to understand the cantilever dynamics in air/liquid and promote the development of the next generation of AFMs. Sensors 2009, 9 It is of significance to understand the cantilever dynamics in air/liquid and promote the development of the next generation of AFMs. (a) 1.2  (b) 1.42  (a) 1.2  (b) 1.42  (c) 1.6  (d) 1.74  References 1. Finot, E.; Passian, A.; Thundat, T. Measurement of mechanical properties of cantilever shaped materials. Sensors 2008, 8, 3497-3541. 1. Finot, E.; Passian, A.; Thundat, T. Measurement of mechanical properties of cantilever shaped materials. Sensors 2008, 8, 3497-3541. 2. Jalili, N.; Laxminarayana, K. A review of atomic force microscopy imaging systems: application to molecular metrology and biological sciences. Mechatronics 2004, 14, 907-945. 2. Jalili, N.; Laxminarayana, K. A review of atomic force microscopy imaging systems: application to molecular metrology and biological sciences. Mechatronics 2004, 14, 907-945. 3. Hersam, M.C. Monitoring and analyzing nonlinear dynamics in atomic force microscopy. Small 2006, 2, 1122-1124. 3. Hersam, M.C. Monitoring and analyzing nonlinear dynamics in atomic force microscopy. Small 2006, 2, 1122-1124. 4. Wei, Z; Zhao, Y.P. Growth of liquid bridge in AFM. J. Phys. D: Appl. Phys. 2008, 40, 4368-4375. 4. Wei, Z; Zhao, Y.P. Growth of liquid bridge in AFM. J. Phys. D: Appl. Phys. 2008, 40, 4368-4375. 4. Wei, Z; Zhao, Y.P. Growth of liquid bridge in AFM. J. Phys. D: Appl. Phys. 2008, 40, 4368-4375. 5. Yacoot, A.; Koenders, L. Aspects of scanning force microscope probes and their effects on di i l t J Ph D A l Ph 2008 41 103001 4. Wei, Z; Zhao, Y.P. Growth of liquid bridge in AFM. J. Phys. D: Appl. Phys. 2008, 40, 4368 4375. 5. Yacoot, A.; Koenders, L. Aspects of scanning force microscope probes and their effects on dimensional measurement. J. Phys. D: Appl. Phys. 2008, 41, 103001. 5. Yacoot, A.; Koenders, L. Aspects of scanning force microscope probes and their effects on dimensional measurement. J. Phys. D: Appl. Phys. 2008, 41, 103001. 6. Lee, S.I.; Howell, S.W.; Raman, A.; Reifenberger, R. Nonlinear dynamics of microcantilevers in tapping mode atomic force microscopy: a comparison between theory and experiment. Phys. Rev. B 2002, 66, 115409. 6. Lee, S.I.; Howell, S.W.; Raman, A.; Reifenberger, R. Nonlinear dynamics of microcantilevers in tapping mode atomic force microscopy: a comparison between theory and experiment. Phys. Rev. B 2002, 66, 115409. 7. Zhao, X.; Dankowicz, H. Characterization of intermittent contact in tapping-mode atomic force microscopy. ASME J. Comput. Nonlin. Dyn. 2006, 1, 109-115. 8. Yagasaki, K. Nonlinear dynamics of vibrating microcantilevers in tapping-mode atomic force microscopy. Phys. Rev. B 2004, 70, 245419. 9. Rutzel, S.; Lee, S.I.; Raman, A. Nonlinear dynamics of atomic-force-microscope probes driven in Lennard-Jones potentials. Proc. R. Soc. Lond. A 2003, 459, 1925-1948. Sensors 2009, 9 3873 Outstanding Youth Foundation of China under Grant No.10325209, and the Specialized Research Fund for State Key Laboratory of Mechanical System and Vibration. Outstanding Youth Foundation of China under Grant No.10325209, and the Specialized Research Fund for State Key Laboratory of Mechanical System and Vibration. Acknowledgments The authors would like to thank Prof. Z. K. Peng for his good suggestions. This work was supported by the National Natural Science Foundation of China under Grant No.10602033, the National References 10. Hashemi, N.; Dankowicz, H.; Paul, M.R. The nonlinear dynamics of tapping mode atomic force microscopy with capillary force interactions. J. Appl. Phys. 2008, 103, 093512. 11. Ashhab, M.; Salapaka, M.V.; Dahleh, M.; Mezic, I. Melnikov-based dynamical analysis of microcantilevers in scanning probe microscopy. Nonlin.Dyn. 1999, 20, 197-220. 12. Basso, M.; Giarre, L.; Dahleh, M.; Mezic, I. Complex dynamics in a harmonically excited Lennard-Jones oscillator: microcantilever–sample interaction in scanning probe microscopes. J. Dyn. Syst. Meas. Control 2000, 122, 240-245. 13. Paulo, A.S.; Garcia, R. Tip-surface forces, amplitude, and energy dissipation in amplitude- modulation (tapping mode) force microscopy. Phys. Rev. B 2001, 64, 193411. 14. Behrend, O.P.; Odoni, L.; Loubet, J.L.; Burnham, N.A. Phase imaging: Deep or superficial? Appl. Phys. Lett. 1999, 75, 2551. 15. Zitzler, L.; Herminghaus, S.; Mugele, F. Capillary forces in tapping mode atomic force microscopy. Phys. Rev. B 2002, 66, 155436. 16. Couturier, G.; Boisgard, R.; Nony, L.; Aime, J.P. Noncontacta tomic force microscopy: Stability criterion and dynamical responses of the shift of frequency and damping signal. Rev. Sci. Instr. 2003, 74, 2726-2734. 17. Zhang, W.M.; Meng, G. Nonlinear dynamical system of micro-cantilever under combined parametric and forcing excitations in MEMS. Sens. Actuat. A: Phys. 2005, 119, 291-299. Sensors 2009, 9 Sensors 2009, 9 3874 18. Zhang, W.M.; Meng, G. Nonlinear dynamic analysis of electrostatically actuated resonant MEMS sensors under parametric excitation. IEEE Sens. J. 2007, 7, 370-380. 19. Motamedi, R.; Wood-Adams, P.M. Influence of fluid cell design on the frequency response of AFM microcantilevers in liquid media. Sensors 2008, 8, 5927-5941. 20. Basak, S.; Raman, A. Hydrodynamic loading of microcantilevers vibrating in viscous fluids. J. Appl. Phys. 2006, 99, 114906. 21. Sader, J.E. Frequency response of cantilever beams immersed in viscous fluids with applications to the atomic force microscope. J. Appl. Phys. 1998, 84, 64-76. 22. Sader, J.E.; Chon, J.W.M.; Mulvaney, P. Calibration of rectangular atomic force microscope cantilevers. Rev. Sci. Instr. 1999, 70, 3967-3969. 23. Matei, G.; Jeffery, S.; Patil, S.; Khan, S.H.; Pantea, M.; Pethica, J.B.; Hoffmann, P.M. Simultaneous normal and shear measurements of nanoconfined liquids in a fiber-based atomic force microscope. Rev. Sci. Instr. 2008, 79, 023706. 24. Bao, M.H.; Yang, H. Squeeze film damping in MEMS. Sens. Actuat. A: Phys. 2007, 136, 3-27. 24. Bao, M.H.; Yang, H. Squeeze film damping in MEMS. Sens. Actuat. A: Phys. 2007, 136, 3-27. 25. Veijola, T.; Lahdenpera, J. References The influence of gas-surface interaction on gas film damping in a silicon accelerometer. Sens. Actuat. A: Phys. 1998, 66, 83-92. 25. Veijola, T.; Lahdenpera, J. The influence of gas-surface interaction on gas film damping in a silicon accelerometer. Sens. Actuat. A: Phys. 1998, 66, 83-92. 26. Song, Y.; Bhushan, B. Atomic force microscopy dynamic modes: modeling and applications. J. Phys.: Condens. Matter. 2008, 20, 225012. 26. Song, Y.; Bhushan, B. Atomic force microscopy dynamic modes: modeling and applications. J. Phys.: Condens. Matter. 2008, 20, 225012. 27. Andrews, M.; Harris, I.; Turner, G.A. comparison of squeeze-film theory with measurements on a microstructure Sens. Actuat. A: Phys. 1993, 36, 79-87. 27. Andrews, M.; Harris, I.; Turner, G.A. comparison of squeeze-film theory with measurements on a microstructure Sens. Actuat. A: Phys. 1993, 36, 79-87. 28. Ostergaard, D. Using a heat transfer analogy to solve for squeeze film damping and stiffness coefficients in MEMS structures. ANSYS Inc.: Canonsburg, PA, USA, January 23, 2003; Available online: http://www.ansys.com/assets/tech-papers/mems-thermal-analogy-fsi- damping.pdf 28. Ostergaard, D. Using a heat transfer analogy to solve for squeeze film damping and stiffness coefficients in MEMS structures. ANSYS Inc.: Canonsburg, PA, USA, January 23, 2003; Available online: http://www.ansys.com/assets/tech-papers/mems-thermal-analogy-fsi- damping.pdf © 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W4240332128	http://oceanrep.geomar.de/8497/1/essd-2-105-2010.pdf	English	null	The CARINA data synthesis project: introduction and overview	null	2,009	cc-by	15,431	The CARINA data synthesis project: introduction and overview R. M. Key1, T. Tanhua2, A. Olsen3,4, M. Hoppema5, S. Jutterstr¨om6, C. Schirnick2, S. van Heuven7, A. Kozyr8, X. Lin1, A. Velo9, D. W. R. Wallace2, and L. Mintrop10,* 1Atmospheric and Oceanic Sciences Program, Princeton University, Princeton, NJ, USA 2Leibniz-Institute f¨ur Meereswissenschaften, Marine Biogeochemie, Kiel, Germany 3Bjerknes Centre for Climate Research, UNIFOB AS, All´egaten 55, 5007 Bergen, Norway 4Department of Chemistry, University of Gothenburg, 412 96 G¨oteborg, Sweden 5Alfred Wegener Institute for Polar and Marine Research, Postfach 120161, 27515 Bremerhaven, Germany 6Institutionen f¨or kemi, G¨oteborgs universitet, G¨oteborg, Sweden 7Univ. of Groningen, Dept. of Ocean Ecosystems, Biological Center, 9750 AA Groningen, The Netherlands 8Carbon Dioxide Information Analysis Center, Oak Ridge, TN, USA 9Instituto de Investigaciones Marinas – CSIC, Eduardo Cabello 6, 36208 Vigo, Spain 10Fachbereich Geowissenschaften, Universitaet Bremen, Germany *now at: MARIANDA Company, Kiel, Germany Received: 11 September 2009 – Published in Earth Syst. Sci. Data Discuss.: 3 December 2009 Received: 11 September 2009 – Published in Earth Syst. Sci. Data Discuss.: 3 December 2009 Revised: 25 February 2010 – Accepted: 2 March 2010 – Published: 18 March 2010 Abstract. The original goal of the CARINA (Carbon in Atlantic Ocean) data synthesis project was to create a merged calibrated data set from open ocean subsurface measurements by European scientists that would be generally useful for biogeochemical investigations in the North Atlantic and in particular, studies involving the carbon system. Over time the geographic extent expanded to include the entire Atlantic, the Arctic and the Southern Ocean and the international collaboration broadened signiﬁcantly. In this paper we give a brief history of the project, a general overview of data included and an outline of the procedures used during the synthesis. The end result of this project was a set of 3 data products, one for each of the listed ocean regions. It is critical that anyone who uses any of the CARINA data products recognize that the data products are not simply concatenations of the originally measured values. Rather, the data have been through an extensive calibration procedure designed to remove measurement bias and bad data. Also a signiﬁcant fraction of the individual values in the data products were derived either by direct calculation or some means of approximation. These data products were constructed for basin scale biogeochemical investigations and may be inappropriate for investigations involving small areal extent or similar detailed analyses. Open Access Earth System Science Data Open Access Earth System Science Data Open Access Earth System Science Data Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License. Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License. 1 Background Historically, the vast majority of chemical oceanographic in- vestigations have focused on problems that had the scale of an ocean basin or smaller. There were multiple reasons for this restricted view that included lack of ﬁnancial resources, lack of manpower, and the fact that very limited data shar- ing occurred between individual researchers. Some data sets were submitted to national data centers, however, many were not, and the level of quality control possible at the national data repositories is limited. The end result was that no really high quality biogeochemical ocean data set with global scope existed. The GEOSECS program (Geochemical Ocean Sections) was conceived in 1967 and carried out during the 1970s. GEOSECS sampling consisted of 312 stations distributed approximately along the center of each major ocean basin. Many parameters were analyzed in addition to the com- mon hydrographic measurements, i.e. pressure, temperature, salinity, oxygen, and the macro nutrients nitrate, silicate and phosphate. Most remarkable about GEOSECS was the ex- tremely high quality of the measurements – in some cases equivalent to the best data being generated today. Also revo- lutionary was the fact the entire data set was available to the public in a reasonably short time. It is not an overstatement to say that GEOSECS revolutionized chemical oceanography. The greatest limitation of GEOSECS is that fact that it only provided a two dimensional picture of chemical distributions in the global ocean. The data were not suﬃcient to generate property distributions on horizontal surfaces. Global prop- erty integrals computed from the data had signiﬁcant errors (Peacock, 2004; Key et al., 2004). WOCE originated as a physical oceanographic program with sampling designed to optimize global transport calcula- tions. The occupied sections were either meridional or zonal and had dense sampling along the sections relative to previ- ous studies (∼30 nm station spacing; 24 to 36 bottle samples per station; high accuracy CTD records). In addition to the common hydrographic measurements a subset of the sam- ples were analyzed for transient tracers (3H, 3He, 13C, 14C, CFC-11 and CFC-12). JGOFS was a process oriented investigation and included repeated sampling at a few locations. The JGOFS locations were chosen for speciﬁc hydrographic and biogeochemical conditions. JGOFS measurements included the common hy- drographic parameters, but focused on less common biogeo- chemical measurements. 1 Background Critical to the CARINA project, JGOFS also funded the analysis of carbon system parameters (total inorganic carbon-DIC, total alkalinity-ALK, pH and/or the partial pressure (or fugacity) of dissolved carbon dioxide) on WOCE cruises. During the 1980s the TTO (Transient Tracers in the Ocean) and SAVE (South Atlantic Ventilation Experiment) programs extended the GEOSECS view to three dimensions for the Atlantic. Station spacing was still sparse, how- ever the individual station locations were chosen so that the combined data could be used to produce property maps on potential density surfaces with reasonable interpolation error (e.g. Kawase and Sarmiento, 1985). The number of measured parameters was signiﬁcantly smaller than for GEOSECS, but the data quality was again remarkably high, and the data were made public. Many of the papers in this special issue discuss total inor- ganic carbon and/or total alkalinity data. In these papers as well as within the chemical oceanographic community there is no standard abbreviation for these two parameters. Total inorganic carbon is abbreviated by DIC, TCO2, CT etc. Total alkalinity is abbreviated with Alk, ALK, AT, TA, etc. Re- gardless of the abbreviation used, in the CARINA papers all are talking about the exact same thing. Eﬀorts to standardize these abbreviations have failed. Two other transitions resulted from these programs. The ﬁrst was that nutrient and oxygen data were reported in mi- cromoles per kilogram rather than in micromoles or millil- itres per liter. This change was based on chemical arguments and has been adopted by subsequent large-scale programs. Unfortunately, this transition has not been universal. Second, the data were presented in a format designed for computer ac- cess. By today’s standards, the formats were far from ideal, but they were carefully thought out and the format “ﬂaws” were largely a result of computer limitations. Concurrent with WOCE sampling came the general accep- tance that human activities – most importantly the release of CO2 into the atmosphere by burning fossil fuels - had the potential to alter global climate. By the end of WOCE one of the largest uncertainties in global climate change stud- ies was the inventory of anthropogenic CO2 stored in the ocean. Accurate quantiﬁcation of this inventory was the pri- mary motivation for GLODAP (Global Ocean Data Analy- sis Project). The CARINA data synthesis project: introduction and overview More information on speciﬁc parts of this project can be found in companion articles in this issue. In particular, Tanhua et al. (2010) and Tanhua (2009) describe the procedures and software used to remove measurement bias from the original data. The three data products and a signiﬁcant volume of supporting information are available from the CARINA web site hosted by the Carbon Dioxide Information Analysis Center (CDIAC: http://cdiac.esd.ornl.gov/oceans/ CARINA/Carina inv.html). Anyone wanting to use the data is advised to get the highest version number of each data product. Incremental versions represent either corrections or additions. The web site documents speciﬁcs of the changes. Correspondence to: R. M. Key (key@princeton.edu) Correspondence to: R. M. Key (key@princeton.edu) Published by Copernicus Publications. Published by Copernicus Publications. R. M. Key et al.: The CARINA data synthesis project: introduction and overview 106 In the late 1980s WOCE (World Ocean Circulation Experi- ment) and JGOFS (Joint Global Ocean Flux Study) began. Unlike the previous studies, both of these had international organization and participation. Both programs had accuracy goals for every measured parameter, both required that the data be released quickly for public use in uniform-format computer-accessible ﬁles, and both had standard reporting units for every measurement. WOCE protocol had the addi- tional requirement that each measurement in a bottle data set (except CTD derived temperature and pressure) be assigned an integer quality ﬂag. The ﬂag values were determined ei- ther by ﬁrst hand knowledge of the analysis, or by “data ex- perts” after a data set was submitted. This data ﬂagging pro- cedure has come to be called “primary quality control” or simply “1st QC”. Primary quality control is largely a mea- sure of the precision of a particular measurement rather than accuracy. The WOCE data ﬂags have been used by many subsequent programs. 2 History of the CARINA project 2 History of the CARINA project Unlike GLODAP, the CARINA project began as an infor- mal collaboration with very limited funding. The project was started by D. Wallace and L. Mintrop, and had an organiza- tional meeting at Delmenhorst, Germany in 1999. Subse- quently, funding was obtained from German JGOFS to sup- port Mintrop who acted as data collector. Participation was voluntary and consisted mostly of European scientists. Par- ticipating scientists were required to submit their historical data sets that included either subsurface carbon system mea- surements or underway surface pCO2 data. The last meeting of this group was held in 2002. By that time the group had accumulated subsurface data from approximately 30 cruises (excluding those that were in GLODAP) and twice that num- ber of underway data sets. The funding ended in March 2003 and, unfortunately, the support level was insuﬃcient to do much more than amass and catalog the submitted data. Once the GLODAP team had completed the 2nd QC work, they produced two data products (Key et al., 2004). The ﬁrst was a set of three merged calibrated data sets, one each for the Atlantic, Indian and Paciﬁc Oceans. These compilations used a simpliﬁed set of quality ﬂags (subset of the WOCE ﬂags), had all questionable/bad data removed, included inter- polated values for missing salinity, oxygen and nutrient data and reduced the carbon measurements to ALK and DIC (by calculation from whatever carbon-pair was measured). The second product was a series of objectively mapped property distributions. The maps used the same grid spacing and depth levels as previous work (e.g. Levitus, 1982 and subsequent revisions) for compatibility. The maps were then integrated to provide inventories (for the region covered by the data) for DIC, ALK, natural 14C, bomb-produced 14C, anthropogenic CO2, CFC-11 and CFC-12 (Table 1, Key et al., 2004). These inventories were not quite global since GLODAP included very little data from the Arctic Mediterranean Seas. Sabine et al. (2004) made reasonable extrapolations to extend the data to the remainder of the global ocean and produced the ﬁrst data-based anthropogenic CO2 global ocean inventory using the method of Gruber (1998). The same data have been used with diﬀerent methods to calculate alternate anthropogenic CO2 inventory estimates (McNeil et al., 2003; Waugh et al., 2006). 1 Background The uniform calibration requirement led to the development (or adop- tion) of various techniques designed to quantify (and subse- quently correct) measurement bias that existed between var- ious cruise data sets. The data bias existed because there were no universal standards for most of the needed mea- surements (e.g. nutrients, oxygen, carbon system measure- ments). The quantiﬁcation of measurement bias has come to be known as secondary quality control or simply “2nd QC”. Details of the GLODAP 2nd QC procedures can be found in the literature (Key et al., 2004; Sabine et al., 2005) and at the CDIAC web site (http://cdiac.esd.ornl.gov/oceans/ glodap/Glodap home.htm). For the carbon system, most of the data bias was eliminated by the availability, part way through the WOCE sampling, of CRMs (Certiﬁed Reference Material) which were devised, prepared and distributed by A. Dickson (Dickson, 1990; Dickson et al., 2003; Dick- son, http://andrew.ucsd.edu/co2qc/index.html). The GLO- DAP team did not have the manpower to do complete 2nd QC on all of the parameters included in the data products, but rather adopted results from previous studies where available (Gouretski and Jancke, 2001; Johnson et al., 2001; C. Mordy and L. Gordon, personal communication to R. Key, 2003). very widely used for varied biogeochemical and physical in- vestigations by modelers and data analysts (Orr et al., 2001, 2005; Feely et al., 2002, 2004; Gnanadesikan et al., 2004; Lee et al., 2006: Matsumoto et al., 2004; Matsumoto, 2007; McNeil et al., 2007; Mikaloﬀ-Fletcher et al., 2006, 2007; Roussenov et al., 2004; Sarmiento et al., 2007; Sweeney et al., 2007; Vazquez et al., 2009; and many others). While quite successful, GLODAP did not cover all ocean areas. The only data in the collection from latitudes north of approximately 60◦N were a few GEOSECS and TTO sta- tions in the Nordic Seas. GLODAP included no data from the Arctic Ocean or the Gulf of Mexico, only a couple of stations in the Caribbean Sea, one GEOSECS station from the Mediterranean Sea, etc. Some of the research referenced above also demonstrated that the data density in the North Atlantic was exceptionally sparse relative to the concentra- tion gradients and complicated physics encountered there. These deﬁciencies were partially responsibility for the CA- RINA project. 1 Background GLODAP was a formally organized and funded TTO and SAVE organizers had planned to extend the pro- grams to the other oceans, however, this never materialized. Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ R. M. Key et al.: The CARINA data synthesis project: introduction and overview 107 collaboration. Most of the GLODAP team members were US scientists, but the project included participation by sci- entists from Australia, Japan, Korea and Europe. To achieve the stated goal, the ﬁrst requirement was a high quality, uni- formly calibrated global data set that included carbon system measurements and ancillary data. The core data for GLO- DAP were provided by WOCE and JGOFS. The uniform calibration requirement led to the development (or adop- tion) of various techniques designed to quantify (and subse- quently correct) measurement bias that existed between var- ious cruise data sets. The data bias existed because there were no universal standards for most of the needed mea- surements (e.g. nutrients, oxygen, carbon system measure- ments). The quantiﬁcation of measurement bias has come to be known as secondary quality control or simply “2nd QC”. Details of the GLODAP 2nd QC procedures can be found in the literature (Key et al., 2004; Sabine et al., 2005) and at the CDIAC web site (http://cdiac.esd.ornl.gov/oceans/ glodap/Glodap home.htm). For the carbon system, most of the data bias was eliminated by the availability, part way through the WOCE sampling, of CRMs (Certiﬁed Reference Material) which were devised, prepared and distributed by A. Dickson (Dickson, 1990; Dickson et al., 2003; Dick- son, http://andrew.ucsd.edu/co2qc/index.html). The GLO- DAP team did not have the manpower to do complete 2nd QC on all of the parameters included in the data products, but rather adopted results from previous studies where available (Gouretski and Jancke, 2001; Johnson et al., 2001; C. Mordy and L. Gordon, personal communication to R. Key, 2003). collaboration. Most of the GLODAP team members were US scientists, but the project included participation by sci- entists from Australia, Japan, Korea and Europe. To achieve the stated goal, the ﬁrst requirement was a high quality, uni- formly calibrated global data set that included carbon system measurements and ancillary data. The core data for GLO- DAP were provided by WOCE and JGOFS. 2 History of the CARINA project The GLODAP data products were released to the sci- entiﬁc community immediately, and have subsequently been In 2004 the original CARINA data collection was trans- ferred to CDIAC. This was about the same time that the North Atlantic GLODAP data deﬁciencies were recognized. Consequently, a copy of the CARINA bottle data was trans- ferred to Princeton for data assessment and quality control. In January 2005 the EU funded CARBOOCEAN program began. This consortium consists of more than 40 research groups and includes the most of original CARINA scientists. CARBOOCEAN is an integrated program with the aim of making an accurate assessment of oceanic sources and sinks of carbon over space and time. It has focus on the Atlantic and Southern Ocean and a time interval of −200 to +200 years from the present. All funded CARBOOCEAN part- ners are required to make public all historical data and new data after a two year proprietary period. During workshops www.earth-syst-sci-data.net/2/105/2010/ Earth Syst. Sci. Data, 2, 105–121, 2010 R. M. Key et al.: The CARINA data synthesis project: introduction and overview R. M. Key et al.: The CARINA data synthesis project: introduction and overvie 108 Figure 1. Station locations for cruises included in the three CARINA data products. The division between the Southern Ocean collection and the Atlantic collection was approximately 30◦S and between the Atlantic and Arctic approximately 60◦N (the Greenland-Scotland Ridge). Several cruises that cross one of the boundaries are included in both collections. Regional maps are available at http://cdiac.esd.ornl.gov/ oceans/CARINA/Carina inv.html and a cruise map and data ﬁle for each cruise at http://cdiac.esd.ornl.gov/oceans/CARINA/Carina table. html. Figure 1. Station locations for cruises included in the three CARINA data products. The division between the Southern Ocean collection and the Atlantic collection was approximately 30◦S and between the Atlantic and Arctic approximately 60◦N (the Greenland-Scotland Ridge). Several cruises that cross one of the boundaries are included in both collections. Regional maps are available at http://cdiac.esd.ornl.gov/ oceans/CARINA/Carina inv.html and a cruise map and data ﬁle for each cruise at http://cdiac.esd.ornl.gov/oceans/CARINA/Carina table. html. held in the ﬁrst two years of CARBOOCEAN, the CARINA project was reactivated and additional data sets collected. table.php) that were ﬁnal and that were in one of the focus regions. 2 History of the CARINA project Since the new CLIVAR data were known to be high quality, those data, along with WOCE results would serve as “master cruises” for the data calibration (2nd QC) phase of the synthesis. The areal expansion of the project led to a ﬂood of new data and a ﬁnal total of 188 cruises. The CARINA station locations are shown in Fig. 1. The CA- RINA web site (http://cdiac.esd.ornl.gov/oceans/CARINA/ Carina inv.html) includes links to the original cruise data ﬁles (via the Cruise Summary Table), the resulting data prod- ucts and publications, and detailed information on the quality control procedures used. In June 2007 the CARBOOCEAN/CARINA scientists met in Laugarvatn, Iceland to discuss methods and responsibili- ties for the CARINA data synthesis. By that time, the CA- RINA collection had grown to approximately 80 cruises. During this meeting the group decided to extend the origi- nal scope of CARINA to include the entire Atlantic, the Arc- tic and the Southern Ocean. Various team and project leader assignments were: – Data collection, 1st QC and production of ﬁnal data products: R. Key and X. Lin – Atlantic Ocean: T. Tanhua 3 Instrumentation – Arctic Ocean: S. Jutterstr¨om Data included in the CARINA data products span almost 30 years of measurements. Rather than attempt to summarize the speciﬁc methods and instruments in this document, we have included this information in the individual cruise ﬁle headers. For many cruises additional information can be found in the individual ﬁnal cruise reports and other docu- mentation provided with the cruise data. In many instances, a full description of the methods and instruments can be found in the footnotes to the Cruise Summary Table at the CARINA web site that refer to speciﬁc publications. Certainly the most important changes in methods and instrumentation are the adoption of CRM for standardization of ALK and DIC mea- surements, the development of the SOMMA-type analyzer – Nordic Seas: A. Olsen – Southern Ocean: M. Hoppema – 2nd QC code development: S. van Heuven – Web site development and maintenance: C. Schirnick – Carbon calculation software: A. Velo – Data archive: A. Kozyr/CDIAC Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ 4 CARINA data assembly and synthesis Here we describe the data collection and synthesis steps used for this project. Many of the procedures used dur- ing CARINA were adopted from GLODAP, however, the number of cruises included in CARINA combined with the additional manpower and funding available from the CAR- BOOCEAN contract allowed improvements. The most sig- niﬁcant changes were: (a) more parameters were subjected to 2nd QC by the project participants; (b) software was designed to automate portions of the 2nd QC procedures; (c) work was coordinated among the diﬀerent groups and within groups by means of a web site; (d) pH was included in the ﬁnal data products along with ALK and DIC; (e) fully formatted versions of all the individual cruise ﬁles were sub- mitted to both CCHDO (CLIVAR & Carbon Hydrographic Data Oﬃce: http://whpo.ucsd.edu/) and CDIAC for archive and distribution; and (f) a signiﬁcant collection of references to literature describing the individual cruise results was com- piled. For all of the CARINA cruises the following conventions were used for station information. Only one location was recorded for each station of each cruise. When multiple casts were collected, the location and date of the ﬁrst cast was used for the entire station. Locations were stored as decimal de- grees with negative values for west longitude and south lati- tude. For many of the cruises bottom depth was not recorded for each station. In these cases bottom depth was ﬁrst approx- imated from a global (0.25 degree resolution) topography. This depth was then compared to the deepest sample pres- sure at the station. Whichever was greater, the topographic depth or the deepest sample pressure +10 was recorded for the water depth. These bottom “depths” are not meant for research purposes, but rather to enable drawing approximate bottom topography for section plots. This eﬀort led to two distinct results. The ﬁrst is a set of individual cruise ﬁles with the measured data converted to common units, having quality ﬂags added for all param- eters, and accompanied by metadata. All of the individual cruise ﬁles are in “WHP-Exchange” format (Swift, 2008). This format is a standard that developed during the 1990s and has since become widely accepted. It is a comma sep- arated data ﬁle with formal column header names and units and that can include metadata within the header. 4.1 Collection and 1st QC (Johnson et al., 1998 and references cited therein) for DIC and the shift from electrode based to spectrophotometric pH determination (Clayton and Byrne, 1993). All three began to be used in the early 1990s. (Johnson et al., 1998 and references cited therein) for DIC and the shift from electrode based to spectrophotometric pH determination (Clayton and Byrne, 1993). All three began to be used in the early 1990s. The most time consuming portion of the CARINA synthesis was data assembly. Investigators who had participated in data collection and/or made the measurements, submitted most of the data sets. Along with the data ﬁle, submitters were asked to supply references to any publication(s) that had re- sulted from the data. Whenever they existed, ﬁnal cruise re- ports were obtained. The remaining data sets were obtained by “discovery” which amounted to scanning publications for mention of other cruises, data discussed at CARBOOCEAN and other meetings and similar. Once discovered, either the chief scientist or another cruise participant was contacted for a copy of the data and any existing documentation. In most cases, a complete copy of the cruise data set was not avail- able. In these instances the missing data were sought from the principal investigator(s) (PI) responsible for that data. Though the eﬀort was not completely successful, we tried to obtain all of the bottle measurements from each cruise. As the data were collected, we also obtained permission from each PI to release his/her data to the public. In a few cases electronic versions of the data did not exist and the results were manually entered into the existing ﬁles. – Data archive: A. Kozyr/CDIAC The team also decided to include data from CLIVAR (Climate Variability and Predictability) repeat hydrog- raphy cruises (http://www.clivar.org/carbon hydro/hydro Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ 109 R. M. Key et al.: The CARINA data synthesis project: introduction and overview www.earth-syst-sci-data.net/2/105/2010/ 4 CARINA data assembly and synthesis The second is a set of 3 data products (Arctic Mediterranean Seas-AMS, Atlantic Ocean-ATL and Southern Ocean-SO) that have been fully calibrated (i.e. measurement bias removed via 2nd QC) and include some calculated values. For CARINA we de- ﬁned Arctic Mediterranean Sea(s) to include the main Arc- tic basin and all adjacent seas southward to approximately 60◦N. Thus the AMS region includes the Nordic Seas (down to the Greenland-Iceland-Scotland Ridge) on the Atlantic side and the Bering Sea on the Paciﬁc side. The format for the data products is simple comma separated records with a single header record deﬁning the included values. The header does not include units since everything is standard (as deﬁned for the Exchange format). Additionally, the data products are purely numeric other than the single header record. For most cruises multiple ﬁles with diﬀerent subsets of the data were collected. The ﬁrst synthesis task, and the most error prone, was merging data from these subsets. File merg- ing is a quick and easy computer matching procedure when- ever adequate sample identiﬁcation is given. However, for most of the CARINA cruises the identiﬁcation information was either incomplete or totally missing. In these instances the data ﬁles were manually merged based on available in- formation. The manual merges, which consist of multiple cut and paste operations were made especially tedious by the fact that “intended bottle depth”, “bottle pressure” and “bot- tle depth” were often used synonymously. In the many in- stances where the cast and bottle information was missing, values were fabricated to ease subsequent discussion of spe- ciﬁc results among various project participants and to make the ﬁles more format consistent with modern oceanographic records. Such fabrication is noted in the metadata header The CARINA data products are compatible with the three GLODAP data products, but they are not identical, diﬀering somewhat in column order and included parameters. We plan to merge CARINA and GLODAP once the initial scientiﬁc analysis of CARINA is completed. www.earth-syst-sci-data.net/2/105/2010/ Earth Syst. Sci. Data, 2, 105–121, 2010 R. M. Key et al.: The CARINA data synthesis project: introduction and overview 110 of the ﬁnal format ﬁles submitted to the data centers. Al- phabetic station names were converted to numeric and un- necessarily complex station numbers were simpliﬁed. These alterations were documented in the ﬁle header information. 4 CARINA data assembly and synthesis Without bottle salinity, identiﬁcation of mis-trips and leaking sample bottles is reduced to an educated guess, at best. An additional problem with many of these data sets was that bot- tle salinity and CTD salinity values were not discriminated. That is, it was impossible to determine which of the two was included in a data ﬁle. When we could not determine if a set of values was CTD or bottle salinity, we assumed that it was bottle salinity. Therefore it is virtually certain that some of the bottle salinity data is actually CTD salinity. See also the discussion below on special steps taken with salinity data during production of the ﬁnal data products. In general, the treatment of salinity data in CARINA could be labeled sloppy. We wouldn’t argue with that, however, this wasn’t due to lack of eﬀort – we did the best we could. We also be- lieve that the salinity data in CARINA are adequate for “nor- mal” chemical oceanographic applications. We do not know whether or not the salinity data will be of suﬃcient quality for detailed physical oceanographic applications. Another complication arose with nitrate data. In ideal cases nitrate and nitrite measurements were reported sepa- rately. In others only nitrate was reported or only the com- bination of nitrate plus nitrite. Finally, in a few instances nitrate plus nitrite was reported along with values for nitrite. For the last example the nitrite values were simply subtracted from the reported nitrate plus nitrite values. For cases where only nitrate plus nitrite was reported we had a choice: carry an additional parameter (i.e. NO3 + NO2 in addition to ni- trate) or simply rename the data nitrate (ignoring the nitrite contribution in the upper water column). Both choices are problematic. We chose the latter for CARINA cruises (both original cruise ﬁles and ﬁnal data products). The next step in the synthesis was 1st QC – the assigning of a data quality ﬂag to each measured value. This is a pro- cess by which individual data points are closely scrutinized. It is a method of improving precision and removing spurious data. Details of this procedure are in Tanhua et al. (2010). Chloroﬂuorocarbon data in the CARINA collection cover the time span from 1982 to 2005 and were originally reported on either the SIO-93 or SIO-98 scale. 4 CARINA data assembly and synthesis data were converted to SWS at 25 ◦C in both the individual cruise ﬁles and in the ﬁnal products. While we were pro- ducing the data products, a new version of the handbook of best practices for ocean carbon measurements was published (Dickson et al., 2007). This handbook suggests that the pre- ferred pH scale is the total hydrogen, however, at that point it was already too late for our project. Velo et al. (2009) give the conversion functions and additional details for this work. Immediately after merging, cruise data were read into the same data system used for the GLODAP collection. There, units were converted to match those used during the WOCE program. Most commonly, this amounted to converting oxy- gen and nutrient data from milliliter per liter and micromole per liter into micromole per kilogram (µmole/kg). Unfortu- nately, there is no standard method for this conversion. For this work the most common method was to use density cal- culated from measured salinity for each sample with an as- sumed lab temperature (default of 22◦C) and pressure (1 at- mosphere). In cases where the concentration was reported in standard units (µmole/kg) the conversion method is un- known, but simple division by a constant assumed density (often 1.025) is common. Regardless of method, this con- version error is less than the measurement errors, so we con- sider this inconsistency to be bothersome, but minor. An- other source of error that we were not able to completely eliminate is the possibility of erroneous units for the nutri- ents, i.e. that data were given in volumetric units instead of the stated gravimetric units, or the vice verse. Both cases would cause an oﬀset of 2–3%. Historically, salinity has been analyzed on every bottle sample from a CTD/Rosette cast. The bottle salinity re- sults were calibrated by analyzing seawater standards. The calibrated bottle salinity values were subsequently used to calibrate the CTD conductivity probe. Also, because bottle salinity can routinely be measured with high precision, the bottle salinity data provide the best check that a sample bot- tle closed properly and at the desired depth (for most ocean regions). That is, bottle salinity is the best way to identify mis-trips and leaking sample bottles for most of the global ocean. On many of the CARINA cruises, bottle salinity was only analyzed with suﬃcient frequency to calibrate the CTD. www.earth-syst-sci-data.net/2/105/2010/ R. M. Key et al.: The CARINA data synthesis project: introduction and overview 111 a16n, P>1000dB 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 −0.08 −0.06 −0.04 −0.02 0 0.02 0.04 0.06 0.08 PHSWS25 mean!=−3.0833e−05±0.01237 Measured−Calculated Mean a16n, P>1000dB 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 −30 −20 −10 0 10 20 30 TCO2 mean!=−0.0033766±5.1022 Measured−Calculated Mean a16n, P>1000dB 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 −30 −20 −10 0 10 20 30 ALK Pressure mean!=−0.0032775±5.3096 Measured−Calculated Mean Figure 2. Comparison of measured and calculated carbon system values from Cruise #86; 33RO20030604. Regardless of which pair was used to calculate the third, the mean diﬀerence is statistically indistinguishable from zero. The standard deviation of the diﬀer- ence is only marginally larger than the precision estimate based on replicate analyses. a16n, P>1000dB 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 −0.08 −0.06 −0.04 −0.02 0 0.02 0.04 0.06 0.08 PHSWS25 mean!=−3.0833e−05±0.01237 Measured−Calculated Mean a16n, P>1000dB 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 −30 −20 −10 0 10 20 30 TCO2 mean!=−0.0033766±5.1022 Measured−Calculated Mean a16n, P>1000dB 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 −30 −20 −10 0 10 20 30 ALK Pressure mean!=−0.0032775±5.3096 Measured−Calculated Mean The zero ﬂag indicates a datum that “could have been mea- sured”, but was approximated in some manner. There are three diﬀerent uses for the zero ﬂag in the data products: – Instances where bottle salinity was missing or bad and consequently was replaced with CTD salinity. – Interpolated values for salinity, oxygen or nutrients. – Interpolated values for salinity, oxygen or nutrients. – Calculated carbon parameters. 4.2 2nd QC While 1st QC is designed to improve the overall precision of a data set, 2nd QC procedures are designed to quantify measurement bias. That is, the goal of 2nd QC is to improve the accuracy of a data set. Measurement bias is rather com- mon with nutrient and oxygen measurements because certi- ﬁed standards are not routinely used. The very best nutri- ent measurements can have precision better than 1%, but the accuracy is seldom better than 2%. The same condition ex- isted for ALK and DIC measurements until the early 1990s when CRM were developed. From GEOSECS to WOCE, ALK and DIC measurement precision improved from 5–10 to 4–5 µmole per kilogram. The best CLIVAR data now have precision of <2 µmole/kg. Prior to CRM development, how- ever, it wasn’t uncommon for these measurements to have a bias of >20 µmole/kg. The use of CRMs has lowered that to <5 µmole/kg. Figure 2. Comparison of measured and calculated carbon system values from Cruise #86; 33RO20030604. Regardless of which pair was used to calculate the third, the mean diﬀerence is statistically indistinguishable from zero. The standard deviation of the diﬀer- ence is only marginally larger than the precision estimate based on replicate analyses. The 2nd QC is based on the initial assumption that abyssal waters are at steady-state. That is, deep water concentrations are invariant over time for a given location. This assump- tion was reasonable for the WOCE cruises included in GLO- DAP since the collection period only spanned a few years and few of the cruise track intersections occurred in regions with strong horizontal abyssal concentration gradients. This is not the case for CARINA. Many publications have clearly demonstrated that the abyssal steady state assumption is false over the time interval spanned by CARINA data and espe- cially for some of the regions sampled by CARINA cruises (i.e. the far North Atlantic, the Labrador Sea and the Nordic Seas). Decadal change due to anthropogenic and natural forcing was one of the CARBOOCEAN/CARINA focus ar- eas, so all of the scientists involved in 2nd QC were aware of the potential to erase real changes when attempting to correct measurement bias. 2004; Sabine et al., 2005) and diﬀerent forms of the inver- sion methods derived by Gouretski and Jancke (2001) and Johnson et al. (2001). The 2nd QC methods are discussed in detail by Tanhua et al. (2010). 4 CARINA data assembly and synthesis All of these (CFC-11, CFC-12, CFC-113, CCl4) were converted to the SIO-98 scale (Prinn et al., 2000). SF6 data are reported on the NOAA- GMD 2000 calibration scale. The 2nd QC procedures (discussed in Tanhua et al., 2010) critically examine data using diﬀerent techniques than 1st QC. The goal of 2nd QC is to quantify measurement bias. In some cases additional spurious data points were identiﬁed during 2nd QC, and the initial ﬂag values altered appropri- ately. Once all of the ﬂag values are ﬁnal, each cruise ﬁle was submitted to national data centers (CCHDO and CDIAC). Data bias identiﬁed during 2nd QC was corrected in the ﬁ- nal data products, but these adjustments were not applied to the individual cruise data sets. Reported pH data were also converted to uniform scale and temperature. The CARINA data span 1977–2005. Over that time pH measurements have been made with radically diﬀer- ent techniques and the results reported on three diﬀerent pH scales: National Bureau of Standards scale (NBS), seawa- ter scale (SWS) and total hydrogen scale (TOT). Values are also reported at various temperatures (measurement temper- ature, some arbitrarily chosen temperature or in situ tempera- ture). The diﬀerence between these scales isn’t too large, but it is signiﬁcantly larger than the precision/accuracy of mod- ern spectrophotometric techniques. All of the measured pH The CARINA data product incorporates one additional ﬂag with value zero (0). This ﬂag was also used in GLODAP. Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ www.earth-syst-sci-data.net/2/105/2010/ 4.3 Construction of the data products Parameter Minimum Oﬀset Salinity (CTD and/or bottle) 0.005 Oxygen 1% Nitrate 2% Phosphate 2% Silicate 2% Alkalinity 6 (µmole/kg) DIC 4 (µmole/kg) pH 0.005 Parameter Minimum Oﬀset Salinity (CTD and/or bottle) 0.005 Oxygen 1% Nitrate 2% Phosphate 2% Silicate 2% Alkalinity 6 (µmole/kg) DIC 4 (µmole/kg) pH 0.005 parameter is biased, the implication is that one of the input parameters is biased. The 2nd QC procedures yield an oﬀset for virtually ev- ery cruise. In some previous studies (Gouretski and Jancke, 2001 and Johnson et al., 2001), in order to be as objective as possible, all of the determined oﬀsets were corrected. This will produce a combined data set with the lowest combined variance between cruises. In GLODAP and CARINA a more subjective approach was used. First, only those oﬀsets that exceeded a predetermined minimum value were considered for correction. Second, all oﬀsets that exceeded the thresh- old were examined by the working groups prior to assigning a ﬁnal adjustment value. This subjective approach was nec- essary because the diﬀerent 2nd QC procedures often gave diﬀerent results and because some of the parameters were expected to change with time. This issue is discussed in de- tail in the accompanying methods paper (Tanhua et al., 2010) and in each of the regional CARINA papers in this issue. The minimum oﬀsets considered for adjustment are given in Ta- ble 1. All of the details of the crossover checks, inversion results and ﬁnal adjustments are available at the CARINA web site. The Cruise Summary Table (CST) contains a wealth of ad- ditional information. Along with the EXPOCODE the sec- ond column also lists aliases. Aliases include names used by the original investigators for the cruise or project and in some cases WOCE line designations (e.g. for cruise #4 the “WOCE SR04e”). The third column (Area) refers to the CARINA region (and data product) with: 1 = ATL, 2 = SO, 3 = ATL & SO, 4 = AMS and 5 = AMS & ATL. The numbers under the parameter columns indicate the number of stations that have the particular measurement. Two entries under the parameter columns have a diﬀerent meaning. Very few cruises in this collection included discrete pCO2 sampling. For these few, a numeric entry is the station count. A “U” entry, however, indicates that underway pCO2 measurements were made. The CARINA work does not include under- way data. R. M. Key et al.: The CARINA data synthesis project: introduction and overview 112 4.2 2nd QC 2nd QC tests were run for salinity, oxygen, nutrients, DIC, ALK, pH, CFC-11, CFC-12, CFC-113 and CCl4. For the carbon system parameters, additional tests were possible using calculated values. For example, if DIC and ALK were measured, calculated pH could be compared to measured pH from another cruise. To demonstrate the valid- ity of this comparison, we compared calculated to measured parameters for one Atlantic cruise that had very high qual- ity measurements for three carbon system parameters (Cruise #86; 33RO20030604; Fig. 2). Regardless of the pair used for the calculation, the mean diﬀerence between the mea- sured and calculated values was statistically indistinguish- able from zero and the standard deviation of the diﬀerence was not much larger than the measurement precision. This comparison provides strong evidence that the calculation er- ror is insigniﬁcant and that calculated carbon parameters can be used for 2nd QC investigations. If a calculated carbon The 2nd QC normally consisted of two steps: quantiﬁ- cation of the relative measurement oﬀsets between diﬀerent cruises and assignment of a adjustment factor to data deemed to have a measurement bias that exceeded a predetermined limit. The ﬁrst step was objective, the second subjective and inﬂuenced by the experience of the scientists involved and the knowledge that real temporal changes were expected for some regions. Oﬀset was determined using variants of the crossover technique developed for GLODAP (Key et al., www.earth-syst-sci-data.net/2/105/2010/ Earth Syst. Sci. Data, 2, 105–121, 2010 R. M. Key et al.: The CARINA data synthesis project: introduction and overview 4.3 Construction of the data products Table 1. Minimum oﬀset values considered for adjustment. Not all cruise to cruise diﬀerences that exceeded the minima were adjusted. In a very few cases (with very precise data) smaller adjustments were made. A table of the adjustments applied to the CARINA data can be found at http://carina.ifm-geomar.de/ (this site is being copied to CDIAC for permanent archive). The CARINA project resulted in three data collections or products: the Arctic Mediterranean Seas (AMS), the At- lantic Ocean and Mediterranean Sea (ATL), and the Southern Ocean (SO). The divisions between the regions were approx- imately 60◦N (the Greenland-Scotland Ridge in the Atlantic and the Aleutians in the Paciﬁc) and 30◦S. Cruises which spanned a division line were generally included in both col- lections. Each cruise in the collection was assigned an EX- POCODE (Swift, 2008). These codes provide an unique identiﬁer and are composed of NODC (National Ocean Data Center) platform code for the research vessel (http://www. nodc.noaa.gov/General/NODC-Archive/platformlist.txt) fol- lowed by the date when the cruise left port. The NODC code is composed of a 2 digit country code and a 2 character (num- ber or letter) ship code. For example a cruise that started on 3 October 1999 aboard the Norwegian vessel Haakon Mosby would have EXPOCODE 58AA19991003. All of the cruises were then sorted by EXPOCODE, numbered sequentially, and a Cruise Summary Table (CST) was created (http://cdiac. esd.ornl.gov/oceans/CARINA/Carina table.html). The last 5 entries in the CST are not single cruises, but cruise collec- tions representing a single investigator (#’s 184 and 185) or a single project (#’s 186–188). Assignment of an EX- POCODE in these 5 cases was inappropriate so they were simply named. The data for these 5 collections were not seg- regated into individual cruise ﬁles because we thought the data more valuable as a collection and because the limited amount of data for each individual cruise did not warrant the increased record keeping that would have been required. The three data products include only the sequential cruise num- ber, not the EXPOCODE so that the data records could re- main purely numeric. Lookup tables are provided along with the data products so that the cruise number can be matched to the EXPOCODE. 4.3 Construction of the data products Underway pCO2 data are being compiled by an- other team (SOCAT; Surface Ocean CO2 Atlas Project; http: //ioc3.unesco.org/ioccp/Synthesis.html#SOCAT). A “C” en- try in the CST under the pH, CT or AT column indicates that In a few instances 2nd QC and associated investigations determined that all of the measurements of some parameter from a cruise could not be adequately adjusted. The reasons varied, but included strongly conﬂicting 2nd QC results, ex- tremely noisy data and similar problems. In these cases the entire set of parameter measurements was discarded from the data product. Instances of this are indicated in the on-line version of the adjustment table by the lower case letter “o” in the ﬂag column for each parameter instead of the normal check mark (√) which indicates acceptable results. If this table is downloaded these two adjustment quality ﬂags are translated into “3” and “2”, respectively. The decision to dis- card an entire set of measurements was made independently from the individual datum 1st QC ﬂags. Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ R. M. Key et al.: The CARINA data synthesis project: introduction and overview 113 Table 2. Translation table for parameter names, ﬂags and units. 4.3 Construction of the data products Data Product Data Product Exchange File Exchange File Units Parameter Name Flag Name Parameter Name or Flag Name Full name of Parameter station STANBR nosamp Number of samples at each station integer day DATE month DATE year DATE latitude LATITUDE decimal degrees longitude LONGITUDE decimal degrees maxdepth DEPTH meters maxsampdepth Pressure of the deepest sample at each station decibar cruiseno CARINA assigned sequential number bottle bf BTLNBR BTLNBR FLAG W cast CASTNO depth Calculated sample depth meters temperature CTDTMP ◦C salinity sf SALNTY SALNTY FLAG W ctdsal ctdsf CTDSAL CTDSAL FLAG W pressure CTDPRS decibars oxygen of OXYGEN OXYGEN FLAG W micomole kg−1 nitrate no3f NITRAT NITRAT FLAG W micomole kg−1 nitrite no2f NITRIT NITRIT FLAG W micomole kg−1 silicate sif SILCAT SILCAT FLAG W micomole kg−1 phosphate po4f PHSPHT PHSPHT FLAG W micomole kg−1 tco2 tco2f TCARBN TCARBN FLAG W micomole kg−1 alk alkf ALKALI ALKALI FLAG W micomole kg−1 phsws25 phsws25f PH SWS PH TMP PH SWS FLAG W cfc11 cfc11f CFC-11 CFC-11 FLAG W picomole kg−1 cfc12 cfc12f CFC-12 CFC-12 FLAG W picomole kg−1 cfc113 cfc113f CFC113 CFC113 FLAG W picomole kg−1 CCl4 CCl4f CCl4 CCl4 FLAG W picomole kg−1 SF6 SF6f SF6 SF6 FLAG W femtomole kg−1 c14 c14f DELC14 DELC14 FLAG W ‰ c13 c13f DELC13 DELC13 FLAG W ‰ h3 h3f TRITUM TRITUM FLAG W TU he3 he3f DELHE3 DELHE3 FLAG W % he hef HELIUM HELIUM FLAG W nanomole kg−1 c14e C14ERR ‰ h3e TRITER TU he3e DELHER % hee HELIER nanomole kg−1 R. M. Key et al.: The CARINA data synthesis project: introduction and overview 113 the values in the compiled data product were calculated from other carbon parameters. The calculated values are not in- cluded in the individual cruise ﬁles submitted to the CDIAC and CCHDO. The last column of the CST (Other) lists other measurements made on that cruise. When we were able to obtain these data, they are included in the original cruise ﬁles. using current methods. The list of retained parameters is given in Table 2. This table also translates the parame- ter names in the products to the “oﬃcial” Exchange format nomenclature and it gives units for the measurements. This naming convention was selected so that the CARINA data products matched the GLODAP data products as closely as possible. The data products do not contain all of the measurements from all of the cruises. www.earth-syst-sci-data.net/2/105/2010/ R. M. Key et al.: The CARINA data synthesis project: introduction and overview R. M. Key et al.: The CARINA data synthesis project: introduction and overview 114 Table 2. Continued. Table 2. Continued. Table 2. Continued. Table 2. Continued. Calculated values included in the data products. These parameters are not listed in standard exchange format ﬁles. Data Product Data Product Full name of Units Parameter Name Flag Name Parameter pf11 CFC-11 Partial Pressure ppt (parts per trillion) pf12 CFC-12 Partial Pressure ppt pf113 CFC113 Partial Pressure ppt pCCl4 CCl4 Partial Pressure ppt pSF6 SF6 Partial Pressure ppt aou aouf Apparent Oxygen Utilization micomole kg−1 theta Potential Temperature ◦C sigma0 Potential Density relative to 0 dB kg m−3 sigma1 Potential Density relative to 1000 dB kg m−3 sigma2 Potential Density relative to 2000 dB kg m−3 sigma3 Potential Density relative to 3000 dB kg m−3 sigma4 Potential Density relative to 4000dB kg m−3 manually calling several programs in sequence with the ap- propriate options set for each program. With the exception of one step, all of the code was developed and runs on the same computer used for archiving the master version of each cruise data ﬁle. All of this code is written in S-Plus (Ver- sion 3.4 release 1 for Sun SPARC; TIBCO Spotﬁre, previ- ously Insightful®). Below, each step of the procedure is brieﬂy described. nally, any necessary adjustments (multiplicative or additive) are taken from the adjustment table and applied. The result is two ﬁles: one with station information and a second with data. The two ﬁles are checked for missing value numbers (−9, −999, etc.) that may have resulted from other software and these are replaced with NA. Care is required with the station ﬁle since −9 is a possible real value for latitude and longi- tude, consequently, a very few latitude and longitude values that were exactly −9 were changed to −9.00001. This change is scientiﬁcally inconsequential. The cruises included in the CARINA data products generally exclude those that were included in GLODAP. This was done primarily to facilitate later merging of these two data products. There are, however, 3 excep- tions: 06MT19941012, 06MT19941115 and 74DI19970807 (Cruise Numbers 12, 13 and 171 respectively). These cruises were added to CARINA because additional parameters crit- ical to the CARINA goals became available after GLODAP was published. R. M. Key et al.: The CARINA data synthesis project: introduction and overview The CARINA 2nd QC, however, made full use of many of the GLODAP cruises and details are given in many of the accompanying publications in this issue. Finally, the compiled data were subjected to a very coarse primary QC to eliminate any highly anomalous data points that had not previously been discovered. This check was made by plotting all values of each parameter against pres- sure. For most parameters a few points were noted. These few anomalous points were removed from the data product. With this procedure, it is far more likely that questionable values were retained than good data eliminated, but the latter is still possible. 1. ﬂag 0, 2, 9, no change to data or ﬂag 4.3 Construction of the data products Rather we narrowed the total list of diﬀerent measurements down to those that were commonly measured or would be useful for carbon system calculations With a few minor changes the CARINA data products were constructed with the same software used for GLODAP. The procedure is semi-automated and execution amounts to www.earth-syst-sci-data.net/2/105/2010/ Earth Syst. Sci. Data, 2, 105–121, 2010 4.3.4 Interpolation Many of the procedures used to interpret biogeochemical data involve various property-property plots or linear least squares ﬁtting procedures. Since the highest priority appli- cation for the CARINA data set was oceanic carbon chem- istry, we did not want to exclude relatively expensive carbon measurements from such analyses only because the sample was not analyzed for salinity, oxygen or one of the nutrients. Consequently, we made the same decision as was made dur- ing the GLODAP eﬀort (Key et al., 2004) and interpolated missing values for salinity, oxygen, nitrate, phosphate and/or silicate where it was reasonable to do so. The GLODAP algorithm was used. That is, a quasi-Hermetian piecewise polynomial was ﬁt to existing data and that ﬁt used to ap- proximate missing values. The distance over which interpo- lation was allowed varied with pressure in the water column and by region. The zones and limits were determined by ex- periment and consensus between Princeton and the four area team leaders. Table 4 summarizes the pressure zones and the maximum allowable data separation for each zone. Ex- trapolation was not allowed. These interpolated values were assigned a zero ﬂag value. 3. ﬂag 6 reset to 2 with no change to data value 3. ﬂag 6 reset to 2 with no change to data value 4. to correct ﬂag errors which occurred at any step, the data are searched for NA and the ﬂag associated with NA is set to 9. The ﬁnal result should be a ﬁle that only has ﬂag values 0, 2, or 9. This procedure is not perfect. It is impossible to pre- dict all the possible typographical errors in ﬁles of this size. While it is trivially easy to identify the unique ﬂag values in the combined data set it can be extremely tedious to iden- tify the exact location of the error and know the appropriate correction. www.earth-syst-sci-data.net/2/105/2010/ R. M. Key et al.: The CARINA data synthesis project: introduction and overview 115 Table 3. Summary of data quality ﬂags used for CARINA cruise ﬁles. For the data products the ﬂag list was reduced to 0, 2 and 9 (see text). Table 4. Interpolation zones and limits. Zones and limits were determined by experimentation. For each interpolated value the ad- jacent measured values (above and below) can be separated by no more than the corresponding limit for the interpolated value to be deemed acceptable. Flag Value Interpretation in CARINA 0 Approximated 1 Not used 2 Good 3 Questionable 4 Clearly bad result 5 Value not reported 6 Average of replicate 7 Not used 8 Not used 9 Not measured Arctic Atlantic Southern Ocean Zone Limit Zone Limit Zone Limit 0–100 25 0–100 25 0–100 25 101–300 75 101–300 75 101–300 75 301–750 150 301–750 205 301–750 150 751–2000 250 751–1500 405 751–2000 505 2001–bottom 500 1501–2500 605 2001–bottom 1005 2501–bottom 1005 the CTD salinity (and ﬂag) was copied into the bottle salin- ity data slot. The rationale for this procedure was to make the data easier to use without incurring errors that would be signiﬁcant for most applications. This procedure probably added noise to the salinity data, but one might expect the noise to be pseudo-random for the entire data set. 4.3.2 Flag simpliﬁcation Program makeocean is the main routine for building merged calibrated data products. Input includes: (1) a list of cruise names, (2) a list of parameters to be included in the data prod- uct, (3) a list of parameters that were considered for adjust- ment and (4) the name of the table that contains all of the var- ious parameter adjustment factors. In sequence, each cruise ﬁle is ﬁrst read and then reduced to the list of measured pa- rameters that are included in the output product. Any param- eter (and accompanying ﬂag) that is in the include list, but not in the cruise data set is generated and ﬁlled with null val- ues (NA; −999 on output). The parameter columns are then sorted into the same order as the input parameter list. Fi- Program ﬂagmod simpliﬁes the full set of WOCE quality control ﬂag values (Table 3) to a minimum subset. The ratio- nale is to make the data products easily usable to the widest audience without losing information that is critical to a large merged data set. The following transformations to the ﬂags (and values) in the merged data ﬁle were made: 1. ﬂag 0, 2, 9, no change to data or ﬂag 2. ﬂag 3, 4, 5 (questionable, bad, not reported), existing data values reset to NA and ﬂags to 9 Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ R. M. Key et al.: The CARINA data synthesis project: introduction and overview R. M. Key et al.: The CARINA data synthesis project: introduction and overview 4.3.6 Carbon calculations All of the various carbon calculations in CARINA used the MATLAB® translation (van Heuven et al, 2009; http://cdiac. esd.ornl.gov/oceans/co2rprt.html) of the code originally de- veloped by Lewis and Wallace (1998, same link). CARINA used the same constants used for GLODAP (most impor- tantly, the Dickson and Millero (1987) reﬁt of Mehrbach et al. (1973), but see also van Heuven et al. (2009)). This decision is supported by signiﬁcant literature (e.g. Lee et al., 1996; Wanninkhof et al., 1998; McElligot et al., 1998; Millero et al., 2002; Mojica Prieto and Millero, 2002). Oth- ers have suggested diﬀerent constants and given new ﬁts to old data, but these studies were either vetted on a re- gional scale rather than globally or oﬀered only very min- imal improvement. The CARINA team carbon experts de- cided that the potential for minor improvement was less im- portant than being consistent with values calculated during GLODAP since data from the two collections will undoubt- edly be used together. extremely close together the function can give spurious re- sults. Consequently, the interpolated values generated with the Hermitian scheme were compared to values derived by simple linear interpolation. In cases where the Hermitian ap- proximation diﬀered from the linear approximation by more than 1%, the linear value was chosen. An example of this is shown in Fig. 3. Even these precautions will not cover all questionable interpolations, therefore, after the interpolation step was completed, the combined (measured + interpolated) parameters were checked and the obvious ﬂiers eliminated from the data set. This check was very crude with the result that the ﬁnal data set undoubtedly contains a few anomalous interpolated values. As an experiment, the data shown in Fig. 3 were also ﬁtted with spline, spline under tension, “csakm” (from the IMSL FORTRAN library; Virtual Numerics, Inc.), and “loess” (from the S-Plus library; see Cleveland and Devlin, 1988) functions. The ﬁrst 3 showed “ringing” equal to or worse than the Hermitian function. The “loess” ﬁt does not ring, but is overly smoothed. For this example an obvious “ﬁx” would be to average the two data points that are so close to each other (near 3000 dB) and use the average as input to the ﬁtting routine. Such an averaging scheme for data that are nearly co-located would be a good modiﬁcation to the in- terpolation software. 4.3.3 Salinity and miscellaneous corrections For CARINA we decided that a sample must have pressure and temperature to have any value. Basically, we assumed that if either of these values was missing then something had gone critically wrong with that sample. Consequently, if ei- ther temperature or pressure was missing, then all data for that sample bottle was set to NA and the ﬂags to 9. Fortu- nately, there were very few instances. Salinity data is also critical, however, the circumstances are diﬀerent. For CARINA we chose bottle salinity in pref- erence to CTD derived salinity. Some original data ﬁles contained bottle measurements only, others contained CTD salinity values only, others contained both, and many ﬁles had salinity values with the source not identiﬁed. When the source was not identiﬁed, we assumed that the values were bottle salinity. While this procedure has proven to be very reliable, it is not perfect. Unusual sample distributions combined with the nature of the ﬁtting function can generate anomalous val- ues. In particular for the CARINA cruises it was not un- common to have multiple samples at very similar pressures for a given station. This situation was virtually never encoun- tered with GLODAP sampling. The Hermite ﬁtting function is not prone to “ring”, however, when adjacent samples are Up to this point the two types of salinity data were both retained and stored separately. Here we made two assump- tions: ﬁrst that any CTD salinity was better than nothing and that any existing salinity was better than what could be in- terpolated. Both assumptions should usually be true even with uncorrected CTD salinity. Consequently, wherever bot- tle salinity was missing and a CTD salinity value existed, www.earth-syst-sci-data.net/2/105/2010/ Earth Syst. Sci. Data, 2, 105–121, 2010 R. M. Key et al.: The CARINA data synthesis project: introduction and overview 116 Pressure Oxygen (umol/kg) 2000 2200 2400 2600 2800 3000 3200 230 235 240 245 250 Measured Hermite Linear Figure 3. Illustration of interpolation. The black dots are measured data. The red boxes and blue x’s are interpolated values at the in- dicated pressures using the Hermitian and linear ﬁtting functions, respectively. Note that there are two measurements near 3000 dB and that these measured values are very nearly identical. The close proximity (in pressure) of these two measurements causes the Her- mitian ﬁtting function to “ring” thus producing the errant interpo- lated value near 3100 dB. 4.3.6 Carbon calculations The problem is that one has to deﬁne “close” and that deﬁnition will certainly vary with pressure and geographic location. If one only had 10 or 100 inter- 4.3.5 Basic calculations Figure 3. Illustration of interpolation. The black dots are measured data. The red boxes and blue x’s are interpolated values at the in- dicated pressures using the Hermitian and linear ﬁtting functions, respectively. Note that there are two measurements near 3000 dB and that these measured values are very nearly identical. The close proximity (in pressure) of these two measurements causes the Her- mitian ﬁtting function to “ring” thus producing the errant interpo- lated value near 3100 dB. In cases such as this, when the two ﬁtting functions produce results that diﬀer by more than 1%, the linear in- terpolation is used. For all the other cases shown the diﬀerence is less than 1% and the approximation from the Hermitian function is used. All of the interpolated points shown in this example pass the “maximum measured data separation distance” test described in the text and in Table 4. The existing data were used to calculate values for poten- tial temperature, potential density relative to 0, 1000, 2000, 3000 and 4000 dBar, and apparent oxygen utilization (AOU) using the same algorithms used for GLODAP. Additionally, sample depth was approximated for all samples using a sim- ple function based on pressure and latitude (in cases where only depth was available, pressure was approximated using a similar function). These parameters were added to each data ﬁle. 4.3.3 Salinity and miscellaneous corrections In cases such as this, when the two ﬁtting functions produce results that diﬀer by more than 1%, the linear in- terpolation is used. For all the other cases shown the diﬀerence is less than 1% and the approximation from the Hermitian function is used. All of the interpolated points shown in this example pass the “maximum measured data separation distance” test described in the text and in Table 4. polations then the interpolation procedure could be visually monitored, however, with more than 84 000 possible interpo- lations that was not practical. Therefore, the required soft- ware development and testing has been left as a future exer- cise. Pressure Oxygen (umol/kg) 2000 2200 2400 2600 2800 3000 3200 230 235 240 245 250 Measured Hermite Linear We are aware that myriad other interpolation algorithms exist. Only those mentioned were tested and we do not im- ply that the method used is the “best” (however one might choose to deﬁne best). We do feel that the interpolation is worthwhile and that the method used is both reasonable and adequate. In the end, the limits over which interpolation is allowed tend to be more important than the ﬁtting algorithm. 4.3.8 Data product parameter accuracy Stated simply, it is impossible to determine the general accu- racy of the various parameters included in the CARINA data products. Precision estimates could be calculated for various subsets of the data, however those results would have limited, if any, value. In lieu of such numbers, we investigated the “internal consistency” of the data products. Details of these estimates are given in Tanhua et al. (2010; Table 3). This exercise clearly demonstrated that the internal consistency of the data product was signiﬁcantly better than for the orig- inal data. Excluding oxygen and nutrient data (since there are no “standards”) the consistency values could loosely be interpreted as an upper limit of accuracy. This approxima- tion is an upper limit since some of the variance included in the internal consistency calculation is due to real change. Conversely, if the 2nd QC procedure removed real change signals rather than measurement bias, then the internal con- sistency calculation would imply that the data in the products are “better” than they really are. The development of CRM for the calibration of ALK and DIC was noted as one of the most important developments with carbon system measurements for GLODAP (Key et al., 2004). The same is true for CARINA. CRM are readily avail- able and reasonably priced. Production of a high quality ALK and/or DIC data set requires frequent CRM analysis. pH measurements were rarely made during the WOCE program and the few measurements that were made were not included in GLODAP. Rather in GLODAP, pH and DIC were used to calculate ALK. With the CARINA collection pH was frequently measured. Additionally, since GLODAP was completed the issue of ocean acidiﬁcation has attracted signiﬁcant attention. Finally, the spectrophotometric mea- surement technique has become common and is far superior to electrode based measurements. One result of this history is that reporting requirements for pH data were not previ- ously standardized. When CARINA began, the most ac- cepted scale for oceanographic measurements was the sea- water scale. During this project, however, agreement was ﬁnally reached that pH data should be reported on the total- hydrogen ion scale at some speciﬁed temperature (generally 25 ◦C). By the time this decision was made, it was too late to change all of the CARINA data sets. 5 Lessons learned Two things are clear. The CARINA project both beneﬁted from and improved upon GLODAP techniques. The most signiﬁcant improvements include development of software to automate much of the 2nd QC work and consequently be- ing able to carry out 2nd QC on a larger subset of the total parameter set. This software also allowed the CARINA team to derive either additive or multiplicative adjustment factors for the various parameters. Experience has shown that multi- plicative adjustments are superior to additive adjustments for oxygen and nutrients in particular (the additive nutrient ad- justments used in GLODAP occasionally generated negative near surface concentrations!). As with GLODAP, CARINA 2nd QC demonstrated that diﬀerent analytical techniques can yield diﬀerent results with respect to data adjustments. We believe that retaining human control is preferable to fully au- tomated analysis for data such as these. For GLODAP, Key et al. (2004) noted that the need for nutrient standards similar to the carbon CRMs. Progress has been made (Aoyama et al., 2008; Aminot and Kirkwood, 1995), but so far, the use of nutrient “CRMs” has not been generally adopted. Analysis of the CARINA data make it abundantly clear that this practice must stop. The commu- nity must adopt a set of CRMs and those “standards” should be used on every cruise. This change in methodology is ab- solutely critical if we are ever to understand subtle changes in nutrient distributions and stoichiometric ratios in a changing ocean environment. Certainly the most glaring shortcoming for many of the cruise data sets was that complete records were not retained with the data. Prior to the WOCE program in the 1990s ﬁ- nal cruise reports were not produced for many cruises. This was particularly prevalent when the cruise was manned by a single group from one institution. This situation was ex- acerbated by the fact that the data from most of the CA- RINA cruises were held exclusively in the collection of in- dividual scientists. By the time the data were released for inclusion in this data product many of the people who had made the measurements were no longer working in the ﬁeld. Fortunately, these practices are slowly ending. The CAR- BOOCEAN program requires that all funded projects report The development of a dedicated web site for the CARINA work was extremely helpful. 4.3.8 Data product parameter accuracy Consequently, all CARINA pH values (both in the cruise ﬁles and in the data products) are reported on the seawater scale at 25 ◦C. 4.3.7 Partial pressure The partial pressures of CFC-11, CFC-12, CFC-113, CCl4 and SF6 were calculated based on the solubility equations given by Warner et al. (1995), Bu and Warner (1995), Bullis- ter and Wisegarver (1998) and Bullister et al. (2002). The partial pressure values and fractional equilibrium relative to the atmosphere at sampling time were extremely useful in the 2nd QC procedures for these parameters (Steinfeldt et Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ 117 R. M. Key et al.: The CARINA data synthesis project: introduction and overview al., 2010). Note that the GLODAP data products included “simple” CFC ages rather than partial pressures. data within 2 years after the cruise. For CLIVAR, shipboard measurements are made public immediately and ﬁnal data are required within 6 months after the cruise (except for shore based measurements). This paradigm shift from “proprietary forever” to rapid public availability carries the risk that an- other scientist will publish data before the PI responsible for the data has a chance. This occurrence is, however, extremely rare. Rapid public scrutiny of data more commonly results in elimination of data errors and new collaborative research opportunities. Timely data reporting ensures that suﬃcient metadata can still be obtained if it is not originally provided. 4.3.8 Data product parameter accuracy www.earth-syst-sci-data.net/2/105/2010/ 6 Conclusions Investigation of more subtle signals such as the expected temporal increase in near surface DIC due to anthropogenic CO2 will require more careful analysis. The CARINA QC and adjustment procedures risk remov- ing real signals from the original data. Without a much larger and higher initial quality data set such removal would be impossible to detect. As others use these data for indepen- dent research projects additional information will be gained. However, temporal signals still exist in the data products. As an example Fig. 4 shows boxplots of near-surface (0–25 dB) nitrate and AOU (apparent oxygen utilization) data from the Nordic Seas region taken from the AMS data product. The data were taken after all adjustments had been applied. No interpolated values were included in this analysis. AOU was used rather than oxygen to remove the temperature depen- dence of oxygen solubility. It is abundantly clear that the seasonal cycle has not been removed from these data. A sim- ilar seasonal cycle exists for the near surface DIC data from this region, however, without removing the seasonal cycle, the expected anthropogenic increase is not readily apparent for these surface waters (it is visible in deep water). Detailed analyses are required to identify subtle signals. Such studies are planned, but not discussed here. each year is due to spatial variability. Even though this test is crude, the increasing concentration trend with time is clearly evident and statistically signiﬁcant at a very high conﬁdence level. The DIC increase rate derived from these combined data (0.33 µmole/kg/yr) is less than that derived from the near bottom data in the Irminger Sea time series (0.8 µmol/kg/yr; cruise #185). Again, detailed investigation will be required to determine if the diﬀerence in increase rate is real or due to the averaging incurred in the trend shown in Fig. 5. The next planned step is to merge CARINA with GLO- DAP. Tests show the two data products to be consistently calibrated. The merge is, however, non-trivial because of diﬀerences in the parameters included and various detail dif- ferences such as sample indexing. The seasonal signal demonstrated in Fig. 4 is so strong that it is not the most convincing demonstration that 2nd QC did not remove real signals from the data products. Figure 5 illustrates a much sterner test. Here, deep water DIC data from the same region as Fig. 4 are summarized by measure- ment year. 6 Conclusions Measured Data Only; Nordic Seas Regio The CARINA data products represent the work of hundreds of scientists. The project has now extended for a decade with the ﬁnal eﬀort requiring half that time. The original goal, to assemble a collection of European data that would be useful to study the inorganic carbon system in the North Atlantic Ocean, was signiﬁcantly expanded and, we believe, success- fully completed. Not only were the data assembled, but the most critical parameters were subjected to very careful anal- ysis to remove various data biases. An independent analy- sis of the CARINA data product would undoubtedly show that overall the data quality of CARINA is not as high as GLODAP. This was expected. The CARINA cruises cover a longer time interval and more importantly the cruises were primarily carried out by individual scientists operating in small groups rather than being the result of a globally orga- nized survey eﬀort. Regardless, the secondary quality control activities have resulted in a data product that is suﬃciently accurate for modern analyses including climate change is- sues. Equally important is the fact that CARINA both sup- plements and extends the global coverage provided by GLO- DAP. Chemical oceanographers now have a very nice data set covering the northern North Atlantic and Nordic Seas, the beginning of coverage for the Arctic Ocean, and signiﬁ- cantly more data for the Southern Ocean. Additionally, while the CARINA calibration techniques diﬀered somewhat from those of GLODAP, the two data sets are thought to be com- patible without alteration for large scale investigations. 0 5 10 15 Nitrate (umol/kg) 0 5 10 15 1 2 3 4 5 6 7 8 9 10 11 12 Month Nitrate (umol/kg) 1 2 3 4 5 6 7 8 9 10 11 12 Month Figure 4. These two boxplots were generated using measured val- ues from the AMS data product. The data selected are from the up- per 25 m of the Nordic Seas region. The box widths are proportional to the number of data included. Even though a substantial fraction of the data were adjusted as part of the 2nd QC work, the seasonal cycle in these two parameters is retained. The near surface DIC data from this region have the same trend. Similar analyses with other parameters and other regions demonstrate that the 2nd QC proce- dure has not “erased” strong temporal signals. 5 Lessons learned This site allowed team mem- bers to easily share data and ideas and provided a location to store all of the QC output and ﬁnal adjustment tables. Now that the project is ﬁnished all of the CARINA website mate- rials are being transferred to CDIAC for archive and public access. www.earth-syst-sci-data.net/2/105/2010/ Earth Syst. Sci. Data, 2, 105–121, 2010 R. M. Key et al.: The CARINA data synthesis project: introduction and overview CARINA AMS Data Measured Data Only; Nordic Seas Region 118 -40 0 20 40 1 2 3 4 5 6 7 8 9 10 11 12 Month AOU (umol/kg) CARINA AMS Data Measured Data Only; Nordic Seas Region 0 5 10 15 1 2 3 4 5 6 7 8 9 10 11 12 Month Nitrate (umol/kg) -40 0 20 40 1 2 3 4 5 6 7 8 9 10 11 12 Month AOU (umol/kg) y; g 0 5 10 15 1 2 3 4 5 6 7 8 9 10 11 12 Month Nitrate (umol/kg) Figure 4. These two boxplots were generated using measured val- ues from the AMS data product. The data selected are from the up- per 25 m of the Nordic Seas region. The box widths are proportional to the number of data included. Even though a substantial fraction of the data were adjusted as part of the 2nd QC work, the seasonal cycle in these two parameters is retained. The near surface DIC data from this region have the same trend. Similar analyses with other parameters and other regions demonstrate that the 2nd QC proce- dure has not “erased” strong temporal signals. Investigation of more subtle signals such as the expected temporal increase in near surface DIC due to anthropogenic CO2 will require more careful analysis. -40 0 20 40 1 2 3 4 5 6 7 8 9 10 11 12 Month AOU (umol/kg) CARINA AMS Data Measured Data Only; Nordic Seas Region -40 0 20 40 1 2 3 4 5 6 7 8 9 10 11 12 Month AOU (umol/kg) www.earth-syst-sci-data.net/2/105/2010/ References Aminot, A. and Kirkwood, D. S.: Report on the results of the ﬁfth ICES Intercomparison Exercise for Nutrients in Seawater, ICES Cooperative Research Report No. 213, 79 pp., 1995. Aoyama, M., Barwell-Clarke, J., Becker, S., Blum, M., Braga E. S., Coverly, S. C., Czobik, E., Dahllof, I., Dai, M. H., Donnell, G. O., Engelke, C., Gong, G. C., Hong, Gi-Hoon, Hydes, D. J., Jin, M. M., Kasai, H., Kerouel, R., Kiyomono, Y., Knockaert, M., Kress, N., Krogslund, K. A., Kumagai, M., Leterme, S., Li, Yarong, Masuda, S., Miyao, T., Moutin, T., Murata, A., Nagai, N., Nausch, G., Ngirchechol, M. K., Nybakk, A., Ogawa, H., van Ooijen, J., Ota, H., Pan, J. M., Payne, C., Pierre-Duplessix, O., Pujo-Pay, M., Raabe, T., Saito, K., Sato, K., Schmidt, C., Schuett, M., Shammon, T. M., Sun, J., Tanhua, T., White, L., Woodward, E. M. S., Worsfold, P., Yeats, P., Yoshimura, T., Youenou, A., and Zhang, J. Z.: 2006 Intercomparison Exercise for Reference Material for Nutrients in Seawater in a Seawater Matrix, Technical Reports of the Meteorological Research Insti- tute No. 58, 104 pp., 2008. chuett, M., Shammon, T. M., Sun, J., Tanhua, T., White, L., Figure 5. This boxplot shows deep water DIC measurements from the same region as Fig. 4. A signiﬁcant fraction of the spread in- dicated by each box is due to spatial variability. In spite of the crude nature of this summary, the average concentration increase over time is statistically signiﬁcant at a very high conﬁdence level. The increase rate derived here is only about half that found for the Irminger Basin alone. For this discussion the important point is that the secondary QC adjustments have not erased subtle large scale temporal signals. Figure 5. This boxplot shows deep water DIC measurements from the same region as Fig. 4. A signiﬁcant fraction of the spread in- dicated by each box is due to spatial variability. In spite of the crude nature of this summary, the average concentration increase over time is statistically signiﬁcant at a very high conﬁdence level. The increase rate derived here is only about half that found for the Irminger Basin alone. For this discussion the important point is that the secondary QC adjustments have not erased subtle large scale temporal signals. Bu, X. and Warner, M. J.: Solubility of chloroﬂuorocarbon 113 in water and seawater, Deep-Sea Res. Pt. I, 42(7), 1151–1161, 1995. Bullister, J. L. R. M. Key et al.: The CARINA data synthesis project: introduction and overview R. M. Key et al.: The CARINA data synthesis project: introduction and overview R. M. Key et al.: The CARINA data synthesis project: introduction and overview 119 2145 2150 2155 2160 2165 2170 1991 1993 1994 1996 1997 1998 2001 2002 2003 CARINA AMS Data Nordic Seas Region - Values Deeper than 2500m Year DIC (umol/kg) Average Increase = 0.33 +/- 0.04 micromole/kg/yr Figure 5. This boxplot shows deep water DIC measurements from the same region as Fig. 4. A signiﬁcant fraction of the spread in- dicated by each box is due to spatial variability. In spite of the crude nature of this summary, the average concentration increase over time is statistically signiﬁcant at a very high conﬁdence level. The increase rate derived here is only about half that found for the Irminger Basin alone. For this discussion the important point is that the secondary QC adjustments have not erased subtle large scale temporal signals. 2145 2150 2155 2160 2165 2170 1991 1993 1994 1996 1997 1998 2001 2002 2003 CARINA AMS Data Nordic Seas Region - Values Deeper than 2500m Year DIC (umol/kg) Average Increase = 0.33 +/- 0.04 micromole/kg/yr 6 Conclusions A signiﬁcant fraction of the data variability for www.earth-syst-sci-data.net/2/105/2010/ Earth Syst. Sci. Data, 2, 105–121, 2010 References and Wisegarver, D. P.: The solubility of carbon tetra- chloride in water and seawater, Deep-Sea Res. Pt. I, 45(8), 1285– 1302, 1998. Bullister, J. L., Wisegarver, D. P., and Menzia, F. A.: The solubility of sulfur hexaﬂuoride in water and seawater, Deep-Sea Res. Pt. I, 49(1), 175–187, 2002. Clayton, T. D. and Byrne, R. H.: Spectrophotometric seawater pH measurements: Total hydrogen ion concentration scale calibra- tion of m-cresol purple and at-sea results, Deep-Sea Res., 40, 2115–2129, 1993. Acknowledgements. The CARINA project was highly collab- orative, however, the participants only represent a small fraction of the scientists and ship’s crew members who were involved in generating the data. We have tried to acknowledge those people in the CST, but there are unintentional omissions. We consider the online version of the CST to be a dynamic document and will correct/add any such errors/omissions. The scope of this project would have been impossible without the generous support of the CARBOOCEAN contract that supported many of the CARINA participants and provided signiﬁcant travel support for Key and Kozyr. Additional support from the International Ocean Carbon Coordination Project IOCCP and the Hanse Institute for Advanced Study (HWK Delmenhorst, Germany) are gratefully acknowledged. Cleveland, W. S. and Devlin, S. J.: Locally weighted regression: An approach to regression analysis by local ﬁtting, J. Am. Statis. Assoc., 83, 596–610, 1988. Dickson, A.: The ocean carbon dioxide system: Planning for qual- ity data, US JGOFS News, 2, 2, 1990. Dickson, A., Afghan, J. D., and. Anderson, G. C.: Reference mate- rials for oceanic CO2 analysis: A method for the certiﬁcation of total alkalinity, Mar. Chem., 80, 185–197, 2003. Dickson, A. G., Sabine, C. L. and Christian, J. R. (Eds.): Guide to best practices for ocean CO2 measurements. PICES Special Pub- lication 3, 191 pp., http://cdiac.esd.ornl.gov/oceans/Handbook 2007.html, 2007. Support: R. M. Key et al.: The CARINA data synthesis project: introduction and overview Chem., 55, 233–245, 1996. Lee, K., Tong, L. T., Millero, F. J., Sabine, C. L., Dickson, A. G., Goyet, C., Park, G.-H., Wanninkhof, R., Feely, R. A., and Key, R. M.: Global relationships of total alkalinity with salinity and tem- perature in surface waters of the world’s ocean, Geophys. Res. Lett., 33, L19605, doi:10.1029/2006GL027207, 2006. Orr, J. C., Maier-Reimer, E., Mikolajewicz, U., Monfray, P., Sarmiento, J. L., Toggweiler, J. R., Taylor, N. K., Palmer, J., Gruber, N., Sabine, C. L., Le Qu´er´e, C., Key, R. M., and Boutin, J.: Estimates of anthropogenic carbon uptake from four three- dimensional global ocean models, Global Biogeochem. Cy., 15, 43–60, 2001. Lewis, E. and Wallace, D. W. R.: Program Developed for CO2 Sys- tem Calculations. ORNL/CDIAC-105. Carbon Dioxide Informa- tion Analysis Center, Oak Ridge National Laboratory, US De- partment of Energy, Oak Ridge, Tennessee, 1998. Orr, J. C., Fabry, V. J., Aumont, O., Bopp, L., Doney, S. C., Feely, R. A., Gnanadesikan, A., Gruber, N., Ishida, A., Joos, F., Key, R. M., Lindsay, K., Maier-Reimer, E., Matear, R., Monfray, P. Mouchet, A., Najjar, R. G., Plattner, G.-K., Rodgers, K. B., Sabine, C. L., Sarmiento, J. L., Schlitzer, R., Slater, R. D., Tot- terdell, I. J., Weirig, M.-F., Yamanaka, Y., and Yool, A.: Anthro- pogenic ocean acidiﬁcation over the twenty-ﬁrst century and its impact on calcifying organisms, Nature, 437, 681–686, 2005. Levitus, S.: Climatological atlas of the world ocean, NOAA Profes- sional Paper #13, Rockville, MD., 191 pp., 1982. Matsumoto, K., Sarmiento, J. L., Key, R. M., Aumont, O., Bullister, J. L., Caldeira, K., Campin, J.-M., Doney, S. C., Drange, H., Dutay, J.-C., Follows, M., Gao, Y., Gnanadesikan, A., Gruber, N., Ishida, A., Joos, F., Lindsay, K., Maier-Reimer, E., Marshall, J. C., Matear, R. J., Monfray, P., Mouchet, A., Najjar, R., Plattner, G.-K., Schlitzer, R., Slater, R., Swathi, P. S., Totterdell, I. J., Weirig, M.-F., Yamanaka, Y., Yool, A., and Orr, J. C.: Evaluation of ocean carbon cycle models with data-based metrics, Geopys. Res. Lett., 31, L07303, doi:10.1029/2003GL018970, 2004. Peacock, S.: Debate over the ocean bomb radiocarbon sink: Closing the gap, Global Biogeochem. Cy., 18, GB2022, doi:10.1029/2003GB002211, 2004. Prinn, R. G., Weiss, R. F., Fraser, P. J. , Simmonds, P. G. , Cun- nold, D. M., Alyea, F. N., O’Doherty, S., Salameh, P., Miller, B. R., Huang, J., Wang, R. H. J., Hartley, D. E., Harth, C., Steele, L. P., Sturrock, G., Midgley, P. R. M. Key et al.: The CARINA data synthesis project: introduction and overview 120 Gruber, N.: Anthropogenic CO2 in the Atlantic Ocean, Global Bio- geochem. Cy., 12, 165–192, 1998. icz, R. N.: Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure, Limnol. Oceanogr., 18, 897–907, 1973. Johnson, G. C., Robbins, P. E., and Huﬀord, G. E.: Systematic adjustments of hydrographic sections for internal consistency, J. Atmosph. Ocean. Tech., 18, 1234–1244, 2001. MikaloﬀFletcher, S. E., Gruber, N., Jacobson, A. R., Doney, S. C., Dutkiewicz, S., Gerber, M., Follows, M., Joos, F., Lindsay, K., Menemenlis, D., Mouchet, A., Muller, S. A., and Sarmiento, Johnson, K. M., Dickson, A. G., Eischeid, G., Goyet, C., Guen- ther, P., Key, R. M., Millero, F. J., Purkerson, D., Sabine, C. L., Schottle, R. G., Wallace, D. W. R., Wilke, R. J., and Winn, C. D.: Coulometric total carbon dioxide analysis for marine studies: as- sessment of the quality of total inorganic carbon measurements made during the US Indian Ocean CO2 Survey 1994–1996, Mar. Chem., 63(1–2), 21–37, 1998. J. L.: Inverse estimates of anthropogenic CO2 uptake, transport, and storage by the ocean, Global Biogeochem. Cy., 20, GB2002, doi:10.1029/2005GB002530, 2006. MikaloﬀFletcher, S. E., Gruber, N., Jacobson, A. R., Doney, S. C., Dutkiewicz, S., Gerber, M., Follows, M., Joos, F., Lindsay, K., Menemenlis, D., Mouchet, A., Muller, S. A., and Sarmiento, J. L.: Inverse estimates of the oceanic sources and sinks of natu- ral CO2 and the implied oceanic carbon transport, Global Bio- geochem. Cy., 21, GB1010, doi:10.1029/2006GB002751, 2007. Kawase, M. and Sarmiento, J. L.: Nutrients in the Atlantic thermo- cline, J. Geophys. Res., 90, 8961–8976, 1985. Key, R. M., Kozyr, A., Sabine, C. L., Lee, K, Wanninkhof, R., Bullister, J. L., Feely, R. A., Millero, F. J., Mordy, C., and Peng, T.-H.: A global ocean carbon climatology: Results from Global Data Analysis Project (GLODAP), Global Biogeochem. Cy., 19, GB4031, doi:10.1029/2004GB002247, 2004. Millero, F. J., Pierrot, D., Lee, K., Wanninkhof, R., Feely, R., and Sabine, C. L.: 2 R. M. Key, and T. Takahashi, Dissociation con- stants for carbonic acid determined from ﬁeld measurements, Deep Sea Res. Pt. I, 49, 1705–1723, 2002. Mojica Prieto, F. J. and Millero, F.J.: The values of pK1 + pK2 for the dissociation of carbonic acid in seawater, Geochim. Cos- mochim. Acta, 66(14), 2529–2540, 2002. Lee, K., Millero, F. J., and Campbell, D. M.: The reliability of the thermodynamic constants for the dissociation of carbonic acid in seawater, Mar. Support: pp R. Key: NOAA grant NA08OAR4320752 and NA08OAR4310820 X. Lin: NOAA grant NA08OAR4310820 A. Olsen: Research Council of Norway grant 178167/S30 (A-CARB) T. Tanhua: EU project CarboOcean (Project 511176) A. Velo: EU FP6 CARBOOCEAN Integrated Project, Contract no. 511176; PGIDIT05OXIC40203PM Xunta de Galicia; CTM200627116E/MAR MEC pp R. Key: NOAA grant NA08OAR4320752 and NA08OAR4310820 X. Lin: NOAA grant NA08OAR4310820 A. Olsen: Research Council of Norway grant 178167/S30 (A-CARB) T. Tanhua: EU project CarboOcean (Project 511176) A. Velo: EU FP6 CARBOOCEAN Integrated Project, Contract no. 511176; PGIDIT05OXIC40203PM Xunta de Galicia; CTM200627116E/MAR MEC Feely, R. A., Sabine, C. L., Lee, K., Berelson, W., Kleypas, J., Fabry, V. J., and Millero, F. J.: Impact of anthropogenic CO2 on the CaCO3 system in the oceans, Science, 305, 362–366, 2004. Gnanadesikan, A., Dunne, J. P., Key, R. M., Matsumoto, K., Sarmiento, J. L., Slater, R. D., and Swathi, P. S.: Oceanic ven- tilation and biogeochemical cycling: Understanding the phys- ical mechanisms that produce realistic distributions of trac- ers annd productivity, Global Biogeochem. Cy., 18, GB4010, doi:10.1029/2003GB002097, 2004. Edited by: V. Gouretski Gouretski, V. V. and Janke, V.: Systematic errors as the cause for an apparent deep water property variability: Global analysis of the WOCE and historical hydrographic data, Prog. Oceanogr., 40, 337–402, 2001. Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ www.earth-syst-sci-data.net/2/105/2010/ R. M. Key et al.: The CARINA data synthesis project: introduction and overview R. M. Key et al.: The CARINA data synthesis project: introduction and overview 121 Oak Ridge National Laboratory, US Department of Energy, Oak Ridge, Tennessee, 110 pp., 2005. Oak Ridge National Laboratory, US Department of Energy, Oak Ridge, Tennessee, 110 pp., 2005. van Heuven, S., Pierrot, D., Lewis, E., and Wallace, D. W. R.: MATLAB Program Developed for CO2 System Calcula- tions. ORNL/CDIAC-105b. Carbon Dioxide Information Anal- ysis Center, Oak Ridge National Laboratory, US Department of Energy, Oak Ridge, Tennessee, http://cdiac.ornl.gov/ftp/co2sys/ CO2SYS calc MATLAB/, 2009. Sarmiento, J. L., Simeon, J., Gnanadesikan, A., Gruber, N., Key, R. M., and Schlitzer, R.: Deep ocean biogeochemistry of sili- cic acid and nitrate, Global Biogeochem. Cy., 21, GB1S90, doi:10.1029/2006GB002720, 2007. Steinfeldt, R., Tanhua, T., Bullister, J. L., Key, R. M., Rhein, M., and Khler, J.: Atlantic CFC data in CARINA, Earth Syst. Sci. Data, 2, 1–15, 2010, http://www.earth-syst-sci-data.net/2/1/2010/. V´azquez-Rodr´ıguez, M., Touratier, F., Lo Monaco, C., Waugh, D. W., Padin, X. A., Bellerby, R. G. J., Goyet, C., Metzl, N., R´ıos, A. F., and P´erez, F. F.: Anthropogenic carbon distributions in the Atlantic Ocean: data-based estimates from the Arctic to the Antarctic, Biogeosciences, 6, 439–451, 2009, http://www.biogeosciences.net/6/439/2009/. Sweeney, C., Gloor, E., Jacobson, A. J., Key, R. M., McKin- ley, G., Sarmiento, J. L., and Wanninkhof, R.: Con- straining global air-sea exchange for CO2 with recent bomb 14C measurements, Global Biogeochem. Cy., 21, GB2015, doi:10.1029/2006BG002784, 2007. Velo, A., P´erez, F. F., Lin, X., Key, R. M., Tanhua, T., de la Paz, M., van Heuven, S., Jutterstr¨om, S., and R´ıos, A. F.: CARINA data synthesis project: pH data scale uniﬁcation and cruise ad- justments, Earth Syst. Sci. Data Discuss., 2, 421–475, 2009, http://www.earth-syst-sci-data-discuss.net/2/421/2009/. Swift, J.: A guide to submitting CTD/hydrographic/tracer data and associated documentation to the CLIVAR and car- bon hydrographic data oﬃce, ver. 4/22/08, UCSD Scripps Institution of Oceanography, http://whpo.ucsd.edu/CCHDO DataSubmitGuide.pdf, 37 pp., 2008. Wanninkhof, R., Lewis, E., Feely, R. A., and Millero, F. J.: The optimal carbonate dissociation constants for determining sur- face water pCO from alkalinity and total inorganic carbon, Mar. Chem., 65, 291–301, 1998. Tanhua, T.: MATLAB program developed for secondary qual- ity control of hydrographic data, http://cdiac.ornl.gov/ftp/oceans/ CARINA/CARINA MATLAB/, Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, US Depart- ment of Energy, Oak Ridge, Tennessee, 2009. Warner, M. J. and Weiss, R. F.: Solubilities of chloroﬂuorocarbons 11 and 12 in water and seawater, Deep Sea Res. Pt. I, 32(12), 1485–1497, 1985. Waugh, D. W., Hall, T. M., McNeil, B. Earth Syst. Sci. Data, 2, 105–121, 2010 R. M. Key et al.: The CARINA data synthesis project: introduction and overview M., and McCulloch, A.: A history of chemically and radiatively important gases in air deduced from ALE/GAGE/AGAGE, J. Geophys. Res., 105, 17751–17792, 2000. Matsumoto, K.: Radiocarbon-based circulation age of the world oceans, J. Geophys. Res., 112, C09004, doi:10.1029/2007JC004095, 2007. McElligott, S., Byrne, R. H., Lee, K., Wanninkhof, R., Millero, F. J., and Feely, R. A.: Discrete water column measurements of CO2 fugacity and pHT in seawater: A comparison of direct measurements and thermodynamic calculations, Mar. Chem., 60, 63–73, 1998. Roussenov, V., Williams, R. G., Follows, M. J., and Key, R. M.: Role of bottom water transport and diapycnic mixing in deter- mining the radiocarbon distribution in the Paciﬁc, J. Geophys. Res., 109, C06015, doi:10.1029/2003JC002188, 2004. McNeil, B. I., Matear, R. J., Key, R. M., Bullister, J. L., and Sarmiento, J. L.: Anthropogenic CO2 uptake by the ocean based on the global chloroﬂuorocarbon data set, Science, 299, 235– 239, 2003. Sabine, C. L., Feely, R. A., Gruber, N., Key, R. M., Lee, K., Bullis- ter, J. L., Wanninkhof, R., Wong, C. S., Wallace, D. W. R., Tilbrook, B., Millero, F. J., Peng, T.-H., Kozyr, A., Ono, T., and Rios, A. F.: The oceanic sink for anthropogenic CO2, Science, 305, 367–370, 2004. McNeil, B. I., Metzl, N., Key, R. M., Matear, R. J., and Cor- biere, A.: An Empirical Estimate of the Southern Ocean air-sea CO2 ﬂux, Global Biogeochem. Cy., 21, GB3011, doi:10.1029/2007GB002991, 2007. Sabine, C. L., Key, R. M., Kozyr, A., Feely, R. A., Wanninkhof, R., Millero, F. J., Peng, T.-H., Bullister, J. L., and Lee, K.: Global Ocean Data Analysis Project: Results and Data. ORNL/CDIAC- 145, NDP-083, Carbon Dioxide Information Analysis Center, Mehrbach, C., Culberson, C. H., Hawley, J. E., and Pytkow- Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/ www.earth-syst-sci-data.net/2/105/2010/ R. M. Key et al.: The CARINA data synthesis project: introduction and overview I., and Key, R.: Anthro- pogenic CO2 in the oceans estimated using transit-time distribu- tions, Tellus, 588, 376–389, 2006. Tanhua, T., van Heuven, S., Key, R. M., Velo, A., Olsen, A., and Schirnick, C.: Quality control procedures and methods of the CARINA database, Earth Syst. Sci. Data, 2, 35–49, 2010, http://www.earth-syst-sci-data.net/2/35/2010/. Earth Syst. Sci. Data, 2, 105–121, 2010 www.earth-syst-sci-data.net/2/105/2010/
W2168415829.txt	https://ccsenet.org/journal/index.php/ies/article/download/5888/4661	en		A Brief Study on the Implication of Constructivism Teaching Theory on Classroom Teaching Reform in Basic Education	International education studies	2,010	cc-by	2,047	International Education Studies Vol. 3, No. 2; May 2010 A Brief Study on the Implication of Constructivism Teaching Theory on Classroom Teaching Reform in Basic Education Qiong Jia Normal College, Shihezi University, Shihezi 832003, China E-mail: cherry_0203@163.com Abstract Constructivism learning theory is the further development as behaviorism arrives at cognitivism. According to its teaching theory: knowledge is uncertain; the learning process of knowledge is also the construction process of knowledge; students are the main body of learning activity and they construct knowledge on their own initiatives; teachers are the helpers and the drivers for students constructing knowledge. These views generate more implications for China’s teaching reform, what affect the reform of learning theory and teaching theory in a sense and turn into the theoretical base for China’s education reform. This paper tries to probe into the implication of constructivism teaching theory on China’s basic education. Keywords: Constructivism, Learning theory, Basic education, Implication Constructivism, as a new cognitive theory, has been introduced in China since late 80s and early 90s in 20th century, exerting a significant effect on the education field. The development from behaviorism to cognitivism to constructivism is not only a leap of epistemology and a progress of learning psychology, and also a revolution of traditional education. Constructivism’s profound and unique recognition to human learning make the traditional teaching thought change thoroughly, generating a deep effect on teaching practice. In recent years, Chinese scholars introduce the constructivism theory sufficiently and make systematic studies, producing amounts of rich research fruits. The author thinks that the constructivism teaching theory has an important guiding effect on China’s basic education reform. This paper tends to probe into the implication of constructivism teaching theory on China’s basic education and teaching. 1. The origin of constructivism Any theory has its basis and background. Constructivism learning theory is not an exception. Its origins mainly include philosophy and psychology. 1.1 The philosophy origin Constructivism, as a new thought, is a new learning philosophy. Some people agree that the first constructivist is Socrates. His “hippcrates” is a successful model for constructivism teaching. Kant’s studies on the integration of rationalism and empiricism indicate kind of constructivism. In his opinion, the subject can not open toward the external world directly. Only by the constructed-internally principal cognitive rules, the subject can organize experiences and develop knowledge. Later, along with the transfer of structuralism methodology toward post-structuralism, the absolute status of rationalism is broken further. Constructivism learning theory comes into being from the development of cognitivism, turning into a new learning theory. 1.2 The psychology origin In perspective of psychology, the first who contribute a lot to the development of constructivism thought and apply it to classes and students’ learning and development are Dewey, Piaget, and Vygotsky. Dewey advances the experimental learning theory, emphasizing on the generation and reform of experiences. Switzerland Famous psychologist Piaget is taken as the forerunner of modern constructivism. In 1972 Piaget puts forward the concepts of “assimilation” and “conformability”. In his opinion, the recognition means the subject affects the object by his unique recognition structure, achieving a balance between the subject and the object. Based on psychological ideas, Piaget thinks that all knowledge has the external origin and students’ cognitive development is actualized naturally in the process of receiving knowledge. During 70s and 80s in 20th century, Russia excellent psychologist Vygotsky founds the base for the formation of modern constructivism. According to his idea, learning is a social construction. Individual learning is under certain history and social background. 2. Basic ideas of constructivism learning theory 2.1 On knowledge Knowledge is only an explanation and an assumption but not the final answer for all questions. In contrast, it will be discarded along with the human process and new assumption will appear. Besides, knowledge can not 197 International Education Studies www.ccsenet.org/ies summarize the world rules precisely. In other words, we can not apply knowledge to certain problems directly. We have to analyze certain issue based on practical conditions. In addition, constructivists agree that knowledge can not live on its physical form and out of specific entity. Although language and signals endow knowledge with certain forms, it does not mean learners have same understandings toward these statements, just as one hundred people will have one hundred different understandings toward Hamlet. These understandings are based on individual learners’ experiences and backgrounds, what is determined by specific learning experience. 2.2 On learning Learning is the process that individuals construct their cognitive structures. “Construction” is a kind of initiative, conscious, and self-organized recognition way. It is the “interaction” between the subject and the object. The learning process is the construction of knowledge. Learning is an initiative construction and the generation of meanings. This process is completed by the interaction of learners’ old and new knowledge. In other words, pure external stimulation is meaningless. Only when learners code, process, and construct their unique understandings based on their previous experiences, can it be real learning. 2.3 On students Students enter classrooms with their rich previous experiences. They hold their opinions toward daily life and even universal issues. Even though they do not know some issues and have no experiences, they may form special explanations and assumptions based on previous experiences and cognitive abilities as some issues appear. That is not illogical guess but logical assumption based on previous experiences. Therefore, teaching should take students’ previous knowledge and experience as the growth point of new knowledge, and introduce students to generate new knowledge from the former. 2.4 On teachers As we emphasize on the students as the subjects, we should change the role of teachers, from the initiator and the indoctrinator into the helper and the driver for students constructing meanings initiatively. In other words, teachers should be the designer of teaching environment, the guider for students’ learning, and the academic consultant for students. It discards the traditional teaching mode that takes teachers as the center, which merely focuses on conveying knowledge, regarding students as the object for receiving knowledge. The new teaching mode takes students as the center, under the guidance of teachers. Teachers organize and guide the whole teaching process. 3. Implication of constructivism theory on classroom teaching in basic education Based on analysis above, we know that constructivism learning theory puts forward new explanations for learning and teaching. According to this theory, students are the subject in teaching. Teachers should offer more humanism cares for students and create a favorable teaching environment for students. It emphasizes on the initiatives and the interaction in teaching. Students should focus on exploration learning and cooperative learning based on previous knowledge and experiences by means of interactive actions. By this way, students can improve their cognitive ability continuously. Teachers can help students form the positive technique, the affection, the attitude, and the habit in learning. All these innovative ideas constitute the complete constructivism teaching theory system, which contribute a lot to modern teaching theory. Constructivism teaching theory has important meanings for China’s basic education that focuses on improving students’ comprehensive quality. (1) Reform the teaching views. Pro. Lan Ye has said: “Today’s teaching reform should change not only the teaching theory but also the teachers’ teaching views, together with teachers’ daily teaching activities.” In the traditional teaching mode, teachers explain, analyze, and introduce too much. Students receive knowledge passively. They have few time and space for thinking. The traditional mode neglects students’ practicing process and just input fixed things into students. As a result, students will refuse to think by themselves. This mode taking teachers’ teaching as the center is not good for developing students’ potentials and their development. In contrast, the constructivism agrees that learning is initiative and positive. In learning, students are the subject of teaching. Without students’ initiative participation, the learning is meaningless. In teaching, teachers are the subject of teaching. Teachers mean to inspire and guide students to learn knowledge effectively. (2) Emphasize on cooperation and communication and train students’ cooperative consciousness. In traditional teaching, the cooperation between teachers and students, students and students are neglects. Constructivism agrees that knowledge is the social construction of individuals and others by negotiation. Therefore, in the process of constructing knowledge, students must cooperate and communicate with others. In a cooperation and communication environment, students can enlarge their views, instead of receiving knowledge passively. It can help them to build up their own knowledge system, cultivate their innovative spirit and problem-solving ability. 198 International Education Studies Vol. 3, No. 2; May 2010 Meanwhile, it can make them respect others’ opinions and ideas. (3) Students’ previous knowledge, experiences, thinking mode, learning habits and methods are the start for teaching. Modern cognitive psychology shows that learning is an interactive process of new knowledge and old knowledge. The former knowledge and techniques stored in the memory system are important internal conditions for generating study activities. Ausubel, in perspective of meaning study, gives priority to students’ original knowledge in learning. He said: “If I conclude all educational psychology into one principle, it will be that the only important factor affecting study is what learners have known. Knowing about this point, the teaching will be effective.” Constructivism learning theory illustrates this point deeply. Learning is an initiative process based on students’ previous knowledge and experiences. Before the class, teachers should prepare for two fields. Firstly, compare and analyze the curriculum standards and textbooks, and confirm the teaching targets. Make best use of textbooks flexibly. Secondly, analyze and understand students, and know students’ previous knowledge and experiences. Therefore, we advocate that teachers should take students’ previous knowledge and experiences as the growth point for new knowledge during the process of teaching. Guide students to produce new knowledge and experience from original ones, achieving the mutual connection of new and old knowledge. (4) Teaching should be changed from authoritative conducting to equal association and communication. In traditional teaching, teachers are at a sovereign authoritative position. They do not respect or care for students as necessary, which leads to a gap between teachers and students, and even stimulates conflicts. Ancient men said: “People with more knowledge are teachers and with morals are examples.” Teachers should give students more respects, one more chance, and one more trust, building a kind and harmonious relations. Teachers should treat students with an equal, kind and considerate attitude, supplying a loose environment for students’ learning. Then, students can learn to communicate with teachers and response to teachers’ praises and criticizes, which can greatly improve the relations between teachers and students. Construct a democratic and equal teacher-student relation. (5) Create a better teaching environment. Constructivism learning theory requires to study in real or semi-real environment, emphasizing on non-structural knowledge or students’ previous experiences. Traditional teaching focuses on structural knowledge but not non-structural knowledge and students’ life experiences. Therefore, in teaching, we should create an interactive learning environment for students by modern teaching media, helping students to explore and discover new knowledge. Students should solve problems by themselves. Build a bridge between new knowledge and students’ previous knowledge, which can improve students’ ability of solving problems. To sum up, constructivism teaching theory has already turned into a hot inside and outside, which advances many new thoughts and views. We should judge and absorb them reasonably, dealing with sorts of relations properly, including the subjectivity and objectivity of knowledge, the transfer and construction of knowledge in learning, teachers’ guidance, and students as the center, and applying relevant theory to basic education teaching properly. References Chen, Qi & Zhang, Jianwei. (1998). An analysis on the Key of Constructivism Instructional Theory. Journal of East China Normal University: Educational Sciences. No.1. Ding, Yuankun. (2003). Constructivism teaching theory and the implication. Journal of Shenyang College of Education. No.3. p165-167. Kong, Xiansui. (2002). The implications of constructivism theory on teaching. Research on Education Tsinghua University. No.1. p132. Ma, Guolin. (2005). Teaching study on the reform of primary education. Future and Development. No.4. p63-64. Yang, Chunhong & Zhang, Chunsheng. (1998). Constructivism and reform of basic education. Journal of Hebei Normal University: Education Studies. No.3. p38. Zheng, Yuxin. Wang, Changpei & Zhang, Guojie. (1994). Views of constructivism theory. Journal of Mathematics Education. No.5. p9-14. Zhong, Zhixian. (2002). Core thoughts of constructivism teaching idea. China Educational Technology. No.2. p17-18. Zou, Yanchun. (2002). On the constructivism learning theory’s development origins and logic starts. Studies in Foreign Education. No.5. 199
https://openalex.org/W4318706061	https://breast-cancer-research.biomedcentral.com/counter/pdf/10.1186/s13058-023-01606-7	English	null	Expression of hormone receptors is associated with specific immunological profiles of the breast cancer microenvironment	Breast cancer research	2,023	cc-by	9,352	Hanamura et al. Breast Cancer Research (2023) 25:13 https://doi.org/10.1186/s13058-023-01606-7 Hanamura et al. Breast Cancer Research (2023) 25:13 https://doi.org/10.1186/s13058-023-01606-7 Breast Cancer Research Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Expression of hormone receptors is associated with specific immunological profiles of the breast cancer microenvironment Toru Hanamura1, Shigehisa Kitano2, Hiroshi Kagamu3, Makiko Yamashita2, Mayako Terao1, Takuho Okamura1, Nobue Kumaki4, Katsuto Hozumi5, Takayuki Iwamoto6, Chikako Honda7, Sasagu Kurozumi8 and Naoki Niikura1* Abstract Background Elucidating the unique immunoregulatory mechanisms in breast cancer microenvironment may help develop new therapeutic strategies. Some studies have suggested that hormone receptors also have immune regula- tory functions, but their mechanisms are not fully understood. In this study, we have comprehensively analyzed the relationship between the expressions of estrogen (ER), progesterone (PgR), and androgen receptors (AR), and the immunological profile in breast cancer. Methods Using publicly available gene expression profile datasets, METABRIC and SCAN-B, the associations between the expressions of hormone receptors and the immune cell compositions in breast cancer tissue, estimated by CIBERSORTx algorithm, were analyzed. We histologically evaluated tumor-infiltrating lymphocytes (hTIL), PD-L1 (hPD- L1) expression, and the infiltration of 11 types of immune cells by flow cytometry (FCM) for 45 breast cancer tissue samples. The relationships between them and the expressions of ER, PgR, and AR of tumor tissues, evaluated immu- nohistochemically, were analyzed. Results Expressions of ESR1, PGR, and AR were negatively correlated with overall immune composition. Expressions of ER and AR, but not that of PgR, were inversely associated with hTIL and hPD-L1 expression. FCM analysis showed that the expressions of ER and AR, but not that of PgR, were associated with decreased total leukocyte infiltration. Both CIBERSORTx and FCM analysis showed that ER expression was associated with reduced infiltration of mac- rophages and CD4+ T cells and that of AR with reduced macrophage infiltration. Conclusion Hormone receptor expression correlates with specific immunological profiles in the breast cancer micro- environment both at the gene and protein expression levels. Conclusion Hormone receptor expression correlates with specific immunological profiles in the breast cancer micro- environment both at the gene and protein expression levels. Keywords Breast cancer, Estrogen receptor, Progesterone receptor, Androgen receptor, Tumor immunity, Microenvironment, Immune cell composition Correspondence: Naoki Niikura niikura@is.icc.u-tokai.ac.jp Full list of author information is available at the end of the article Correspondence: Naoki Niikura niikura@is.icc.u-tokai.ac.jp Full list of author information is available at the end of the article Correspondence: Naoki Niikura niikura@is.icc.u-tokai.ac.jp Full list of author information is available at the end of the article Background Breast cancer is the most commonly occurring can- cer in women. Despite recent advances in multimodal treatment, advanced and recurrent cases are challeng- ing to cure [1], and the urgent need to develop innova- tive treatment strategies has to be addressed. Owing to the development of immune checkpoint inhibitors (ICIs) that reinvigorate the adaptive immune response in the tumor microenvironment and the successful application of these ICIs in various neoplasms, tumor immunology has recently gained renewed interest across multiple can- cers [2, 3]. ICIs have also been applied for breast cancer treatment, but their effectiveness is limited, likely due to the immunosuppressive tumor microenvironment [4–9]. Elucidating the unique immunomodulatory mechanisms of the breast cancer microenvironment will provide sig- nificant insights into the development of new therapeutic strategies. Hormone dependency is one of the prominent bio- logical features of breast cancer. Approximately 70% of breast cancer cells express the estrogen receptor (ER), resulting in ER-dependent growth of breast cancer [1]. Therefore, treatment strategies that inhibit ER function are frequently used as postoperative adjuvant therapy for patients with early-stage breast cancer and as sys- temic therapy for metastatic cases [1]. The progesterone receptor (PgR) is clinically considered a complementary marker of hormone dependency in breast cancer [1, 10] because it is driven partly, but not exclusively, by ER- mediated transcriptional events [11, 12]. PgR is a binding partner and major modifier of ER-mediated processes, suggesting its additional role in breast cancer other than its identification as an ER activity marker [13, 14]. The androgen receptor (AR) is a nuclear transcription factor with a diverse range of biological actions, mainly in the development and maintenance of the male reproductive system [15]. It is widely expressed in all breast cancer subtypes to varying extents, with approximately 60–80% of the cases being AR-positive [16–18]. Furthermore, according to The Human Protein Atlas, AR expression, at both gene and protein levels, is second-highest in breast cancer, after prostate cancer, among various malignan- cies [19]. Although the function of AR in breast cancer depends on the tumor subtype, treatment, and other factors, it is suggested to have a tumor-promoting role [20–22], thereby attracting attention as a new therapeutic target for breast cancer treatment [23, 24]. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hanamura et al. Breast Cancer Research (2023) 25:13 Hanamura et al. Breast Cancer Research (2023) 25:13 Page 2 of 13 interfering with human leukocyte antigen-II expression in ER-positive breast cancer cell lines [27]. These findings suggest that estrogen signaling in the tumor microenvi- ronment regulates anti-tumor immunity [2]. In agree- ment with this, hormone receptor-positive breast cancer is characterized by low infiltration of tumor-infiltrating lymphocytes (TILs) and minimal response to ICIs [5, 9, 28–32]. To the best of our knowledge, the immuno- logical function of PgR in breast cancer has not yet been reported. However, limited studies have shown a rela- tionship between PgR expression and tumor immunity in breast cancer; PgR expression is inversely associated with programmed death-ligand 1 (PD-L1) expression in epithelial cells or the stroma and the infiltration of CD8+ T and CD20+ B cells [9, 33, 34]. Further, immune regula- tory functions of AR signals have been demonstrated via in vivo models of various autoimmune diseases and some malignancies [35]. Moreover, in breast cancer, AR expres- sion is inversely correlated with immune cell infiltration and cytotoxic immune activity, suggesting an immuno- suppressive effect of AR signals [36–38]. Background f Despite fragmentary evidence on sex steroid hormone signals and tumor immunity, the interactions between hormone receptors and immune cells are not well under- stood because of the complexity of the immune milieu in the breast cancer microenvironment and limited reports on systematic evaluation of immune cell composition in breast cancer tissue [39]. In this study, we systematically analyzed the relationship between the expression of sex steroid hormone receptors such as ER, PgR, and AR and the immunological profiles of breast cancer tissues. Our results demonstrated that hormone receptor expression, at both gene and protein levels, correlates with specific immunological profiles of the breast cancer microen- vironment, strongly suggesting their direct or indirect immunomodulatory role. CIBERSORTx ll Immune cell composition in breast cancer tissue was determined from gene expression profiles using a bioin- formatics algorithm, CIBERSORTx: https://cibersortx. stanford.edu/ (accessed on 25/2/2022) [44]. Briefly, “LM22” representing the profiling of 22 functionally defined human immune cell types [45] was applied as a signature matrix file. The non-log-transformed gene expression data from METABRIC and SCAN-B were applied to the mixture file. The program was run in the absolute mode with 100 permutations. A B-mode batch correction was applied, and quantile normalization was set as disabled. Absolute scores representing the over- all immune content and absolute abundance of each immune cell fraction in the mixture were produced by the algorithm. Cases with a CIBERSORTx p-value < 0.05 were filtered and selected for subsequent analysis. Abso- lute scores and absolute abundance of each cell type in a mixture were used for correlation analysis with the expression values of the indicated genes that were log2-transformed. Gene set enrichment analysis (GSEA)h The gene expression values in the METABRIC dataset were log2-transformed before performing GSEA [46, 47]. Hallmark gene set collections (50 gene sets) representing specific well-defined biological states or processes were obtained from MsigDB v7.1: https://www.gsea-msigdb. org/gsea/msigdb/ (accessed on 13/5/2020) and applied to GSEA using GSEA software v4.0: https://www.gsea- msigdb.org/gsea/msigdb/ (accessed on 21/11/2019). While performing GSEA, the permutations were set at 1000 with phenotype as the permutation type. Expression values of the indicated genes were used as phenotype labels, and Pearson’s correlation was set as the metric for ranking genes. Thresholds for nominal p-value and false discovery rate (FDR) q-value were set at < 0.05 and < 0.25, respectively. According to user guidelines, the SCAN-B dataset was not applied to GSEA because of the incom- patible normalization method used for this analysis. Histological evaluation of hormone receptors and tumor immunity‑related biomarkers y Rabbit monoclonal antibodies for ER (SP1) and PgR (1E2) were purchased from Ventana Medical Systems Japan Inc. (Tokyo, Japan) and for AR (AR27) from Leica Biosystems Inc. (Wetzlar, Germany). The immunohis- tochemistry (IHC) staining was performed using the VENTANA BenchMark ULTRA automated IHC device (Roche Diagnostics, Basel, Switzerland) for ER and PgR and BOND-III automated IHC device (Leica Biosystems Inc.; Wetzlar, Germany) for AR. The antigen–antibody complex was visualized using diaminobenzidine and counterstained with hematoxylin. The nuclear staining of ER, PgR, and AR in carcinoma cells was counted, and the percentage of immunoreactive cells was determined. ER and PgR were determined as positive when nuclear staining-positive cells were ≥ 10%. [48]. However, the accepted cutoff value for AR expression is not known; since the median value of nuclear staining-positive cells for AR was 60% in the present study, AR was considered positive at ≥ 60%. We diverted the data from histologi- cal analysis of the expression of TIL and PD-L1 from our previous study, referred to as hTIL and hPD-L1, respec- tively [39]. According to the International TILs Working Group guidelines [49], the percentages of TILs in stro- mal tissue sections stained with hematoxylin and eosin (H&E) were evaluated and categorized into three grades: low (0–10%), intermediate (10–40%), and high (40–90%). PD-L1 expression was assessed by IHC using an anti-PD- L1 antibody (SP142; Spring Bioscience, Pleasanton, CA, USA). Tumors with ≥ 1% immune cells showing cyto- plasmic and/or membrane PD-L1 staining were deter- mined to be PD-L1 positive [50]. A previous study report accounted that in a case, hTIL and hPD-L1 could not be evaluated because the tumor tissue was insufficient for analysis. Materials and methods Gene expression profile datasets Two publicly available gene expression profile datasets of patients with breast cancer used in this study were the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) [40, 41] cohort (n = 1904) and the Sweden Cancerome Analysis Network-Breast (SCAN-B) [42, 43] cohort (n = 3273). Gene expression data of METABRIC and SCAN-B, generated by micro- array and RNA-sequencing, were downloaded from the cBioPortal: https://www.cbioportal.org/ (accessed on 20/2/2019) and Gene Expression Omnibus: https://www. ncbi.nlm.nih.gov/geo/ (accessed on 16/6/2019), respec- tively. The inclusion criteria and clinicopathological information for each cohort have been provided in the original papers. The METABRIC cohort included patients Some recent studies have suggested that sex steroid hormones and their receptor signaling have immune regulatory functions. In vitro studies have shown that estrogen can expand the regulatory T-cell fraction and reduce the function of antigen-presenting cells [25, 26]. In addition, estrogen can promote immune tolerance by Page 3 of 13 Hanamura et al. Breast Cancer Research (2023) 25:13 and an association between histologically assessed expression of TIL and PD-L1 and the immunological profile of the tumor microenvironment was reported [39]. The inclusion criteria and patient characteristics have also been described previously [39], and this data- set was used for further analyses. Briefly, 47 breast cancer samples were obtained, regardless of clinicopathologi- cal factors or treatment histories, except for patients with distant metastases or complete clinical responses to neoadjuvant chemotherapy. None of the patients had received irradiation or endocrine therapy before surgery. Clinicopathological data were collected by reviewing the case records. Two cases were excluded from the analysis, and the reasons are mentioned in section "TIL prepara- tion/ FCM analysis". with primary invasive breast cancer, including < 1% meta- static breast cancer. However, information on the use of preoperative chemotherapy was not provided. On the contrary, the SCAN-B cohort consisted of patients with non-metastatic primary invasive breast cancer and included the cases of preoperative chemotherapy. Both cohorts included all breast cancer subtypes, and all sam- ples were obtained from primary lesions. Statistical analyses GraphPad Prism ver. 9.1.0 software was used for statisti- cal analyses and graph preparation. The gene expression and FCM data were tested using the D’Agostino–Pearson normality test, which showed non-normal distribution in almost all the datasets. Correlation analyses between groups were performed using Spearman’s rank correla- tion coefficient. \|r-value\|> 0.3 and a significant p-value was defined as a positive or negative correlation [52, 53]. The Mann–Whitney U test and Fisher’s exact test were used to compare continuous and categorical vari- ables between the two unpaired groups, respectively. The p-value < 0.05 was defined as statistically significant. In the previous study [39], the FCM data contained outli- ers. Here, although all analyses were performed without omitting outliers, we identified the outliers using the robust regression and outlier removal method, excluded them, and performed all statistical analyses to ensure the reliability of our analyses. Statistics with omitted outliers are shown in each figure along with the original data. Correlation of histologically assessed TIL and expression of PD‑L1 with protein expression of ER and AR The status of ER, PgR, AR, PD-L1 (hPD-L1), and TIL (hTIL) in breast cancer tissue was histologically evaluated by IHC and H&E staining using the samples obtained at our facility from 45 patients with breast cancer. A sum- mary of clinicopathological findings according to hor- mone receptor status is shown in Additional file 1: Tables S1–S3. The correlation between hormone receptor expression and immune-related biomarkers was further analyzed at the protein level. Expression of both hTIL and hPD-L1 showed a negative correlation with the pro- tein expression of ER and AR, but not with that of PgR, probably due to the small number of PgR-positive cases (Fig. 2a–f). Results TIL preparation/ FCM analysis FCM data from breast cancer tissue samples were obtained from a previously reported study, which con- tains the detailed method [39]. To perform the analysis, fresh breast cancer tissues were mechanically dissociated and filtered using a 70-micron cell strainer. From the fil- tered cell suspension, mononuclear cell components were separated by density-gradient centrifugation and sub- jected to FCM analysis. Samples stained with an antibody cocktail were detected using LSR II Fortessa with the flu- orescence-activated cell sorting Diva software (BD Bio- sciences). All analyses were performed using the FlowJo software v10.6.1 (BD Biosciences). The list of antibodies used, gating strategy, and definitions of immune cell frac- tions have been described in the previous report [39]. According to the staining profile of the surface antigen, evaluated by FCM, the cells were classified as follows: leukocytes, total T cells (total T), CD4+ T cells (CD4+ T), CD8+ T cells (CD8+ T), B cells (B), monocytes/mac- rophages (Mo/Mφ), non-classical monocytes (CD16+ Mo), myeloid-derived suppressor cells (MDSCs), den- dritic cells (DCs), myeloid dendritic cells (mDCs), natu- ral killer (NK) cells, minor NK cells, and natural killer T cells (NKT). As mentioned in section "Patients", two cases were excluded because they had a low number of living cells (count < 1000) in the FCM analysis of tumor tissue. Leukocyte density was defined as the count of total CD45+ cells per weight of tumor tissue (count/g), as described in a previous paper [51]. Similarly, we deter- mined the count of each immune cell fraction per weight of the tissue fragment (count/g). Gene expression levels of hormone receptors in breast cancer tissue Gene expression of estrogen receptor 1 (ESR1), PGR, and AR negatively correlated with the absolute score in the METABRIC cohort (r > − 0.3, p < 0.05); however, the negative correlation was weak for PGR in the SCAN-B cohort (r = − 0.284, p < 0.05) (Fig. 1a–f). ESR1 expression levels inversely correlated with Mφ M1 and M0, CD4 + T (memory activated), CD8+ T, and CD4+ T (memory resting) in at least one dataset (r < − 3, p < 0.05) (Fig. 1g). Likewise, PGR expression levels inversely correlated with Mφ M1 and M0 and CD4+ T (memory activated) in at least one dataset (r < − 3, p < 0.05) (Fig. 1h). Similarly, AR expression levels inversely correlated with Mφ M0 and M1 in at least one dataset (r < − 3, p < 0.05) (Fig. 1i). These results suggest that the expression of hormone receptors ESR1, PGR, and AR is inversely associated with the total immune content and the infiltration of specific immune cell fractions in the tumor tissue. Patients In a previous study, immune cell composition of breast cancer tissue was evaluated using flow cytometry (FCM) Page 4 of 13 Hanamura et al. Breast Cancer Research (2023) 25:13 Hanamura et al. Breast Cancer Research (2023) 25:13 Hanamura et al. Breast Cancer Research Association of hormone receptor status with leukocyte density and tumor‑infiltrating immune cells in breast cancer tissueh The relationship between each hormone receptor status, density of leukocytes, and each immune cell fraction in breast cancer tissue was analyzed. ER positivity was asso- ciated with decreased leukocyte density and reduced infiltration of total T, CD4+ T, Mo/Mφ, MDSC, DC, and mDC in breast cancer tissue (Fig. 3a–m). PgR positivity was associated with decreased infiltration of DC but not with leukocyte density or other immune cell fractions (Fig. 4a–m). AR positivity was associated with decreased leukocyte density and infiltration of CD4+ T, Mo/Mφ, CD16+ Mo, MDSC, DC, mDC, and minor NK cells (Fig. 5a–m). These results suggest a strong association of Hanamura et al. Breast Cancer Research (2023) 25:13 Hanamura et al. Breast Cancer Research Page 5 of 13 i b l l f h i Fig. 1 Association between gene expression levels of hormone receptors and immunological profile of the breast cancer tissue. a–f Scatterplots showing a correlation between the gene expression levels of estrogen receptor 1 (ESR1), progesterone receptor (PGR), and androgen receptor (AR), and absolute score estimated by CIBERSORTx. g–i Graphs showing the correlation coefficient (r-value) between indicated gene expression and the absolute amount of various immune cell fractions estimated by CIBERSORTx. Immune cell fraction data showing consistently significant p-values in the METABRIC and SCAN-B datasets have been displayed in ascending order of r-value Hanamura et al. Breast Cancer Research (2023) 25:13 Page 6 of 13 Fig. 2 Histological assessment of the expression of tumor-infiltrating lymphocytes (TILs) and programmed death-ligand 1(PD-L1) based on protein expression status of estrogen receptor (ER) and androgen receptor (AR). a–f Graphs showing the number of patients with positive or negative immune-related markers according to hormone receptor status. Fisher’s exact test was used to compare categorical variables between two groups; actual p-values are shown in the figure Fig. 2 Histological assessment of the expression of tumor-infiltrating lymphocytes (TILs) and programmed death-ligand 1(PD-L1) based on protein expression status of estrogen receptor (ER) and androgen receptor (AR). a–f Graphs showing the number of patients with positive or negative immune-related markers according to hormone receptor status. Fisher’s exact test was used to compare categorical variables between two groups; actual p-values are shown in the figure the expression of ER and AR with decreased infiltration of leukocytes and specific immune cell fractions such as CD4+ T and Mo/Mφ into breast cancer tissue. cells, while AR expression with reduced macrophage infiltration. Association of hormone receptor status with leukocyte density and tumor‑infiltrating immune cells in breast cancer tissueh These results were consistent at the gene and protein expression levels. The present analysis showed that gene expression of hormone receptors was inversely correlated to total immune cell infiltration into the tumor microenviron- ment (Fig. 1a–f). This was further verified by perform- ing GSEA on the METABRIC dataset, which analyzed the relationship between the expression levels of hor- mone receptors and hallmark gene set collection (50 gene sets) representing specific well-defined biological states or processes. Expression levels of ESR1, PGR, and AR showed a significant positive correlation with gene sets representing estrogen response, such as estrogen Discussionh This study systematically analyzed the relationship between hormone receptor expression (at both gene and protein levels) and the immunological profile of breast cancer tissues, including multiple immune cell fractions. Expression of ER and AR in breast cancer tissues was associated with a decreased infiltration of immune cells into the tumor microenvironment. More specifically, ER expression was associated with decreased infiltration of macrophages and CD4+ T Hanamura et al. Breast Cancer Research (2023) 25:13 Page 7 of 13 Fig. 3 Association between estrogen receptor (ER) status and subsets of tumor-infiltrating immune cells. a Total leukocyte density (count/g) in breast cancer tissue according to ER status. b–m Count of each immune cell fraction per unit weight of the tissue (count/g) according to ER status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p > 0.05 (not significant); p < 0.05; p < 0.01; p < 0.001 Fig. 3 Association between estrogen receptor (ER) status and subsets of tumor-infiltrating immune cells. a Total leukocyte density (count/g) in breast cancer tissue according to ER status. b–m Count of each immune cell fraction per unit weight of the tissue (count/g) according to ER status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p > 0.05 (not significant); p < 0.05; p < 0.01; p < 0.001 receptors, such as ESR1, PGR, and AR, correlate with immunosuppressive phenotypes of breast cancer. response early and estrogen response late (Additional file 1: Figure S1a–c). On the other hand, ESR1 and AR expression levels showed a significant negative cor- relation with multiple gene sets representing immu- nological processes such as inflammatory response, allograft rejection, complement, and interferon-gamma response (Additional file 1: Figure S1d, f). Likewise, a significant negative correlation was observed between PgR expression level and gene sets of inflammatory response, allograft rejection, and interferon-gamma response (Additional file 1: Figure S1e). These find- ings suggest that the gene expression levels of hormone Several preclinical studies have suggested that ER signaling may suppress the immune response of breast cancer [2, 25–27], and ER-positive breast cancers have shown reduced TIL infiltration and minimal response to ICI [5, 9, 28–32]. This is consistent with the present findings, suggesting an immunomodulatory function of ER. Discussionh Although numerous studies have analyzed the rela- tionship between a single immune cell lineage and hor- mone receptor expression [32, 54–57], few reports have systematically investigated the multiple immune cell Hanamura et al. Breast Cancer Research (2023) 25:13 Page 8 of 13 Fig. 4 Association between progesterone receptor (PgR) status and subsets of tumor-infiltrating immune cells. a Total leukocyte density (count/g) in breast cancer tissue according to PgR status. b–m Count of each immune cell fraction per unit weight of the tissue (count/g) according to PgR status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p > 0.05 (not significant); p < 0.05; p < 0.01; p < 0.001 Fig. 4 Association between progesterone receptor (PgR) status and subsets of tumor-infiltrating immune cells. a Total leukocyte density (count/g) in breast cancer tissue according to PgR status. b–m Count of each immune cell fraction per unit weight of the tissue (count/g) according to PgR status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p > 0.05 (not significant); p < 0.05; p < 0.01; p < 0.001 composition of breast cancer tissues [51, 58–60]. The present study reports the first systematic analysis dem- onstrating that ER expression is preferentially associated with reduced infiltration of macrophages and CD4+ T cells (Fig. 1g, 3c, 3f). This result is consistent with ear- lier reports that show a negative correlation between ER expression and intratumoral infiltration of macrophages and CD4+ T cell [54–57]. Further, an inverse correla- tion between ER expression and CD8+ T cells has been reported previously [32, 54]; however, in our analysis, this correlation was weaker than that for CD4+ T cells, both at the gene and protein levels (Figs. 1g and 3). In addition, PGR expression showed a weaker correlation with total immune content than ESR1 and AR, as estimated by CIB- ERSORTx. Moreover, the correlation between PgR status and hTIL and hPD-L1 was insignificant at the protein level, probably due to the small number of PgR-positive samples. To validate these findings, a greater number of samples are required to be analyzed. Discussionh AR signaling is suggested to have immunomodula- tory functions in some malignancies, including breast cancer [35–38]; however, its immunological role in breast cancer has not been fully validated. To the best of our knowledge, a single study has investigated the relationship between immune cell composition and AR expression in breast cancer using the CIBERSORT Hanamura et al. Breast Cancer Research (2023) 25:13 Page 9 of 13 Fig. 5 Association between androgen receptor (AR) status and subsets of tumor-infiltrating immune cells. a Total leukocyte density (count/g) in breast cancer tissue according to AR status. b–m Count of each immune cell fraction per unit weight of the tissue (count/g) according to AR status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p > 0.05 (not significant); p < 0.05; p < 0.01; p < 0.001 Fig. 5 Association between androgen receptor (AR) status and subsets of tumor-infiltrating immune cells. a Total leukocyte density (count/g) in breast cancer tissue according to AR status. b–m Count of each immune cell fraction per unit weight of the tissue (count/g) according to AR status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p > 0.05 (not significant); p < 0.05; p < 0.01; p < 0.001 Fig. 5 Association between androgen receptor (AR) status and subsets of tumor-infiltrating immune cells. a Total leukocyte density (count/g) in breast cancer tissue according to AR status. b–m Count of each immune cell fraction per unit weight of the tissue (count/g) according to AR status. A tabulated summary of the statistics values and recalculated statistics values excluding outliers is shown on the upper and lower left sides of the figure, respectively. ns p > 0.05 (not significant); p < 0.05; p < 0.01; *p < 0.001 We analyzed the PD-L1 expression in each immune cell fraction by FCM analysis, similar to our previous study [39]. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13058-023-01606-7. The online version contains supplementary material available at https://doi. org/10.1186/s13058-023-01606-7. Additional file 1: Fig. S1. Association of typical biological processes with the expression of hormone receptors estimated using the gene expres- sion profile of breast cancer. (a-f) Summary of results of GSEA performed on the METABRIC datasets is shown. Biological processes positively or inversely correlated with each gene expression are shown in the descend- ing order of the absolute value of the normalized enrichment score (NSE) with absolute values of the log-transformed nominal p-values and FDR q-values. Thresholds of the nominal p-value and FDR q-value were set to < 0.05 and < 0.25, respectively, and the boundaries are shown in the graph by dashed lines. Fig. S2. Programmed death-ligand 1 (PD-L1) posi- tive ratio in each immune cell fraction according to estrogen receptor (ER) status. (a-k) the percentage of PD-L1 positive cells in each immune cell fraction by ER status is shown. A summary of statistics value is shown at left side of figure. A summary of the recalculated statistics values exclud- ing outliers is shown in the lower left of the figure. Fig. S3. Programmed death-ligand 1 PD-L1 positive ratio in each immune cell fraction according to progesterone receptor (PgR) status. (a-k) the percentage of PD-L1 positive cells in each immune cell fraction by PgR status is shown. A sum- mary of statistics value is shown at left side of figure. A summary of the recalculated statistics values excluding outliers is shown in the lower left of the figure. Fig. S4. Programmed death-ligand 1 (PD-L1) positive ratio in each immune cell fraction according to androgen receptor (AR) status. (a-k) the percentage of PD-L1 positive cells in each immune cell fraction by AR status is shown. A summary of statistics value is shown at left side of figure. A summary of the recalculated statistics values excluding outliers is shown in the lower left of the figure. Fig. S5. Reanalysis of Figs. 2–4 using 1% cut-off point for ER and PgR. (a-d) Correlation of hTIL and hPD-L1 with protein expression of ER and PgR were reanalyzed using the cut-off points 1% for ER and PgR. (e, f) Association between ER / PgR status and subsets of tumor-infiltrating immune cells were reanalyzed using the cut-off points 1% for ER and PgR. Discussionh Findings of this study suggest that the hormone recep- tor signals may primarily affect specific immune cell line- ages. In particular, the expression of ER and AR showed a negative correlation not only with total immune content in the tumor microenvironment but also with certain immune cell subsets such as macrophages and CD4+ T cells. This indicates that specific immune cell lineages may be primary targets of the immunoregulatory func- tion of the hormone receptor signals. Our next goal is to verify this hypothesis using an in vitro or in vivo analysis model. Abbreviations ER Estrogen receptor PgR Progesterone receptors AR Androgen receptor hTIL Histologically evaluated tumor-infiltrating lymphocytes hPD-L1 Histologically evaluated programmed death ligand 1 FCM Flow cytometry ICIs Immune checkpoint inhibitors METABRIC Molecular Taxonomy of Breast Cancer International Consortium SCAN-B Sweden Cancerome Analysis Network-Breast GSEA Gene Set Enrichment Analysis IHC Immunohistochemistry H&E Hematoxylin and eosin Mo/Mφ Monocytes/macrophages CD16+ Mo Non-classical monocytes MDSCs Myeloid-derived suppressor cells DCs Dendritic cells mDCs Myeloid dendritic cells NK Natural killer cells NKT Natural killer T cells In our previous study [39], a relatively small number of samples were used for FCM analysis, and compared to those of the general breast cancer cohort, the clinico- pathological characteristics of tumors were biased with larger invasive tumor sizes, more ER-negative cases, and higher Ki67 cases. Similarly, in this study, the small sam- ple size prevented us from performing subgroup analyses based on the tumor subtype. Therefore, the inclusion of a greater number of samples and more detailed analyses are required in future studies. In this exploratory analy- sis, the cut-off value for ER and PgR positivity was set at ≥ 10% according to our previous study [39]. However, in a recent clinical guideline update [63], 1% is recom- mended as a cutoff value for ER- and PgR-positive cells because of limited but present data on endocrine therapy benefit for cancers with 1% to 10% of cells staining ER positive. Therefore, the analysis of Figs. 2, 3, and 4 was repeated using the cutoff points 1% for ER and PgR, as shown in Additional file 1: Figure S5a–h. When the cut- off value is set to 1%, for Fig. 2, the expression of hPD-L1 showed a negative correlation with the protein expres- sion of PgR (Fig S5d). Similarly, for Fig. Discussionh According to hormone receptor status, we found that ER positivity was associated with decreased PD-L1 expression in Mo/Mφ and mDC (Additional file 1: Figure S2d, h), PgR positivity with increased PD-L1 expression in MDSCs and decreased PD-L1 expression in NK cells (Additional file 1: Figure S3f, j), and AR positivity with increased PD-L1 expression in CD8+ T and decreased PD-L1 expression in Mo/Mφ cells (Additional file 1: Fig- ure S4b, d). PD-L1 expression reflects ongoing (or active) immune responses in addition to immunosuppression via the PD-1/PD-L1 pathway [60–62]. Thus, ER positivity We analyzed the PD-L1 expression in each immune cell fraction by FCM analysis, similar to our previous study [39]. According to hormone receptor status, we found that ER positivity was associated with decreased PD-L1 expression in Mo/Mφ and mDC (Additional file 1: Figure S2d, h), PgR positivity with increased PD-L1 expression in MDSCs and decreased PD-L1 expression in NK cells (Additional file 1: Figure S3f, j), and AR positivity with increased PD-L1 expression in CD8+ T and decreased PD-L1 expression in Mo/Mφ cells (Additional file 1: Fig- ure S4b, d). PD-L1 expression reflects ongoing (or active) immune responses in addition to immunosuppression via the PD-1/PD-L1 pathway [60–62]. Thus, ER positivity algorithm [38]. Tumors with high AR expression were reported to be associated with pro-cancer regulatory T cells, and those with low AR expression were associ- ated with anti-cancer immune cells, such as CD4, CD8, gamma delta T cells, and memory B cells in ER-positive breast cancer. In our study, CIBERSORTx was run in absolute mode and included all subtypes in the analysis; therefore, a simple comparison was not possible. High AR-expressing tumors showed reduced macrophage infiltration into the tumor microenvironment; this find- ing was consistent with CIBERSORTx and FCM analy- sis results. In the present study, expressions of ER and AR were inversely correlated with hPD-L1 expression (Fig. 2b, f). Hanamura et al. Breast Cancer Research (2023) 25:13 Page 10 of 13 and AR positivity may reflect specific immune response in the breast cancer microenvironment. expression. These data suggest that hormone recep- tor signaling may suppress tumor immunity through a specific mechanism. Additionally, our data showed that certain immune cell lineages might get more strongly affected by hormonal signals than others, providing useful suggestions for further analysis of the effects of hormonal signals on tumor immunity. Supplementary Information (g, h) Recalculated statistics values excluding outliers are shown for ER and PgR status. Table S1. Clinical-pathological Discussionh 3, the significant association between ER positivity and some immune cell compositions were lost (i.e., leukocyte density, infiltration of total T, CD4+ T, Mo/Mφ) (Fig S5e). For Fig. 4, PgR positivity gained significant association with decreased leukocyte density and reduced infiltration of total T, CD4+ T, MDSC, DC (Fig S5f). No meaningful changes were observed in other results. Only 2 cases each had changes in ER and PgR status due to changes in the cutoff values. Therefore, we speculate that the discrepancies in the analysis results are a consequence of the small sample size. Declarations 7. Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, Carter SL, Stewart C, Mermel CH, Roberts SA, et al. Mutational heteroge- neity in cancer and the search for new cancer-associated genes. Nature. 2013;499(7457):214–8. Availability of data and materials 6. Ali HR, Glont SE, Blows FM, Provenzano E, Dawson SJ, Liu B, Hiller L, Dunn J, Poole CJ, Bowden S, et al. PD-L1 protein expression in breast cancer is rare, enriched in basal-like tumours and associated with infiltrating lymphocytes. Ann Oncol. 2015;26(7):1488–93. 6. Ali HR, Glont SE, Blows FM, Provenzano E, Dawson SJ, Liu B, Hiller L, Dunn J, Poole CJ, Bowden S, et al. PD-L1 protein expression in breast cancer is rare, enriched in basal-like tumours and associated with infiltrating lymphocytes. Ann Oncol. 2015;26(7):1488–93. The datasets generated and/or analyzed during the current study are available upon reasonable request from the corresponding author. Author contributions TH analyzed and interpreted the data and wrote the manuscript. MT, BT, TO, and NN collected clinical samples from the participants. HK (FCM), SK and MY (processing of the MCF data), NK, CH, and SK (the histological assessment) per- formed the experiments. KH processed samples for the FCM and supervised the writing of the manuscript. NN, TI, and SK developed the concept, designed the study, and supervised the writing of the manuscript. All authors have read and approved the final version of this manuscript for publication. Competing interests 10. Harbeck N, Penault-Llorca F, Cortes J, Gnant M, Houssami N, Poort- mans P, Ruddy K, Tsang J, Cardoso F. Breast cancer. Nat Rev Dis Primers. 2019;5(1):66. Hiroshi Kagamu has an advisory role in ImmuniT Research Inc, received hono- raria from AstraZeneca K.K., Ono Pharmaceutical Co. Ltd., and Bristol-Myers Squibb Co. Ltd. Shigehisa Kitano received honoraria from Ono Pharmaceutical Co., Bristol-Myers Squibb Co., Ltd., AstraZeneca K.K., Chugai Pharmaceutical Co., Ltd., Pfizer Japan Inc., and MSD Co. Ltd, received research funding from Astellas Pharma Inc., Gilead Sciences Inc., Eisai Co., Ltd., Regeneron Pharma- ceuticals Inc., Boehringer Ingelheim GmbH, Daiichi Sankyo Co., Ltd., Ono Pharmaceutical Co., Takara Bio Inc., PACT Pharma Inc., Chugai Pharmaceutical Co., Ltd., and MSD Co., Ltd. Naoki Niikura received honoraria from AstraZeneca K.K., Daiichi Sankyo Co. Ltd., Pfizer Japan Inc., Eisai Co. Ltd., and Nippon Kayaku Co. Ltd. Sasagu Kurozumi has received honoraria from Eli Lilly and Company, Daiichi Sankyo co. ltd, Taiho Pharmaceutical co. ltd, MSD K.K., AstraZeneca K.K., Chugai Pharmaceutical, Ltd., Dinow Inc., Eisai Co., Ltd. and Novartis Japan. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. 11. Hewitt SC, Korach KS. Progesterone action and respon alphaERKO mouse. Steroids. 2000;65(10–11):551–7. 12. Horwitz KB. The molecular biology of RU486. Is there a role for antipro- gestins in the treatment of breast cancer? Endocr Rev. 1992;13(2):146–63 13. Trabert B, Sherman ME, Kannan N, Stanczyk FZ. Progesterone and breast cancer. Endocr Rev. 2020;41(2):320–44. 14. Truong TH, Lange CA. Deciphering steroid receptor crosstalk in hormone- driven cancers. Endocrinology. 2018;159(12):3897–907. 15. Davey RA, Grossmann M. Androgen receptor structure, function and biol- ogy: from bench to bedside. Clin Biochem Rev. 2016;37(1):3–15. 16. Basile D, Cinausero M, Iacono D, Pelizzari G, Bonotto M, Vitale MG, Ger- ratana L, Puglisi F. Androgen receptor in estrogen receptor positive breast cancer: Beyond expression. Cancer Treat Rev. 2017;61:15–22. 17. Collins LC, Cole KS, Marotti JD, Hu R, Schnitt SJ, Tamimi RM. Androgen receptor expression in breast cancer in relation to molecular phenotype: results from the Nurses’ Health Study. Mod Pathol. 2011;24(7):924–31. Acknowledgements We would like to thank Editage (www.editage.com) for English language editing. Received: 11 July 2022 Accepted: 17 January 2023 Funding 3. Riley RS, June CH, Langer R, Mitchell MJ. Delivery technologies for cancer immunotherapy. Nat Rev Drug Discov. 2019;18(3):175–96. This research was funded by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Project No.18K16266 to NN, and No.20K22856 to TH), and the 2021 Tokai University School of Medicine Research Aid (to TH). This work was also supported by Chugai Pharmaceutical Co. Ltd. 4. Emens LA. Breast cancer immunotherapy: facts and hopes. Clin Cancer Res. 2018;24(3):511–20. 5. Goldberg J, Pastorello RG, Vallius T, Davis J, Cui YX, Agudo J, Waks AG, Keenan T, McAllister SS, Tolaney SM, et al. The immunology of hormone receptor positive breast cancer. Front Immunol. 2021;12: 674192. 5. Goldberg J, Pastorello RG, Vallius T, Davis J, Cui YX, Agudo J, Waks AG, Keenan T, McAllister SS, Tolaney SM, et al. The immunology of hormone receptor positive breast cancer. Front Immunol. 2021;12: 674192. References 1. Harbeck N, Gnant M. Breast cancer. Lancet (London, England). 2017;389(10074):1134–50. 2. Dieci MV, Griguolo G, Miglietta F, Guarneri V. The immune system and hormone-receptor positive breast cancer: is it really a dead end? Cancer Treat Rev. 2016;46:9–19. Conclusions In the present study, the gene expression levels of hormone receptors correlated with immunosuppres- sive phenotypes of breast cancer. The expression level of ER and AR proteins was associated with decreased tumor-infiltrating immune cells and decreased PD-L1 Page 11 of 13 Page 11 of 13 Hanamura et al. Breast Cancer Research (2023) 25:1 and Endocrine Surgery, Okayama University Hospital, 2‑5‑1 Shikata‑cho, Kitaku, Okayama Prefecture 700‑8558, Japan. 7 Department of General Surgical Science, Gunma University Graduate School of Medicine, 39‑22, Showa‑machi 3‑chome, Maebashi‑shi, Gunma Prefecture 371‑8511, Japan. 8 Department of Breast Surgery, International University of Health and Welfare, 4‑3, Kozuno- mori, Narita‑shi, Chiba Prefecture 286‑8686, Japan. characteristics by ER status. Table S2. Clinical-pathological characteristics by PgR status. Table S3. Clinical-pathological characteristics by AR status. Ethics approval and consent to participate This study was conducted at Tokai University Hospital and approved by the Ethics Committee, which conforms to the provisions of the Declaration of Hel- sinki (Accepted project No. 16R-279 and 20R-049). Patients were enrolled from May 2015 to April 2019, and written informed consent was obtained from all the patients to publish this paper. 8. Thomas A, Routh ED, Pullikuth A, Jin G, Su J, Chou JW, Hoadley KA, Print C, Knowlton N, Black MA, et al. Tumor mutational burden is a determi- nant of immune-mediated survival in breast cancer. Oncoimmunology. 2018;7(10): e1490854. 9. Wimberly H, Brown JR, Schalper K, Haack H, Silver MR, Nixon C, Bossuyt V, Pusztai L, Lannin DR, Rimm DL. PD-L1 expression correlates with tumor- infiltrating lymphocytes and response to neoadjuvant chemotherapy in breast cancer. Cancer Immunol Res. 2015;3(4):326–32. 9. Wimberly H, Brown JR, Schalper K, Haack H, Silver MR, Nixon C, Bossuyt V, Pusztai L, Lannin DR, Rimm DL. PD-L1 expression correlates with tumor infiltrating lymphocytes and response to neoadjuvant chemotherapy in breast cancer. Cancer Immunol Res. 2015;3(4):326–32. and Endocrine Surgery, Okayama University Hospital, 2‑5‑1 Shikata‑cho, Kitaku, Okayama Prefecture 700‑8558, Japan. 7 Department of General Surgical Science, Gunma University Graduate School of Medicine, 39‑22, Showa‑machi 3‑chome, Maebashi‑shi, Gunma Prefecture 371‑8511, Japan. 8 Department of Breast Surgery, International University of Health and Welfare, 4‑3, Kozuno- mori, Narita‑shi, Chiba Prefecture 286‑8686, Japan. Author details 1 f The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature. 2012;486(7403):346–52. 22. Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Piet- enpol JA. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Investig. 2011;121(7):2750–67. 23. Barton VN, Gordon MA, Richer JK, Elias A. Anti-androgen therapy in triple- negative breast cancer. Ther Adv Med Oncol. 2016;8(4):305–8. 42. Brueffer C, Vallon-Christersson J, Grabau D, Ehinger A, Häkkinen J, Hegardt C, Malina J, Chen Y, Bendahl P-O, Manjer J, et al. Clinical value of RNA sequencing-based classifiers for prediction of the five conven- tional breast cancer biomarkers: a report from the population-based multicenter Sweden Cancerome Analysis Network—breast initiative. JCO Precis Oncol. 2018;2:1–18. 23. Barton VN, Gordon MA, Richer JK, Elias A. Anti-androgen therapy in triple- negative breast cancer. Ther Adv Med Oncol. 2016;8(4):305–8. 24. Vasiliou SK, Diamandis EP. Androgen receptor: a promising therapeutic target in breast cancer. Crit Rev Clin Lab Sci. 2019;56:200–23. 24. Vasiliou SK, Diamandis EP. Androgen receptor: a promising therapeutic target in breast cancer. Crit Rev Clin Lab Sci. 2019;56:200–23. 25. Polanczyk MJ, Hopke C, Vandenbark AA, Offner H. Estrogen-mediated immunomodulation involves reduced activation of effector T cells, potentiation of Treg cells, and enhanced expression of the PD-1 costimu- latory pathway. J Neurosci Res. 2006;84(2):370–8. 43. Saal LH, Vallon-Christersson J, Häkkinen J, Hegardt C, Grabau D, Winter C, Brueffer C, Tang M-HE, Reuterswärd C, Schulz R, et al. The Sweden Cancerome Analysis Network - Breast (SCAN-B) Initiative: a large-scale multicenter infrastructure towards implementation of breast cancer genomic analyses in the clinical routine. Genome Med. 2015;7(1):20–20. 26. Prieto GA, Rosenstein Y. Oestradiol potentiates the suppressive function of human CD4 CD25 regulatory T cells by promoting their proliferation. Immunology. 2006;118(1):58–65. 44. Newman AM, Steen CB, Liu CL, Gentles AJ, Chaudhuri AA, Scherer F, Khodadoust MS, Esfahani MS, Luca BA, Steiner D, et al. Determining cell type abundance and expression from bulk tissues with digital cytometry. Nat Biotechnol. 2019;37(7):773–82. 27. Mostafa AA, Codner D, Hirasawa K, Komatsu Y, Young MN, Steimle V, Drover S. Activation of ERα signaling differentially modulates IFN-γ induced HLA-class II expression in breast cancer cells. PLoS ONE. 2014;9(1): e87377. 45. Newman AM, Liu CL, Green MR, Gentles AJ, Feng W, Xu Y, Hoang CD, Diehn M, Alizadeh AA. Robust enumeration of cell subsets from tissue expression profiles. Nat Methods. 2015;12(5):453–7. 28. Author details 1 f 18. Gonzalez LO, Corte MD, Vazquez J, Junquera S, Sanchez R, Alvarez AC, Rodriguez JC, Lamelas ML, Vizoso FJ. Androgen receptor expresion in breast cancer: relationship with clinicopathological characteristics of the tumors, prognosis, and expression of metalloproteases and their inhibi- tors. BMC Cancer. 2008;8:149. 1 Department of Breast Oncology, Tokai University School of Medicine, 143 Shimokasuya, Isehara‑shi, Kanagawa Prefecture 259‑1193, Japan. 2 Division of Cancer Immunotherapy Development, Center for Advanced Medical Development, The Cancer Institute Hospital of JFCR, 3‑8‑31, Ariake, Koto, Tokyo 135‑8550, Japan. 3 Division of Respiratory Medicine, Saitama Medical University International Medical Center, 1397‑1, Yamane, Hidaka‑shi, Saitama Prefecture 350‑1298, Japan. 4 Department of Pathology, School of Medicine, Tokai University, 143 Shimokasuya, Isehara‑shi, Kanagawa Prefecture 259‑1193 Japan. 5 Department of Immunology, Tokai University School of Medicine, 143 Shimokasuya, Isehara‑Shi, Kanagawa Prefecture 259‑1193, Japan. 6 Breast 19. Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson Å, Kampf C, Sjöstedt E, Asplund A, et al. Proteomics. Tissue- based map of the human proteome. Science. 2015;347(6220):1260419. Page 12 of 13 Page 12 of 13 Hanamura et al. Breast Cancer Research (2023) 25:13 Hanamura et al. Breast Cancer Research (2023) 25:13 20. Cochrane DR, Bernales S, Jacobsen BM, Cittelly DM, Howe EN, D’Amato NC, Spoelstra NS, Edgerton SM, Jean A, Guerrero J, et al. Role of the androgen receptor in breast cancer and preclinical analysis of enzaluta- mide. Breast cancer research : BCR. 2014;16(1):R7. 39. Hanamura T, Kitano S, Kagamu H, Yamashita M, Terao M, Tsuda B, Oka- mura T, Kumaki N, Hozumi K, Harada N, et al. Immunological profiles of the breast cancer microenvironment represented by tumor-infiltrating lymphocytes and PD-L1 expression. Sci Rep. 2022;12(1):8098. i lymphocytes and PD-L1 expression. Sci Rep. 2022;12(1):8098. 21. Fujii R, Hanamura T, Suzuki T, Gohno T, Shibahara Y, Niwa T, Yamaguchi Y, Ohnuki K, Kakugawa Y, Hirakawa H, et al. Increased androgen recep- tor activity and cell proliferation in aromatase inhibitor-resistant breast carcinoma. J Steroid Biochem Mol Biol. 2014;144 Pt B:513–22. 40. Pereira B, Chin SF, Rueda OM, Vollan HK, Provenzano E, Bardwell HA, Pugh M, Jones L, Russell R, Sammut SJ, et al. The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic land- scapes. Nat Commun. 2016;7:11479. 41. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, Speed D, Lynch AG, Samarajiwa S, Yuan Y, et al. Author details 1 f Variation in the incidence and magnitude of tumor-infiltrating lymphocytes in breast cancer subtypes: a systematic review. JAMA Oncol. 2016;2(10):1354–60. 33. Mahmoud SM, Paish EC, Powe DG, Macmillan RD, Grainge MJ, Lee AH, Ellis IO, Green AR. Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer. J Clin Oncol. 2011;29(15):1949–55. 51. Buisseret L, Garaud S, de Wind A, Van den Eynden G, Boisson A, Solinas C, Gu-Trantien C, Naveaux C, Lodewyckx JN, Duvillier H, et al. Tumor-infiltrat- ing lymphocyte composition, organization and PD-1/ PD-L1 expression are linked in breast cancer. Oncoimmunology. 2017;6(1): e1257452. 34. Mahmoud SM, Lee AH, Paish EC, Macmillan RD, Ellis IO, Green AR. The prognostic significance of B lymphocytes in invasive carcinoma of the breast. Breast Cancer Res Treat. 2012;132(2):545–53. 52. Mukaka MM. Statistics corner: a guide to appropriate use of correlation coefficient in medical research. Malawi Med J. 2012;24(3):69–71. 53. Akoglu H. User’s guide to correlation coefficients. Turkish journal of emer- gency medicine. 2018;18(3):91–3. 35. Trigunaite A, Dimo J, Jorgensen TN. Suppressive effects of androgens on the immune system. Cell Immunol. 2015;294(2):87–94. 54. Seo AN, Lee HJ, Kim EJ, Kim HJ, Jang MH, Lee HE, Kim YJ, Kim JH, Park SY. Tumour-infiltrating CD8+ lymphocytes as an independent predictive factor for pathological complete response to primary systemic therapy in breast cancer. Br J Cancer. 2013;109(10):2705–13. 36. van Rooijen JM, Qiu SQ, Timmer-Bosscha H, van der Vegt B, Boers JE, Schroder CP, de Vries EGE. Androgen receptor expression inversely cor- relates with immune cell infiltration in human epidermal growth factor receptor 2-positive breast cancer. Eur J Cancer. 2018;103:52–60. 55. Lu X, Yang R, Zhang L, Xi Y, Zhao J, Wang F, Zhang H, Li Z. Macrophage colony-stimulating factor mediates the recruitment of macrophages in triple negative breast cancer. Int J Biol Sci. 2019;15(13):2859–71. 37. Kim S, Moon BI, Lim W, Park S, Cho MS, Sung SH. Feasibility of classifica- tion of triple negative breast cancer by immunohistochemical surrogate markers. Clin Breast Cancer. 2018;18(5):e1123–32. 38. Okano M, Oshi M, Butash AL, Asaoka M, Katsuta E, Peng X, Qi Q, Yan L, Takabe K. Estrogen receptor positive breast cancer with high expres- sion of androgen receptor has less cytolytic activity and worse response to neoadjuvant chemotherapy but better survival. Int J Mol Sci. 2019;20(11):2655. 56. Quigley DA, Tahiri A, Lüders T, Riis MH, Balmain A, Børresen-Dale A-L, Bukholm I, Kristensen V. Author details 1 f Loi S, Sirtaine N, Piette F, Salgado R, Viale G, Van Eenoo F, Rouas G, Francis P, Crown JP, Hitre E, et al. Prognostic and predictive value of tumor- infiltrating lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 02–98. J Clin Oncol. 2013;31(7):860–7. 46. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, et al. PGC-1alpha- responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34(3):267–73. 29. Sobral-Leite M, Van de Vijver K, Michaut M, van der Linden R, Hooijer GKJ, Horlings HM, Severson TM, Mulligan AM, Weerasooriya N, Sanders J, et al. Assessment of PD-L1 expression across breast cancer molecular sub- types, in relation to mutation rate, BRCA1-like status, tumor-infiltrating immune cells and survival. Oncoimmunology. 2018;7(12): e1509820. 47. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005;102(43):15545–50. 30. Nanda R, Chow LQ, Dees EC, Berger R, Gupta S, Geva R, Pusztai L, Pathiraja K, Aktan G, Cheng JD, et al. Pembrolizumab in Patients With Advanced Triple-Negative Breast Cancer: Phase Ib KEYNOTE-012 Study. J Clin Oncol. 2016;34(21):2460–7. 48. Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive fac- tors in breast cancer by immunohistochemical analysis. Modern Pathol. 1998;11(2):155–68. 49. Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, Wienert S, Van den Eynden G, Baehner FL, Penault-Llorca F, et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: rec- ommendations by an International TILs Working Group 2014. Ann Oncol. 2015;26(2):259–71. 31. Rugo HS, Delord JP, Im SA, Ott PA, Piha-Paul SA, Bedard PL, Sachdev J, Le Tourneau C, van Brummelen EMJ, Varga A, et al. Safety and antitumor activity of pembrolizumab in patients with estrogen receptor-positive/ human epidermal growth factor receptor 2-negative advanced breast cancer. Clin Cancer Res. 2018;24(12):2804–11. 50. Schmid P, Adams S, Rugo HS, Schneeweiss A, Barrios CH, Iwata H, Dieras V, Hegg R, Im SA, Shaw Wright G, et al. Atezolizumab and nab- paclitaxel in advanced triple-negative breast cancer. N Engl J Med. 2018;379(22):2108–21. 32. Stanton SE, Adams S, Disis ML. Author details 1 f Age, estrogen, and immune response in breast adenocarcinoma and adjacent normal tissue. Oncoimmunology. 2017;6(11):e1356142–e1356142. 57. Sousa S, Brion R, Lintunen M, Kronqvist P, Sandholm J, Mönkkönen J, Kellokumpu-Lehtinen PL, Lauttia S, Tynninen O, Joensuu H, et al. Human Page 13 of 13 Hanamura et al. Breast Cancer Research (2023) 25:13 Hanamura et al. Breast Cancer Research (2023) 25:13 breast cancer cells educate macrophages toward the M2 activation status. Breast Cancer Res. 2015;17(1):101. breast cancer cells educate macrophages toward the M2 activation status. Breast Cancer Res. 2015;17(1):101. 58. Garaud S, Buisseret L, Solinas C, Gu-Trantien C, de Wind A, Van den Eynden G, Naveaux C, Lodewyckx JN, Boisson A, Duvillier H, et al. Tumor infiltrating B-cells signal functional humoral immune responses in breast cancer. JCI Insight. 2019;5(18): e129641. 59. Duechler M, Peczek L, Zuk K, Zalesna I, Jeziorski A, Czyz M. The hetero- geneous immune microenvironment in breast cancer is affected by hypoxia-related genes. Immunobiology. 2014;219(2):158–65. 60. Solinas C, Garaud S, De Silva P, Boisson A, Van den Eynden G, de Wind A, Risso P, Rodrigues Vitória J, Richard F, Migliori E, et al. Immune checkpoint molecules on tumor-infiltrating lymphocytes and their association with tertiary lymphoid structures in human breast cancer. Front Immunol. 2017;8:1412. 61. Pardoll DM. The blockade of immune checkpoints in cancer immuno- therapy. Nat Rev Cancer. 2012;12(4):252–64. 62. Cimino-Mathews A, Thompson E, Taube JM, Ye X, Lu Y, Meeker A, Xu H, Sharma R, Lecksell K, Cornish TC, et al. PD-L1 (B7–H1) expression and the immune tumor microenvironment in primary and metastatic breast carcinomas. Hum Pathol. 2016;47(1):52–63. 63. Allison KH, Hammond MEH, Dowsett M, McKernin SE, Carey LA, Fitzgib- bons PL, Hayes DF, Lakhani SR, Chavez-MacGregor M, Perlmutter J, et al. Estrogen and progesterone receptor testing in breast cancer: ASCO/CAP guideline update. J Clin Oncol. 2020;38(12):1346–66. Publisher’s Note S Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from:
https://openalex.org/W1971536330	http://www.scirp.org/journal/PaperDownload.aspx?paperID=25579	English	null	Towards Cloud to Device Push Messaging on Android: Technologies, Possibilities and Challenges	International journal of communications, network and system sciences/International journal of communications, network, and system sciences	2,012	cc-by	7,528	1. Introduction The goal of cloud computing is to provide the appearance of unlimited scalability and storage for less money than the in-house data centers [1]. Its success is based on an economy of scale and the relative ease of administration of services, as an entire cloud-based data center could be configured through, for example, a series of web pages. Many businesses are today looking at cloud computing as a viable and cost-effective alternative to hosting their own data centers internally, with large IT companies like Microsoft, Google, and IBM all having initiatives relat- ing to cloud computing [2]. Popular cloud-based plat- forms include Microsoft Azure, Amazon EC2 and the Google App Engine. In this paper we present our experience from working closely with push messaging technologies. Specifically, we compare different technologies available for the An- droid platform, from the standard library provided by Google to commercial options. In total we will look at four alternatives that all provide similar push messaging features. We believe this gives an in-depth look at the state of the art in integration between cloud computing and the Android platform not found in existing research. On the server side we have used the Google App Engine as the cloud-based platform. In 2011 Gartner reported that cloud computing, and mobile applications and media tablets are on the top 10-list of strategic technologies [3]. In this paper we will investigate how these technologies can cooperate through push messaging, where a content provider publishes in- formation to a subscriber. We have focused on combin- ing cloud computing with mobile applications through the use of push technology on the Android platform. We focused on push messaging on Android because it is an important aspect for application developers. Push messaging is in many situations a vital aspect of the us- ability and functionality of an application. Additionally, as stated by Gartner [4], the popularity of the Android platform means that this area of research highlights chal- lenges that affect a considerable amount of software de- velopers. Before the platform support for push messaging was added to Android it was common to use a polling me- chanism. This worked by making the application con- stantly poll the server for updates. There are several drawbacks with this alternative, especially the challenge of configuring the frequency of poll-requests sent. ABSTRACT In this paper we look at different push messaging alternatives available for Android. Push messaging provides an im- portant aspect of server to device communication, and we specifically focus on the integration of cloud computing with mo- bile devices through the use of push-based technologies. By conducting a benchmarking test, we investigate the performance of four relevant push technologies for the Android platform, namely C2DM, XMPP, Xtify and Urban Airship. The compari- son focuses on three aspects of the libraries: 1) The stability; 2) Response times; and 3) Energy consumption. The test is con- ducted on both WLAN and 3G, and includes several mobile device types. Additionally, we also integrate with the Google App Engine to provide the cloud integration server that is responsible for sending push messages to the mobile devices. Keywords: Cloud Computing; Android; Push Messaging; Cloud Integration; C2DM; XMPP; Urban Airship; Xtify other possibility is to push messages using SMS (Short Message Service). Android is able to receive and inter- cept SMS messages, but they come with their own limi- tations like availability, cost and message size. Copyright © 2012 SciRes. nt. J. Communications, Network and System Sciences, 2012, 5, 839-849 ttp://dx.doi.org/10.4236/ijcns.2012.512089 Published Online December 2012 (http://www.SciRP.org/journal/ijcn Towards Cloud to Device Push Messaging on Android: Technologies, Possibilities and Challenges Jarle Hansen1, Tor-Morten Grønli1,2, Gheorghita Ghinea1,2 1School of Information Systems, Computing and Mathematics, Brunel University, London, UK 2The Norwegian School of Information Technology, Oslo, Norway Email: jarle@jarlehansen.net, tmg@nith.no, george.ghinea@brunel.ac.uk Jarle Hansen1, Tor-Morten Grønli1,2, Gheorghita Ghinea1,2 1School of Information Systems, Computing and Mathematics, Brunel University, London, UK 2The Norwegian School of Information Technology, Oslo, Norway Email: jarle@jarlehansen.net, tmg@nith.no, george.ghinea@brunel.ac.uk Jarle Hansen1, Tor-Morten Grønli1,2, Gheorghita Ghinea1,2 1School of Information Systems, Computing and Mathematics, Brunel University, London, UK 2The Norwegian School of Information Technology, Oslo, Norway Email: jarle@jarlehansen.net, tmg@nith.no, george.ghinea@brunel.ac.uk Received October 5, 2012; revised November 2, 2012; accepted November 14, 2012 2. Related Work Cloud computing is becoming increasingly popular. Fea- tures like elasticity, scalability and a new cost-model are providing new and interesting opportunities for many companies. It has proven particularly useful for small and medium enterprises that have a large variation in their computing needs [5]. However, not all businesses will benefit from moving their data centres to the cloud, e.g. when there are government regulations not allowing sen- sitive data to be stored with an external cloud provider [6]. In similar research, the Bakabs application, created for Android and iOS, Paniagua et al. [13] use C2DM and the Apple Push Notification Service (APNS) to implement push messaging. The application aims to provide a man- agement tool that allows information to be retrieved about the web applications’ traffic and then launch or stop cloud instances based on the current load. The use of push messaging was included to allow for the cloud- based services to send messages asynchronously back to the handset, thus eliminating the need for the client to wait for a response. In an attempt to help decision makers identify their concerns with moving all or parts of their computing needs to the cloud, Khajeh-Hosseini et al. [7] has created a Cloud Adoption Toolkit. With this toolkit, they argue that one can identify the potential benefit or drawback from moving the IT infrastructure and applications to a cloud provider. One of the topics investigated, as part of the bench- marking test we conducted, is energy consumption. Re- search in this area includes the work done by Flinn and Satyanarayanan [14]. They specifically look at energy- aware systems, where they show that a solution for dy- namic balancing of energy consumption and application quality is an essential part of comprehensive energy ma- nagement solutions. Similarly, Rivoire et al. [15] present a research effort in the area of energy efficiency. They propose JouleSort, an external sort benchmark for evalu- ating the energy efficiency of various devices, from lap- tops, desktops and servers. When selecting a cloud platform, there are three main service models to select from [8]: 1) Software as a Service (SaaS), the consumer uses the cloud provider’s applications running on a cloud in- frastructure; 2) Platform as a Service (PaaS), the consumer is able to deploy (either customer created or acquired) applica- tions onto the cloud infrastructure. 1. Introduction An- Accordingly, the main contribution of our research is to investigate the following topic: Compare four push- messaging technologies for Android, which are inte- grated with a cloud-computing environment, in regards to the stability of responses, response times and energy consumption. IJCNS J. HANSEN ET AL. 840 The paper is organised as follows: We begin with a look at related work before presenting a short introduc- tion of the investigated technologies. A description and review of the benchmarking test is shown, and finally the conclusion is presented towards the end. for example that threads cannot outlive the request that creates it and a limit of 10 concurrent request threads [11]. However, by enforcing these Google is able to pro- vide very high scalability [9]. In work more closely related to ours, Minstrel [12] has been developed to provide a push-based messaging sys- tem. The system utilises the publish/subscribe paradigm and has been extended to support mobile devices. Min- strel and the standard push messaging library for Android, which is called Cloud to Device Messaging (C2DM), have several similarities in how they are built, including the publish/subscribe model, where subscribers must re- gister at the content publisher to receive the messages. Copyright © 2012 SciRes. 2. Related Work In our benchmarking test we considered a total of six alternative technologies that offer push messaging for Android. These libraries are currently the main competi- tors in the market. SMS was not considered as part of the push messaging libraries, this was because of limitations such as availability and cost. Specifically, we conducted the test on a tablet device, Samsung Galaxy Tab 10.1, which does not support SMS. Nonetheless, getting highly reliable and repeatable re- sults from a real-world environment is difficult [5]. This is especially so when using cloud-based resources, where there are many components, hardware and software, working together and it can be difficult to isolate the specific parts of the overall system one wants to test. However, we still believe there is value in doing a benchmarking test of different push messaging technolo- gies integrated with cloud computing. There is value in providing performance results for the different technolo- gies because it gives the developers information on the strengths and weaknesses of the popular push-messaging alternatives on the platform. The libraries we found particularly interesting are: C2DM, Urban Airship, Xtify, XMPP, MQTT (Message Queue Telemetry Transport) and Deacon. Although we believe these technologies present the most promising and useful push messaging libraries on Android, we cannot completely rule out the possibility of other inter- esting options we were not able to find. Of these alternatives, we did not go into detail for two specific libraries, namely MQTT and Deacon. MQTT was not included because we wanted to investigate push- messaging technologies that can be easily integrated into the cloud, and specifically on the Google App Engine. MQTT is useful for connections that require a small code footprint and where network bandwidth is limited [16]. It does require a message broker hosted on a separate server. We did not find an easy way to integrate this ser- vice with the Google App Engine. Also, it is important to note that we are integrating ex- ternal components, the mobile devices, into the tests and not just using a cloud-based system, making it even more challenging to use a simulation tool that will provide realistic results. We have focused our work on combining mobile de- vices with cloud computing. In this context, Binnig et al. 2. Related Work The consumer does not manage or control the cloud platform/infrastructure; 3) Infrastructure as a Service (IaaS), the consumer can provision processing, storage, networks, and other computing resources that can be utilised to deploy and run the applications. The research described effort by Rivoire et al. [15] is quite different from ours. Firstly, we focus on one spe- cific element, namely push messaging technologies run- ning on an Android device. We chose to focus on the Android platform because it is well integrated with other Google technologies and, more importantly, it is an open platform making it ideal for experimentation. Secondly, we are not trying to identify the performance of disk I/O, CPU capacity and so on, but we compare the stability, response times and energy consumption of the important push messaging technologies. Generally, our test is much more specific, targeting specific mobile operating system and push-messaging technologies, in contrast to the gen- eral-purpose benchmark proposed by Rivoire et al. [15]. In our work we focus on the Google App Engine, which is a PaaS service model making it possible for developers to run their own applications on Google’s infrastructure [9]. We used the Google App Engine be- cause it provides good support for the technologies we wanted to test, such as a close integration with C2DM and XMPP, and also a simple and easy administration feature. The Google App Engine is a cloud-based PaaS service, the platform is pre-configured by Google and provides a much higher abstraction than an IaaS (Infrastructure as a Service) platform like Amazon EC2, where almost the entire stack from kernel and upwards can be controlled [10]. The Google App Engine is also well integrated with other Google services like e-mail and authentication. The platform imposes certain limitations on the developers, In the context of performance testing, Calheiros et al. [5] have examined the performance of cloud computing alternatives. They present a simulation toolkit, called CloudSim, making it possible to model and simulate IJCNS IJCNS J. HANSEN ET AL. 841 well integrated with the Google App Engine and is there- fore included in our test. cloud computing systems and applications. They argue that it is impossible to perform benchmarking experi- ments in a repeatable, dependable and scalable environ- ment using real-world cloud-based platforms. With Cloud- Sim, they have created a tool making it fast and easy to configure and run these tests. 3.1. XMPP The first push-messaging technology we wanted to in- clude in our benchmarking test was the XMPP protocol. It is created for real-time communication [18] and for streaming XML [19]. The technology behind XMPP was created in 1998 and then refined in the Jabber open source community in 1999 and 2000, before it was for- malised by IETF (The Internet Engineering Task Force) in 2002 and 2003 [20]. It is commonly used in Instant Messaging (IM) and has been used by Google Talk, Jab- ber and other IM networks. The next section will introduce the different push mes- saging technologies. 2. Related Work [1] argue that a benchmarking test in a cloud computing environment should address the following issues: The second technology, The Deacon Project [17], is an open source project providing push notifications to Java and Android applications. We felt that this project was the least mature technology of the options we considered, as it is currently in beta release. The project also states that it is created for users wanting to run push notifica- tions on their own server and support Android versions lower than 2.2, whereas C2DM requires at least Android 2.2. None of these requirements matched what we want- ed to investigate, which included a close integration with a cloud-based server application and devices running on at least the 2.2 version of Android. 1) Adaptability of the system, the ability to adapt to changing load in terms of scalability and cost; 2) Conduct the benchmarking tests from different lo- cations; 3) Access more dynamic “Web 2.0”-like applications including multimedia content. 3) Access more dynamic “Web 2.0”-like applications including multimedia content. These pointers provide valuable insight into what a cloud computing benchmark test should include. We want to use some of the general ideas from this list, but we also want to stress the differences between what we are benchmarking, where a mobile client and cloud inte- gration is involved, with the pure cloud-based bench- marking test that was described by Binnig et al. [1]. Par- ticularly interesting for our test is to include the stability, response times and energy consumption of the system and also results from different networks. Copyright © 2012 SciRes. 3. Push Messaging on Android The technologies used in our experiment all deal with the integration of cloud computing and mobile applications. Not all push messaging technologies investigated are directly related to cloud computing, such as XMPP (Ex- tensible Messaging Presence Protocol). However, it is XMPP is offered as a service on the Google App En- gine, making it possible to write cloud-based applications IJCNS IJCNS J. HANSEN ET AL. 842 a basic overview of C2DM. We believe the C2DM li- brary provides a good basis for a standard push messag- ing technology. However, we identified certain limita- tions with C2DM that we wanted to simplify and provide more features than the standard solution. This will be presented in more detail in a later section. on the Google infrastructure that is able to communicate with users or applications. Accordingly, we integrated with XMPP through the Google App Engine infrastruc- ture. Our Android client used an XMPP library called asmack [20], which is a patched version of smack created for Android. Smack offers an XMPP library and is a pure Java implementation [21]. There will also be a market for different solutions, of- fering more comprehensive services and novel features not covered by the C2DM technology, such as a webpage for administration and multi-platform support. These features are targeted by other commercial technologies, such as Urban Airship and Xtify. XMPP on the Google App Engine has a daily limit of 1 GB data sent and 100,000 invitations with the free de- fault limit [22]. More resources can be purchased, with paid applications incurring a minimum spend of $2.10 per week. 3.4. Xtify The test was conducted in two main iterations. In it- eration 1 we started by looking at the response times and stability for each push-based alternative. Moreover, the test was performed on two network types, namely WIFI and 3G. The message size sent was 450 bytes on all technologies and the tests were run on and off over sev- eral days with messages sent every 5 minutes. Similar to Urban Airship, Xtify is a commercial option that provides push messaging for Android. Xitfy also supports the Android, Blackberry and iOS platforms. For Android it uses the C2DM technology offered by Google and adds features like registration management, notifica- tion handling, notification inbox, rich notification sup- port and the ability to send messages based on user loca- tion. For iteration 1 we included the following devices in the benchmarking test: Samsung Galaxy Tab 10.1 (SG), HTC Evo (HE), and HTC Nexus One (HN). The Just Push-package from Xtify costs $199 per month, and includes support for 30,000 devices and unlimited notifications. Each additional device over this limit costs $0.01 [27]. For iteration 2, we wanted to compare the energy con- sumption of the various push-messaging technologies. This test included the same message size (450 bytes), but we only used one device, which was SG, and we also increased the message frequency to 10 minutes. This device was selected to get a more comprehensive test because the SG device has a significantly larger battery (7000 mAh) when compared to for example the HE (1500 mAh). Both Urban Airship and Xtify support C2DM, and in addition they have their own proprietary Android push notification service [28]. These proprietary alternatives are used in the benchmarking test to provide a way of comparing C2DM-based applications with other push messaging technologies. For Xtify this is implemented on their infrastructure with the XMPP protocol [28]. It is important to note that even though two technologies in our benchmarking test are based on XMPP, we use XMPP directly when integrating with the Google App Engine. When testing Xtify we use their API and infra- structure. Xtify recommends using XMPP in cases where one for example needs to communicate frequently with the mobile devices over a short period of time. In other cases it recommends using the C2DM alternative it pro- vides. 3.2. Cloud to Device Messaging Cloud to Device Messaging (C2DM) was made available from Android 2.2, where the goal was to make it easier for mobile applications to sync data with servers [23]. The technology is used in several standard Google ap- plications including Gmail, Contacts and Calendar. When messages are received on the Android client, the system will wake up the application via an Intent broad- cast, and pass the message data [13]. The message limit is set to 1024 bytes and developers are encouraged to send short messages, essentially notifying the mobile application that updated information can be retrieved from the server. C2DM is a free service, and the maxi- mum number of messages that can be sent is approxi- mately 200,000 per day [24]; however this can be in- creased if there is a need for more resources. Urban Airship provides a commercial option for sending push notifications on Android, Blackberry and the iOS platform [25]. It makes it easier for developers to create applications for multiple device types since it provides a single API for all the supported platforms. It consists of a library that is added to the project to hide all the low level complexity related to push messaging. In addition to push notifications, Urban Airship provides features such as rich push, push composer, reports, in-app pur- chase and subscriptions. Urban Airship also offers a proprietary push-messag- ing platform called Helium that supports Android 1.6 and newer. With newer phones (minimum Android 2.2) it supports the use of C2DM. The pro priceplan costs $199 per month, and includes support for up to 10,000 users and unlimited push messages, with an additional $0.01 per user over this limit [26]. Google offers standard libraries for Android that makes it possible to use C2DM directly. Figure 1 shows Figure 1. C2DM overview. Figure 1. C2DM overview. IJCNS Copyright © 2012 SciRes. J. HANSEN ET AL. 843 4. Benchmark Test We created a benchmarking test to compare different push-messaging technologies on Android. The system consisted of a mobile client that in sequence invoked all the different push messaging technologies and record the time used. On the server side we have a Google App En- gine server application that sends messages when re- quested to do so from the client. This application is also responsible for storing all the data received from the mo- bile application. Copyright © 2012 SciRes. 3.4. Xtify This benchmark test lasted about 6 days for C2DM and Urban Airship, while the XMPP test only lasted about 2 days because of certain limitations in the plat- form, which we will describe in more detail later in this paper. When doing our pilot-tests we noticed that the screen would consume a considerable amount of battery power and made it difficult to find any differences between the push-messaging technologies. For this reason we turned the screen on the device off when testing the energy consumption. Additionally, we also disabled the auto- sync feature to prevent applications using network com- munication resources on the device. These steps were taken to try to eliminate other factors that might impact the energy consumption on the device. In our research reported here, we have included all of these technologies and performed a benchmarking test to see how they perform. 4.2. Results Table 1 presents the overall results from the benchmark- ing test. We start by looking at the numbers for both the SG and HE. Both devices ran on the same WIFI network, and we were also able to provide a fairly equal number of messages for each technology providing a good basis for the comparison. The final technology we tested was Xtify, and it comes very close to the overall performance of C2DM. It pro- vides more stable results than Urban Airship and with slightly better average response time than C2DM. We were unable to conduct the benchmarking test with Xitfy on other devices than HE, because of limitations in the account we used. Additionally, since we were only able to send a limited number of messages this technology was not included in iteration 2. As can be seen in the table below, the results are rela- tively consistent, even though the differences between the technologies (see the standard deviation) were big- gest on the SG. The average response time for XMPP was the shortest, with C2DM on second and finally Ur- ban Airship. There is a difference of 276.12 ms (SG) and 156.98 (HE) between XMPP, which had the shortest re- sponse times, and Urban Airship with the longest re- sponse times. As seen in both Figure 2 (results from SG) and 3 (results from HE), the difference is mostly due to spikes in the response from Urban Airship. The max time used for Urban Airship was 5337 ms (SG) and 3601 ms (HE), whereas both XMPP and C2DM provided consid- erably more stable results in the benchmarking test. These spikes were more frequent on the results gathered from SG than with HE, however, this trend was evident The results for SG and HE are presented in Figures 2 and 3 on the next page, and it is easy to the spikes in the response times for Urban Airship as previously men- tioned. For the final test, iteration 2, we wanted to investigate the energy consumption of the different push-messaging technologies. In this part of the benchmark test, we ran the same application as before, but we increased the time Table 1. Test results. 4.1. Test Procedure Both iterations, as explained above, followed these main steps: 1) The Android client registers with the server. This is done differently for each technology, for example C2DM will send a registration id to the device; 2) A timer is started on the client, followed by a mes- sage being sent to the server requesting a new push mes- sage; In our research we wanted to compare the performance of C2DM with other push-based technologies integrated in a cloud environment. We defined three main charac- teristics that are important for push-based technologies: g 3) The server application receives the message and immediately sends out a message consisting of 450 bytes to the mobile device. This will happen for each technol- ogy type;  Response times, what are the response times for the different push messaging technologies? gy yp 4) When the message is received, the Android client stops the timer and registers the result. This result is then sent back to a result-servlet that is part of the server application, which will permanently store the information;  Stability, are the response times providing stable re- sults over the time we run the test?  Energy consumption, how efficient, in regards to bat- tery power, is the various push-messaging technologies? IJCNS J. HANSEN ET AL. 844 5) Finally, the process waits 5/10 minutes before con- tinuing with the sending the next message. on all the devices included in the test, which is confirmed by looking at the standard deviation. Overall the C2DM results were stable and the per- formance results recorded showed an average response time of 466.82 ms (SG) over 281 messages and 401.89 ms (HE) over 174 messages. Comparing the response time for C2DM on SG and HE, there is only a difference of 23.01 ms in the average response times. The HE had fewer messages received, with 174 compared to 281 for the tablet. In our tests we compare the following technologies: 1) XMPP; 2) Urban Airship Helium; 3) Xtify (proprietary push messaging infrastructure) and 4) C2DM. All push messaging alternatives are integrated with a cloud-based Google App Engine server application. It is important to note that we did not include other technologies built on top of C2DM, like Urban Airship or Xtify with C2DM enabled, because they send messages in the same way as standard C2DM. 4.1. Test Procedure XMPP had the most stable results in our test, with a standard deviation of 172.91 (SG) and 67.84 (HE). Urban Airship did appear to have more stability issues than the rest and these issues surfaced several times during the test. When comparing the results from different WIFI networks and 3G, the same pattern emerges. The 3G re- sponse times are higher, but this is to be expected since they will have less bandwidth than the WIFI connection. Copyright © 2012 SciRes. IJCNS 4.2. Results Device Tech Number of messages Average response time (ms) Standard deviation C2DM 281 466.82 203.76 Urban Airship 279 619.43 708.72 Samsung Galaxy Tab 10.1 —Android 3.1 —WIFI XMPP 280 343.31 172.91 C2DM 174 401.89 95.40 Urban Airship 172 473.88 321.97 HTC Evo —Android 2.3 —WIFI XMPP 168 316.90 67.84 C2DM 17 502.47 59.68 Urban Airship 37 814.27 943.24 HTC Nexus One —Android 2.3 —3G XMPP 30 436.60 286.10 HTC Evo —Android 2.3 —WIFI Xtify 213 432.92 250.09 Table 1. Test results. 845 J. HANSEN ET AL. Figure 2. Results for C2DM, Urban Airship and XMPP on Samsung Galaxy Tab (WIFI). Figure 3. Results for C2DM, Urban Airship and XMPP on HTC Evo (WIFI). Figure 2. Results for C2DM, Urban Airship and XMPP on Samsung Galaxy Tab (WIFI). Figure 2. Results for C2DM, Urban Airship and XMPP on Samsung Galaxy Tab (WIFI). Figure 3. Results for C2DM, Urban Airship and XMPP on HTC Evo (WIFI). Figure 2. Results for C2DM, Urban Airship and XMPP on Samsung Galaxy Tab (WIFI). Figure 3. Results for C2DM, Urban Airship and XMPP on HTC Evo (WIFI). the developer account we had created. the developer account we had created. between messages to 10 minutes. Another difference was that instead of running each technology in sequence, we only recorded one technology at a time. This was done because we wanted to provide results based on the bat- tery level for each technology, and also to expand the test over a longer period of time. As previously described, the client ran the test by sending requests to the cloud-based server, but as part of this iteration we also added a fea- ture that triggered a new request from the mobile device for each change in battery level. By doing this, we were able to record the messages and also the corresponding battery level. With both C2DM and Urban Airship we were able to provide fairly equal number of messages, with 862 and 858 messages sent respectively. However, with XMPP we were unable to send more than 295 messages because of quotas and limits in the Google App Engine [29]. The results are presented in Figure 4, where we have added trend lines for each result to make it easier to see the dif- ferences between the technologies. As can be seen in Figure 4, both C2DM and Urban Airship provided the best results, using less energy than XMPP. Copyright © 2012 SciRes. 4.2. Results We did expect this because it is recommended to use XMPP in scenarios where one needs to communicate frequently over a short period of time, as stated by Xtify In this test we included C2DM, Urban Airship, and XMPP. Xtify was not included due to limitations with IJCNS IJCNS 846 J. HANSEN ET AL. Figure 4. Energy consumption results. Figure 4. Energy consumption results. mobile client. A restart of the Android client would solve the problem, but this scenario happened multiple times. This is why some of the devices have very few test re- sults for certain technologies, for instance the HN had issues with C2DM. This only happened on the Android 2.3 mobile devices, whereas the Android 3.1 tablet was very stable over the entire test period. It would be inter- esting and useful for future work to focus on these stabil- ity issues, by including Android version 4 devices in the tests to see if the problems are fixed or at least improved in this newer version of the operating system. However, at the time of writing, an official version of Android 4 is not released for the devices used in the benchmark test. [30]. This aspect is also verified by the normal use of XMPP, for instance Instant Messaging applications such as Google Talk. The two other technologies, Urban Airship and C2DM, provided fairly similar results. However, from the last 50% and towards 0%, Urban Airship did provide slightly better results. It is difficult to draw any final conclusions of the difference between these technologies because the change is fairly small, and further testing is needed. However, we still find these results interesting, and espe- cially the big difference between C2DM/Urban Airship and XMPP. We would certainly recommend using either C2DM or Urban Airship over XMPP for applications not dependent on frequent messages being pushed from the server to the devices. Additionally, both Urban Airship and Xtify were tested on development servers. In the case of Xtify, we had to run this separately because there is a limit of 300 messages sent when using a basic account. The test con- sisted of a total of 213 messages, because we had to setup and test the system before running the actual ex- periment. For the energy consumption test we were un- able to include Xtify because of the limitation in the number of messages. 4.2. Results Table 2 presents a short summary of the advantages and disadvantages of the libraries we tested. The overall results show that the C2DM, which is the standard An- droid library offered by Google, provided good per- formance compared to the alternatives. Moreover, it also performed well in the energy consumption test, espe- cially compared to XMPP. We found that C2DM pro- vided the best overall results in the three categories in- vestigated, namely response time, stability and energy consumption. Moreover, we used a fairly coarse grained scale, with battery percentage, when registering the energy con- sumption. There are other research efforts that have measured energy efficiency with a power meter, which is used in Rivoire et al. [15]. This will in most cases pro- vide more reliable and accurate result. However, we be- lieve there is still value in our general results and we were also able to push a fairly significant number of messages with: 862 (C2DM), 858 (Urban Airship) and 295 (XMPP). Copyright © 2012 SciRes. Copyright © 2012 SciRes. 4.4. Simple-C2DM We implemented a new open source library called Sim- ple-C2DM. It was created specifically to simplify the development of applications using C2DM on the Android platform. Our library builds on top of standard C2DM and provides additional features. The second option, which in our opinion is the best, is to generate the XML-tags automatically using an annota- tion processor. This part of the Simple-C2DM library was created as an experimental feature to figure out if we were able to automatically generate these XML-tags without the need to manually type in the package name. Because this uses an annotation processor, there is a con- figuration step involved when setting up the development environment. After this is completed, the annotation processor automatically runs when the project is com- piled. An in-depth look at the Simple-C2DM features and functionality is outside the scope of this paper, however, a short introduction is given to provide useful informa- tion on how the development tools and API problems with C2DM can be improved. These features can also be useful for other push messaging technologies. Figure 5 presents an overview of how Simple-C2DM is integrated in the client and server application. Figure 5 presents an overview of how Simple-C2DM is integrated in the client and server application. There were two main reasons why we wanted to pro- vide a new implementation: There were two main reasons why we wanted to pro- vide a new implementation: Table 2. Result summary. Technology Advantages The standard push-messaging technology offered by Google. C2DM Provided stable response times and low energy consumption compared to for example XMPP. Provides several features not found in the free alternatives. Urban Airship Our results show that the energy consumption was considerably less than for XMPP. Works well in situations where one needs to communicate frequently over a short period of time. XMPP Provided good and stable response times in our benchmark test. Xtify For Xtify we were not able to collect the same amount of data, however, the results were very similar to C2DM in terms of response time. Disadvantages Certain features, such as sending messages to multiple clients, is not supported. The development environment and API could be better (see Section 4.4 for more information on this issue). We recorded varying response times. In certain cases we saw quite a big increase in response times for some requests. Offers certain free features, but is mainly a commercial product. Uses more battery power than the other technologies. Disadvantages Certain features, such as sending messages to multiple clients, is not supported. The development environment and API could be better (see Section 4.4 for more information on this issue). We recorded varying response times. In certain cases we saw quite a big increase in response times for some requests. Offers certain free features, but is mainly a commercial product. Uses more battery power than the other technologies. Advantages Uses more battery power than the other technologies. Offers certain free features, but is mainly a commercial product. our opinion is the AndroidManifest.xml-file, and espe- cially the required configuration that needs to be pro- vided. In Simple-C2DM we tried to solve this problem by offering two alternatives. If the developer does not want to, or cannot for some reason, use code generation, we created a manifest-generator hosted on the Google App Engine. This is a webpage that will take the pack- age-name as input and generate the needed XML-tags. related to the lack of flexibility and certain useful fea- tures. We created an open source library, called Simple- C2DM, to address these issues. We will give a short presentation of this library in the next section. 4.3. Limitations The benchmarking test, as described previously, has some limitations, and this was specifically apparent on some of the mobile devices. We had certain issues run- ning all of the push messaging technologies over a period of time. This would result in certain messages not being received. The test would run reliably for period of time, before the messages were no longer registered on the We experienced a few issues with the C2DM API that we wanted to improve. These were specifically issues IJCNS J. HANSEN ET AL. 847 Table 2. Result summary. Technology Advantages Disadvantages The standard push-messaging technology offered by Google. Certain features, such as sending messages to multiple clients, is not supported. C2DM Provided stable response times and low energy consumption compared to for example XMPP. The development environment and API could be better (see Section 4.4 for more information on this issue). Provides several features not found in the free alternatives. We recorded varying response times. In certain cases we saw quite a big increase in response times for some requests. Urban Airship Our results show that the energy consumption was considerably less than for XMPP. Offers certain free features, but is mainly a commercial product. Works well in situations where one needs to communicate frequently over a short period of time. Uses more battery power than the other technologies. XMPP Provided good and stable response times in our benchmark test. Xtify For Xtify we were not able to collect the same amount of data, however, the results were very similar to C2DM in terms of response time. Offers certain free features, but is mainly a commercial product. 5. Conclusions 1) To create a higher level of abstraction for certain key features; 1) To create a higher level of abstraction for certain key features; In this paper we investigated four cloud-integrated push- messaging technologies for the Android platform. We wanted to specifically target technologies that could be easily integrated with cloud computing, and in our test we used the Google App Engine as the cloud-based plat- form. We ran the tests on both WLAN and 3G, and pro- vided results from several Android devices. 2) To provide features not available in standard C2DM. 2) To provide features not available in standard C2DM. Starting with the higher level of abstraction, we solved this by introducing support for annotations. Annotations provide metadata that will not directly affect program semantics and are usually handled by tools and libraries. The main feature we found particularly useful and im- portant by introducing annotation support was flexibility. Developers were no longer strictly forced to follow a specific pattern in the source code. Starting with the higher level of abstraction, we solved this by introducing support for annotations. Annotations provide metadata that will not directly affect program semantics and are usually handled by tools and libraries. Our benchmarking test consisted of a client installed on the mobile devices and a server application running on the Google App Engine. The client had fixed time intervals where it would request a push message from the server. When this requested message was received, it recorded the time used. For the second part of our test, the energy consumption experiment, we used the same basic client but increased the time between each message The second task we wanted to introduce in Simple- C2DM was new features not available in the standard implementation. One of the major issues with C2DM in IJCNS IJCNS 848 J. HANSEN ET AL. Figure 5. Simple-C2DM. Figure 5. Simple-C2DM. and included a feature that was able to record the battery level, in percentage, on the device. sumption test, without the XMPP limitations and the in- clusion of Xtify, would provide a useful future direction to our research. The benchmarking test investigated each technology in regards to stability, response times and energy consump- tion. The results from the tests identified that XMPP pro- vides the best result for response time and stability. However, it is also the library with the worst results for energy consumption. Copyright © 2012 SciRes. 5. Conclusions The next technology, Urban Air- ship, suffers from spikes in the response times, but does provide good results for the energy consumption test. [6] R. C. Elsenpeter, T. Velte and A. Velte, “Cloud Comput- REFERENCES [1] C. Binnig, D. Kossmann, T. Kraska and S. Loesing, “How Is the Weather Tomorrow? Towards a Benchmark for the Cloud,” Proceedings of the 2nd International Work- shop on Testing Database Systems, ACM, New York, 2009, pp. 9:1-9:6. For Xtify we recorded very similar results to C2DM and Urban Airship with the stability and response times. However, we were not able to collect the same amount of data as with the other technologies, and were therefore unable to include the technology in the energy consump- tion test. Overall, when including all aspects of the test from the three technologies we were able to test thor- oughly, we found that C2DM provides the best results. This is especially so if one does not need messages pushed frequently over a short period of time. In these cases XMPP also a good alternative, because it provided the best response times in our evaluation. [2] L. J. Mei, W. K. Chan and T. H. Tse, “A Tale of Clouds: Paradigm Comparisons and Some Thoughts on Research Issues,” IEEE Asia-Pacific Services Computing Confer- ence, Yilan, 9-12 December 2008, pp. 464-469. [3] Gartner, “Gartner Identifies the Top 10 Strategic Tech- nologies for 2011,” 2011. http://www.gartner.com/it/page.jsp?id=1454221 [4] Gartner, “Gartner Says Worldwide Smartphone Sales Soared in Fourth Quarter of 2011 with 47 Percent Growth,” 2012. http://www.gartner.com/it/page.jsp?id=1924314 [5] R. N. Calheiros, R. Ranjan, A. Beloglazov, C. A. F. De Rose and R. Buyya, “CloudSim: A Toolkit for Modeling and Simulation of Cloud Computing Environments and Evaluation of Resource Provisioning Algorithms,” Soft- ware: Practice and Experience, Vol. 41, No. 1, 2011, pp. 23-50. doi:10.1002/spe.995 In future work we would like to investigate how the payload differs due to different message formats. Addi- tionally, including devices with Android 4 in the bench- marking test can also be a useful extension to our work. Finally, conducting a more comprehensive energy con- [6] [6] R. C. Elsenpeter, T. Velte and A. Velte, “Cloud Comput- IJCNS J. HANSEN ET AL. 849 ing, A Practical Approach,” McGraw-Hill Osborne Media, New York, 2009. 2007, pp. 365-376. doi:10.1145/1247480.1247522 Copyright © 2012 SciRes. 2007, pp. 365-376. doi:10.1145/1247480.1247522 [16] MQTT.org, “MQ Telemetry Transport,” MQ Telemetry Transport, 2011. http://mqtt.org [7] A. Khajeh-Hosseini, D. Greenwood, J. W. Smith and I. Sommerville, “The Cloud Adoption Toolkit: Supporting Cloud Adoption Decisions in the Enterprise,” Software: Practice and Experience, Vol. 42, No. 4, 2012, pp. 447- 465. doi:10.1002/spe.1072 [17] The Deacon Project, “The Deacon Project,” 2012. http://deacon.daverea.com/about [18] XMPP Standards Foundation, “About XMPP,” 2011. http://xmpp.org/about-xmpp [8] P. Mell and T. Grance, “The NIST Definition of Cloud Computing,” 2011. http://csrc.nist.gov/publications/nistpubs/800-145/SP800- 145.pdf [19] M. Ohja, “Server Push with Instant Messaging,” Proceed- ings of the 2009 ACM Symposium on Applied Computing, ACM, New York, 2009, pp. 653-658. [20] Asmack, “Asmack,” 2012. http://code.google.com/p/asmack [20] Asmack, “Asmack,” 2012. http://code.google.com/p/asmack [9] Google, “What Is Google App Engine?” 2011. http://code.google.com/appengine/docs/whatisgoogleappe ngine.html [9] Google, “What Is Google App Engine?” 2011. [21] Smack, “Smack API,” 2012. http://www.igniterealtime.org/projects/smack [10] M. Armbrust, A. Fox, R. Griffith, A. D. Joseph, R. H. Katz, A. Konwinski, G. Lee, D. A. Patterson, A. Rabkin and M. Zaharia, “Above the Clouds: A Berkeley View of Cloud Computing,” 2009. http://www.eecs.berkeley.edu/Pubs/TechRpts/.../EECS-2 009-28.pdf [22] Google, “Quotas,” 2012. http://code.google.com/appengine/docs/quotas.html [23] Google, “Android Cloud to Device Messaging Frame- work,” 2011. http://code.google.com/android/c2dm [11] Google, “The Java Servlet Environment,” 2012. https://developers google com/appengine/docs/java/r [11] Google, “The Java Servlet Environment,” 2012. https://developers.google.com/appengine/docs/java/runtime [24] Google, “Android Cloud to Device Messaging: Quotas,” 2012. http://code.google.com/android/c2dm/quotas.html [12] I. Podnar, M. Hauswirth and M. Jazayeri, “Mobile Push: Delivering Content to Mobile Users,” Proceedings of 22nd International Conference on Distributed Computing Systems Workshops, Vienna, 2-5 July 2002, pp. 563-568. [25] Urban Airship, “Push Notifications,” 2012. http://urbanairship.com/products/push-notifications [26] Urban Airship, “Pricing,” 2012. http://urbanairship.com/pricing [13] C. Paniagua, S. N. Srirama and H. Flores, “Bakabs: Man- aging Load of Cloud-Based Web Applications from Mo- biles,” Proceedings of the 13th International Conference on Information Integration and Web-Based Applications and Services, ACM, New York, 2011, pp. 485-490. [27] Xtify, “Xtify Available Packages,” 2012. http://www.xtify.com/pricing.html [28] Xtify, “SDK Implementation Guides,” 2012. http://developer.xtify.com/display/sdk/SDK+Implementation+ Guides [14] J. Flinn and M. Satyanarayanan, “Managing Battery Life- time with Energy-Aware Adaptation,” ACM Transactions on Computer Systems, Vol. 22, No. 2, 2004, pp. 137-179. doi:10.1145/986533.986534 [29] Google, “Quotas and Limits,” 2012. https://developers.google.com/appengine/docs/java/xmpp/ overview#Quotas_and_Limits [15] S. Rivoire, M. A. Shah, P. Ranganathan and C. Kozyrakis, “JouleSort: A Balanced Energy-Efficiency Benchmark,” Proceedings of the 2007 ACM SIGMOD International Conference on Management of Data, ACM, New York, [30] Xtify, “Xtify Android XMPP Rich Notification Guide,” 2012. http://developer.xtify.com/display/sdk/Xtify+Android+X MPP+Rich+Notification+Guide IJCNS Copyright © 2012 SciRes.
https://openalex.org/W2150359400	https://cdr.lib.unc.edu/downloads/6969z742g	English	null	An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis	Human molecular genetics online/Human molecular genetics	2,014	cc-by	9,573	An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis Derek W. Morris1,{, Richard D. Pearson2,{, Paul Cormican1,{, Elaine M. Kenny1, ColmT.O’Dushlaine3,Louis-Philippe LemieuxPerreault2,4,EleniGiannoulatou2,DanielaTropea1, Brion S. Maher5, Brandon Wormley5, Eric Kelleher1, Ciara Fahey1, Ines Molinos1, Stefania Bellini1, Matti Pirinen2, Amy Strange2, Colin Freeman2, Dawn L. Thiselton5, Rachel L. Elves5, Regina Regan6, Sean Ennis6, Timothy G. Dinan7, Colm McDonald8, Kieran C. Murphy9, Eadbhard O’Callaghan10,{, John L. Waddington11, Dermot Walsh12, Michael O’Donovan13, Detelina Grozeva13, Nick Craddock13, Jennifer Stone3, Ed Scolnick3, Shaun Purcell3,14, Pamela Sklar3,14, Bradley Coe15, Evan E. Eichler15, Roel Ophoff16, Jacobine Buizer17, Jin Szatkiewicz18, Christina Hultman19, Patrick Sullivan18, Hugh Gurling20,{, Andrew Mcquillin20, David St Clair21, Elliott Rees13, George Kirov13, James Walters13, Douglas Blackwood22, Mandy Johnstone22, Gary Donohoe1, International Schizophrenia Consortium, SGENE1 Consortium, Francis A. O’Neill23, Wellcome Trust Case Control Consortium 2}, Kenneth S. Kendler5, Michael Gill1, Brien P. Riley5, Chris C. A. Spencer2,§ and Aiden Corvin1,§,∗ Kenneth S. Kendler5, Michael Gill1, Brien P. Riley5, Chris C. A. Spencer2,§ and Aiden Corvin1,§,∗ 1Department of Psychiatry and Neuropsychiatric Genetics Research Group, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland, 2Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK, 3Broad Institute and Center for Human Genetics Research of Massachusetts General Hospital, Boston, MA 02142, USA, 4Montreal Heart Institute, Universite´ de Montre´al, Montre´al, Que´bec H1T 1C8, Canada, 5Departments of Psychiatry and Human Genetics, Virginia Institute of Psychiatric and Behavioral Genetics, Virginia Commonwealth University, Richmond, VA 23284, USA, 6School of Medicine and Medical Science, University College Dublin, Ireland, 7Department of Psychiatry, University College Cork, Cork, Ireland, 8Department of Psychiatry, National University of Ireland, Galway, University Road, Galway, Ireland, 9Department of Psychiatry, RCSI Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland, 10DETECT Early Intervention in Psychosis Services, Dun Laoghaire, Co. Human Molecular Genetics, 2014, Vol. 23, No. 12 3316–3326 doi:10.1093/hmg/ddu025 Advance Access published on January 28, 2014 Human Molecular Genetics, 2014, Vol. 23, No. 12 3316–3326 doi:10.1093/hmg/ddu025 Advance Access published on January 28, 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. # The Author 2014. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. †The authors wish it to be known that, in their opinion, the ﬁrst three authors should be regarded as joint First Authors. ‡Deceased ∗To whom correspondence should be addressed at: Department of Psychiatry and Neuropsychiatric Genetics Research Group, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland. Tel: +353 8962467; Fax: +353 8963405; Email: acorvin@tcd.ie §These authors co-lead the study. }Membership is listed in Supplementary Material. § ‡Deceased. } # The Author 2014. Published by Oxford University Press. An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis Dublin, Ireland, 11Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland, 12Health Research Board, 73 Lower Baggot St, Dublin 2, Ireland, 13MRC Centre for Neuropsychiatric Genetics andGenomics,and Neuroscience andMentalHealthResearch Institute,CardiffUniversity, Heath Park, CardiffCF4 4XN, UK, 14The Mount Sinai Hospital, New York, NY 10029, USA, 15University of Washington School of Medicine, Howard Hughes Medical Institute, Seattle, WA 98195, USA, 16Department of Human Genetics, UCLA School of Medicine, Los Angeles, CA 90095, USA, 17Rudolf Magnus Institute, Universityof Utrecht, 3584 CG Utrecht, Netherlands, 18Universityof North Carolina, Chapel Hill, NC 27599-7264, USA, 19Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, SE-171 77 Stockholm, Sweden, 20Molecular Psychiatry Laboratory, Mental Health Sciences Unit, University College London, London WC1E 6BT, UK, 21Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen †The authors wish it to be known that, in their opinion, the ﬁrst three authors should be regarded as joint First Authors. ‡Deceased ∗To whom correspondence should be addressed at: Department of Psychiatry and Neuropsychiatric Genetics Research Group, Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland. Tel: +353 8962467; Fax: +353 8963405; Email: acorvin@tcd.ie Human Molecular Genetics, 2014, Vol. 23, No. 12 3317 AB25 2ZD, UK, 22Division of Psychiatry, University of Edinburgh, Royal Edinburgh Hospital, Edinburgh EH10 5HF, UK and 23Department of Psychiatry, Queen’s University, Belfast BT7 1NN, Northern Ireland Identifying rare, highly penetrant risk mutations may bean important step in dissectingthe molecular etiology of schizophrenia.We conducted a gene-based analysis of large (>100 kb), rare copy-number variants (CNVs) in the WellcomeTrust CaseControl Consortium2 (WTCCC2) schizophrenia sampleof 1564 casesand 1748controlsall from Ireland, and further extended the analysis to include an additional 5196 UK controls. We found association with duplications at chr20p12.2 (P 5 0.007) and evidence of replication in large independent European schizo- phrenia (P 5 0.052) and UK bipolar disorder case-control cohorts (P 5 0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identiﬁed 22 carriers in 11 707 cases and 10 carriers in 21 204 controls [meta-analysis Cochran–Mantel–Haenszel P-value 5 2 3 1024; odds ratio (OR) 5 11.3, 95% CI 5 3.7, 1]. Nineteen of the 22 cases and 8 of the 10 controls carried duplica- tions starting at 9.68 Mb with similar breakpoints across samples. INTRODUCTION sample of 1564 cases and 1748 controls, all from Ireland. We ﬁnd evidence of association with duplications at 20p12.2 and further support in a large independent European sample. All of the carriers were from the British Isles and an extended analysis including 32 911 Irish/UK subjects provided further association support. Byhaplotype analysisand sequencing,we show that the CNV is not the result of repeated de novo events and is an inher- ited risk mutation potentially inherited from a single European ancestor. This accords with emerging evidence for rare CNVs that are population-speciﬁc and associated with substantial illness risk (21,22), consistent with the ‘Clan Genomics’ concept (23). The mutation involves a tandem duplication of 148 951 bp at chr20:9,684,767–9,833,717(hg18) overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also known as PAK5). Other PAK family members modulate synaptic network development through a signaling pathway regulated by the schizophrenia risk gene DISC1 (24). We demonstrate that PAK7 is co-expressed with DISC1 in developing brain and further investigation of the role of the mutation in synaptic mechanisms salient to schizophrenia is warranted. Schizophrenia [MIM 181500] is a poorly understood, but severe, heritable mental disorder with a lifetime risk of ≏1%. The emer- ging genetic architecture includes a spectrum of risk variation from rare mutations of large effect, to common risk variants of small effect [odds ratio (OR) ,1.15] which collectively account foratleast25%ofsusceptibility(1–3).Throughgenome-wideas- sociation study (GWAS) and subsequent meta-analysis, more than 20 independent common loci have been conﬁrmed, but there are likely to be many more (4–6). A much smaller number of rare mutations of moderate or large effect have been identiﬁed, butthesewillbeparticularlyimportantinfacilitatingdissectionof the risk phenotype in model systems (7). The list of rare, highly penetrant schizophrenia mutations (OR ¼ 2–30) includes recurrent de novo copy-number variants (CNVs) involving deletions or duplications of large, genomic regions (.100 kb) (8,9), but also the accumulation of different CNV events at speciﬁc loci implicating single genes (NRXN1 [MIM 600565] (10–12) and VIPR2 [MIM 601970]) (13,14). Almost all of the conﬁrmed CNVs are also risk factors for other psychiatricordevelopmentalphenotypes[e.g.intellectualdisabil- ity, attention deﬁcit hyperactivity disorder (ADHD), autism] (15,16). This is in keeping with epidemiological and GWAS data supporting shared genetic liability between schizophrenia and other psychiatric disorders, in particular bipolar disorder (17–19). An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis By haplotype analysis and sequencing, we identiﬁed a tandem ∼149 kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P 5 2.5 3 10221), indicative of a singleancestralduplicationevent.Weconﬁrmedthebreakpointsin8/8carrierstestedandfoundco-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a po- tential molecular mechanism involving aberrant synapse development and plasticity. INTRODUCTION As current ﬁndings explain a modest proportion of total schizophrenia susceptibility, expanding the number of risk mutations will be important in understanding molecular etiology but also the relationships between these clinical disorders (20). -sided CMH test used for replication and combined analyses due to multisite nature of samples. NS, non-signiﬁcant (P . 0.1). ludes non-Irish and non-UK cases (n ¼ 1434) and controls (n ¼ 1283) from the ISC Replication 1 sample. udes 2507 additional UK controls from the People of the British Isles (POBI study) with two PAK7 duplication carriers which were unavailable at the time of discovery analysis. the population frequency of what are rare events, we examined the three novel loci in an extended control sample (n ¼ 6944) in- cludingUKcontrolsfrom WTCCC2 [2533controlsfrom theUK National Blood Service (NBS) and 2663 from the UK 1958 Birth Cohort (58BC)]. We identiﬁed chr1q21.1 risk duplications in four cases, chromosome 15q13.3 deletions in three cases and a single case with the large 22q11.2 deletion with no similar events seen in controls from the discovery analysis. Details of our ﬁndings at all previously reported schizophrenia CNV risk loci, with locus coordinates, are provided in Supplementary Material, Table S3a. Further, we provide details of all large (.100 kb), rare CNVs identiﬁed in our discovery sample in cases and controls (Supplementary Material, Table S3b). aFisher’s exact test (two-tailed P-value). bOne-sided CMH test used for replication and combined analyses due to multisite nature of samples. NS, non-signiﬁcant (P . 0.1). cExcludes non-Irish and non-UK cases (n ¼ 1434) and controls (n ¼ 1283) from the ISC Replication 1 sample. dIncludes 2507 additional UK controls from the People of the British Isles (POBI study) with two PAK7 duplication carriers which were unavailable at the time of discovery analysis. Replication phase association evidence at chr20p12.2 Three loci were carried forward for replication (Table 1) in the independent International Schizophrenia Consortium (ISC) dataset of 3111 cases and 2267 controls from the UK, Portugal, Sweden and Bulgaria (8). Only the chr20p12.2 locus containing p21 Protein-Activated Kinase 7 (PAK7, also known as PAK5) showed evidence for association in the replication sample, with six duplication events, all in UK cases, and none in controls (P ¼ 0.052). Because this is a rare event, and absent in the control population, the reported association result was the most signiﬁcant P-value obtainable without a larger sample size. As all of the carriers were identiﬁed in samples from the British Isles, we investigated the locus for additional evidence of association in an independent sample of bipolar disorder in the WTCCC1 (n ¼ 1697) (25) and University College London (UCL) (n ¼ 546) samples (26). The controls included independ- ent UK controls from the UCL study (n ¼ 510) and 10 259 cases ascertained for nonpsychiatric disorders in WTCCC1. We iden- tiﬁed four duplication carriers in cases and ﬁve carriers in the control sample, provided further nominal evidence of associ- ation (P ¼ 0.047). ( ) WeperformedacombinedanalysisofallIrish/UKsubjectsin- cludingadditionalpsychosiscasesfromtheCLOZUKstudy(27) (n ¼ 6223) and independent controls from the WTCCC2 UK control sample (n ¼ 7703) (28,29). Combining the evidence across studies, under the assumption of the same effect on risk in each strata, gave a meta-analysis Cochran–Mantel–Haenszel (CMH) P-value ¼ 2 × 1024 (OR ¼ 11.3, 95% CI ¼ 3.7, 1) with 22 carriers in 11 707 psychosis cases and 10 carriers in 21 204 controls. Discovery phase evidence of association at four loci We observed evidence of association (P , 0.05) at four loci in the Irish discovery dataset of 1564 cases and 1748 controls; 20p12.2(PAK7 [MIM 608038]); 2cen-q13 (ANKRD36B, COX5B [MIM 123866]; ACTR1B [MIM 605144]); 3p25.1 (MRPS25 [MIM 611987], ZFYVE20 [MIM 609511]) and a pre- viously conﬁrmed schizophrenia risk locus [chr1q.21 (CHD1L [MIM 613039]) locus (8,9)]. Association results for the three novel loci are presented in Table 1. To improve our estimate of We report a gene-based analysis of large (.100 kb), rare [,1% minor allele frequency (MAF)] CNVs in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia Human Molecular Genetics, 2014, Vol. 23, No. 12 3318 Table 1. Association analysis of large duplications at three loci in psychosis case–control samples Locus Discovery Replication 1 Replication 2 Combined analysis Chr Position Gene(s) Irish SZ cases (n ¼ 1564) Irish controls (n ¼ 1748) P-valuea UK 58C + NBS controls (n ¼ 5196) P-valuea ISC SZ cases (n ¼ 3111) ISC controls (n ¼ 2267) P-valueb BPD cases (n ¼ 2243) UK controls (n ¼ 10 769) P-valueb Irish and UK cases (n ¼ 11 707)c (including 6223 CLOZUK samples) Irish and UK controls (n ¼ 21 204)d (including 7703 WTCCC2 controls) P-valueb OR (95% CI) 20p12.2, PAK7 5 0 0.023 3 0.007 6 0 0.052 4 5 0.047 22 10 0.0002, 11.3, (3.7, 1) 2cen-q13, COX5B ANKRD36B ACTRIB 12 1 0.001 9 0.0001 3 2 NS 3p25.1, ZFYVE20 MRPS25 6 0 0.011 1 0.0002 0 1 NS aFisher’s exact test (two-tailed P-value). bOne-sided CMH test used for replication and combined analyses due to multisite nature of samples. NS, non-signiﬁcant (P . 0.1). cExcludes non-Irish and non-UK cases (n ¼ 1434) and controls (n ¼ 1283) from the ISC Replication 1 sample. dIncludes 2507 additional UK controls from the People of the British Isles (POBI study) with two PAK7 duplication carriers which were unavailable at the time of discovery analysis. Evidence for a single founder risk mutation at PAK7 Based on the array data, we examined the inferred breakpoints for the chr20p12.2 duplications, and the estimated start and stop coordinates for the 32 duplication carriers (Fig. 1, Supple- mentary Material, Table S5). Nineteen of the 22 duplication car- riers in the cases and 8 of 10 carriers from the controls exhibited duplications of 132–146.5 kb with very similar start positions (≏9.68 Mb), apparently similar breakpoints across samples and no evidence of ﬂanked segmental duplications. The evi- dence for association at the locus in the Irish/UK sample appeared to be driven by these duplication cases. The remaining ﬁve individuals carried duplications occurring on different Human Molecular Genetics, 2014, Vol. 23, No. 12 331 3319 Figure1.Inferredpositionofduplicationeventsatthechr20p12.2locusinbuildhg18.SZ,schizophrenia.BD,bipolardisorder.SeeSupplementaryMaterial,TableS5 for exact start/stop coordinates. Duplications in controls are colored light blue and SZ or BP cases are colored dark blue. Figure1.Inferredpositionofduplicationeventsatthechr20p12.2locusinbuildhg18.SZ,schizophrenia.BD,bipolardisorder.SeeSupplementaryMaterial,TableS5 for exact start/stop coordinates. Duplications in controls are colored light blue and SZ or BP cases are colored dark blue. Figure2.Capillarysequenceof theuniqueregioninthemiddleofthetwocopiesofthetandemrepeatsequenceat thePAK7duplication.Theﬁrstcopyofthesequence ends at chr20:9,833,717(hg18) and the second copy starts with sequence from chr20:9,684,767(hg18), thereby deﬁning the breakpoints. A 15 bp sequence is present between the two copies. Figure1.Inferredpositionofduplicationeventsatthechr20p12.2locusinbuildhg18.SZ,schizophrenia.BD,bipolardisorder for exact start/stop coordinates. Duplications in controls are colored light blue and SZ or BP cases are colored dark blue. Figure1.Inferredpositionofduplicationeventsatthechr20p12.2locusinbuildhg18.SZ,schizophrenia.BD,bipolardisorder.SeeSupplementaryMaterial,TableS5 for exact start/stop coordinates. Duplications in controls are colored light blue and SZ or BP cases are colored dark blue. Figure2.Capillarysequenceof theuniqueregioninthemiddleofthetwocopiesofthetandemrepeatsequenceat thePAK7duplication.Theﬁrstcopyofthesequence ends at chr20:9,833,717(hg18) and the second copy starts with sequence from chr20:9,684,767(hg18), thereby deﬁning the breakpoints. A 15 bp sequence is present between the two copies. Figure2.Capillarysequenceof theuniqueregioninthemiddleofthetwocopiesofthetandemrepeatsequenceat thePAK7duplication.Theﬁrstcopyofthesequence ends at chr20:9,833,717(hg18) and the second copy starts with sequence from chr20:9,684,767(hg18), thereby deﬁning the breakpoints. A 15 bp sequence is present between the two copies. compared with the level of relatedness of random Irish indivi- duals, arguing against a very recent event (see Supplementary Material, Fig. S1). haplotype backgrounds and are likely to represent different molecular events. To explore whether this could represent a single ancestral mu- tationevent,weexamined haplotypesharingintheﬁveIrishcase samples in linkage disequilibrium (LD) blocks (termed Hap Block 1 and Hap Block 2) immediately 5′ and 3′ of the inferred duplication. Haplotypes were phased in PLINK with a probabil- ity .0.98. All ﬁve Irish cases carried a copy of the same haplo- type in both Hap Block 1 (CCTT, f ¼ 0.188) and Hap Block 2 (TCAA, f ¼ 0.208) (Supplementary Material, Tables S6 and S7). Evidence for a single founder risk mutation at PAK7 We extended theanalysis to include 18 of the22 UK carriers (as these had been genotyped on the same Affymetrix platform) and found that all carried copies of the CCTT and TCAA haplo- types ﬂanking the duplication (Supplementary Material, Table S8). Given the low level of LD between these blocks (D′ ¼ 0.22, r2 ¼ 0.02) the probability of making these observa- tions if the duplication was the result of repeated de novo muta- tions is extremely low (P ¼ 2.5 × 10221), providing support for a single ancestral duplication event. We did not ﬁnd an increased level of relatedness among the Irish PAK7 duplication carriers , g ) Next, we conﬁrmed the duplication event in one individual (IRL_101) using an Agilent custom designed comparative genomic hybridization (CGH) microarray with high probe density in the PAK7 region. The breakpoints identiﬁed by CGH (chr20:9,684,902–9,833,151) map closely to those predicted by theSNParrayanalyses(chr20:9,685,413–9,831,947;Supplemen- taryMaterial,Fig.S2).Theeventoverlapsexon1andenhancerand promoter regions adjacent to exon 1 in all four known PAK7 transcripts, and exon 2 in two alternative transcripts and is absent from the Database of Genomic Variants (http://projects. tcag.ca/variation/). By capillary sequencing this individual, we identiﬁed a tandem duplication of 148 951 bp at chr20:9,684, 767–9,833,717(hg18) with a 15 bp sequence inserted between theﬁrstandsecondcopiesoftherepeatedsequence(Fig.2).Byse- quencing the ﬁve remaining Irish carriers and two of the UK car- riers (SCOT_101, SCOT_201), we conﬁrmed that all shared exactly the same breakpoints with the same 15 bp sequence Human Molecular Genetics, 2014, Vol. 23, No. 12 3320 Figure 3. Familial pedigrees for patients from three families (IRL_6, IRL_1, SCOT_1) who were found to be positive for PAK7 duplications. The index indi- vidual in each family screened is indicated with an arrow. Individuals positive (PAK7 + VE) or negative (PAK7 2 VE) for PAK7 are indicated. Filled symbols indicate individuals with psychotic disorders (SCZ, schizophrenia; SCA, schizoaffective disorder; BPA, bipolar affective disorder; PDNOS, psych- otic disorder not otherwise speciﬁed). Partial vertically shaded symbol indicates depressive disorder (DEP, depression). Partial horizontal shaded symbol indi- cates learning disability (LD). Cause of death where known: SUIC, suicide; BA, birth asphyxia; CT, cerebral tumor. European populations. Details of the cohorts analyzed are included in Supplementary Material, Table S1. The estimated control frequency for the mutation in the UK/Irish population is low (f ¼ 0.037%) and most of these cohorts were underpow- ered to provide an accurate frequency estimate for what is a rare event. Functional investigation of PAK7 insertedbetweenthetwocopiesoftherepeatsequence,suggesting that this represents the same duplication event. The p21-activated kinases (PAKs) are a family of serine/threo- nine protein kinases, which are regulated by the Rho family of small G proteins and are involved in multiple intracellular sig- naling pathways. Six PAK genes are expressed in human and based on their regulatory functions are classiﬁed into Group I (PAK 1-3) and Group II (PAK4-6) members (30). Group I PAKs are activated by RAC-PAK signaling to promote axon connectivity, and synapse formation, in the developing brain in a pathway regulated by another schizophrenia risk gene DISC1 [MIM:60521] (31). Aberrant synaptic network develop- mentand maintenance represents aplausiblemolecularmechan- ism for psychosis susceptibility and mutations involving other Group I PAKs (PAK2 (the 3q29 microdeletion syndrome locus [MIM 609425]) and PAK3 [MIM:300558]) are known to be associated with neurodevelopmental syndromes characterized by psychosis (32). PAK7 is a brain-speciﬁc isoform within the cell it is localized to ﬁlipodia, where it has been shown to promote the induction of neurite outgrowth, ﬁlopodium forma- tionandsynapticvesicletrafﬁcking(33).TheGroupIIPAKs,in- cluding PAK7, differ structurally from Group I members and their mechanism of activation requires clariﬁcation. Assessment of additional family members of carriers We wereable to accessadditional family membersof two cases to test for evidence of co-segregation of the duplication with illness (see Fig. 3). In the family of IRL-101, there were three other sur- viving siblings, none of whom had a diagnosis of schizophrenia or arelatedmajormentaldisorder.Onesister(IRL_102)agreedtobe tested. She had experienced a depressive episode after bereave- mentanddidnotcarrytheduplication.Bygoingbacktotheorigin- alGWASdata,weidentiﬁedanotherIrishcase(IRL_601)thathad been excluded from the primary analysis because they are related toIRL_101basedonaproportionofidentity-bydescentestimated at 0.033. IRL_601 is a female patient with a history of Schizo- affective Disorder.She is the youngestina sibship of ﬁve children of which four were affected with a major psychotic disorder. We conﬁrmed that two affected brothers (IRL_603 (bipolar I dis- order), IRL_604 (psychotic disorder, not otherwise speciﬁed)) also carry the PAK7 duplication. The remaining affected brother [IRL_602 (bipolar I disorder)] died by suicide. We tested one of the eight offspring of IRL_601, and this unaffected individual didnotcarrytheduplication.IntheSCOT_1family,weconﬁrmed that the duplication was inherited from the mother (SCOT_103). Neither of the parents was affected clinically, but there was a family history of depression in both families, and a brother with a history of psychosis did not carry the duplication (SCOT_104). q To testwhether PAK7 maybe undersimilar regulatory control to Group I PAKs during early brain development, we investi- gated the relationship between PAK7 and DISC1. Examining Brainspan data (brainspan.org), we identify signiﬁcant negative correlation between DISC1 and PAK7 expression in whole human brain throughout life (r2 ¼ 20.297 to 0.867). The stron- gest correlations are for the ﬁrst and last trimester of pregnancy and the ﬁrst 5 years of life, with the level of correlation falling through adulthood. In a co-immunoprecipitation experiment, we conﬁrmed interaction between PAK7 and DISC1 in synapto- neurosomal preparations from full mouse brain at postnatal day 8–10 [(P8–10); Fig. 4A]. Next, we ran the reciprocal Evidence for a single founder risk mutation at PAK7 We failed to identify any carriers in large Icelandic (.72 000 individuals) and Finnish samples (≏3800 indivi- duals), but did ﬁnd carriers in populations from the Eastern USA, the Netherlands, Sweden and Denmark. We were able to unambiguously phase haplotypes in ﬁve US control carriers with available Affymetrix data, conﬁrming that all of these indi- viduals shared the same haplotype background as the Irish/UK cases, suggesting that they carry the same mutational event al- though the samples were not available for conﬁrmatory sequen- cing. The other European samples were genotyped on a different array platform and where we could deﬁnitively phase haplo- types; this conﬁrmed the same haplotype background. These data suggest that the mutation is present in other European popu- lations. The limited number of cases available precluded formal association analyses, but we identiﬁed four additional cases with the duplication in Dutch, Danish and Swedish cohorts. Informa- tion on the clinical characteristics, illness course, co-morbidity and family history for all mutation case carriers is provided in Supplementary Material, Table S9. Figure 3. Familial pedigrees for patients from three families (IRL_6, IRL_1, SCOT_1) who were found to be positive for PAK7 duplications. The index indi- vidual in each family screened is indicated with an arrow. Individuals positive (PAK7 + VE) or negative (PAK7 2 VE) for PAK7 are indicated. Filled symbols indicate individuals with psychotic disorders (SCZ, schizophrenia; SCA, schizoaffective disorder; BPA, bipolar affective disorder; PDNOS, psych- otic disorder not otherwise speciﬁed). Partial vertically shaded symbol indicates depressive disorder (DEP, depression). Partial horizontal shaded symbol indi- cates learning disability (LD). Cause of death where known: SUIC, suicide; BA, birth asphyxia; CT, cerebral tumor. Figure 3. Familial pedigrees for patients from three families (IRL_6, IRL_1, SCOT_1) who were found to be positive for PAK7 duplications. The index indi- vidual in each family screened is indicated with an arrow. Individuals positive (PAK7 + VE) or negative (PAK7 2 VE) for PAK7 are indicated. Filled symbols indicate individuals with psychotic disorders (SCZ, schizophrenia; SCA, schizoaffective disorder; BPA, bipolar affective disorder; PDNOS, psych- otic disorder not otherwise speciﬁed). Partial vertically shaded symbol indicates depressive disorder (DEP, depression). Partial horizontal shaded symbol indi- cates learning disability (LD). Cause of death where known: SUIC, suicide; BA, birth asphyxia; CT, cerebral tumor. Is the mutation present in other European ancestry populations? To assess whether the mutation was exclusive to the British Isles or present elsewhere in Europe, we examined additional 3321 Human Molecular Genetics, 2014, Vol. 23, No. 12 Figure 4. PAK7 and DISC1 interact in mouse brain synapses. (A) Immunoprecipitation of PAK7 and DISC1 from synaptoneurosomal extracts of adult mouse brains. The extract was immunoprecipitated for PAK7 and the eluate was immunostained for DISC1. (B) Reciprocal experiment described in A: immunoprecipitation of DISC1 followed by immunostaining of the eluate with antiPAK7. In both A and B, the samples immunoprecipitated with control IgG showed no signal, showing the speciﬁcity of the PAK7–DISC1 interaction. An additional control in (C) shows that the brain extracts before immunoprecipitation have the same amount of loading control (b-actin). (D–N) Immunostaining for PAK7 (green) and DISC1 (red) in brain sections containing Hippocampus and Cortex of P7 (D–I) and Adult (J–O) mice. The experiment shows an overlapping (yellow) of PAK7 and DISC1 in puncta, conﬁrming a synaptic interaction between the two proteins. The co-localization is particularly evident in the adult (J–O) preparation. Figure 4. PAK7 and DISC1 interact in mouse brain synapses (A) Immunoprecipitation of PAK7 and DISC1 from synaptoneurosomal extracts of adult mouse brains Figure 4. PAK7 and DISC1 interact in mouse brain synapses. (A) Immunoprecipitation of PAK7 and DISC1 from synaptoneurosomal extracts of adult mouse brains. The extract was immunoprecipitated for PAK7 and the eluate was immunostained for DISC1. (B) Reciprocal experiment described in A: immunoprecipitation of DISC1 followed by immunostaining of the eluate with antiPAK7. In both A and B, the samples immunoprecipitated with control IgG showed no signal, showing the speciﬁcity of the PAK7–DISC1 interaction. An additional control in (C) shows that the brain extracts before immunoprecipitation have the same amount of loading control (b-actin). (D–N) Immunostaining for PAK7 (green) and DISC1 (red) in brain sections containing Hippocampus and Cortex of P7 (D–I) and Adult (J–O) mice. The experiment shows an overlapping (yellow) of PAK7 and DISC1 in puncta, conﬁrming a synaptic interaction between the two proteins. The co-localization is particularly evident in the adult (J–O) preparation. experiment and demonstrated immunoprecipitation of DISC1 followed by immunostaining of the eluate with antiPAK7 (Fig. 4B). Finally, we explored the regional and temporal evi- dence for co-expression in brain regions implicated in schizo- phrenia etiology (hippocampus and cortex). In this analysis, we co-immunostained PAK7 and DISC1 in P7 and adult (.P60) mice. Is the mutation present in other European ancestry populations? We ﬁnd clear overlap in perisomal puncta, which conﬁrms that the two proteins interact at the synapse, par- ticularly in adult mouse brain (Fig. 4D–O). This suggests that PAK7 is developmentally co-expressed with DISC1 and may have a speciﬁc functional role at the synapse. different, generally de novo mutations at a single locus (e.g. NRXN1, VIPR2). At the PAK7 locus, we identiﬁed a single founder event with a 15 bp sequence inserted between the two copies of the tandem duplication. From the 1000 Genomes Project data, most tandem duplications of this type are formed by fork stalling and template switching as detailed in the micro-homology-mediated break-induced replication model (34,35). We identiﬁed 2653 genes affected by a CNV (.100 kb but ,10 Mb) in at least one individual in our study. However, only 382 genes had the four or more overlapping CNVs required to achieve P , 0.05 in our discovery sample. Based on our data, the overall contribution of CNVs to psychosis susceptibility risk is difﬁcult to estimate as we could not exclude rarer or smaller events. Ongoing CNV analysis of the Psychiatric Genomics Consortium dataset will be important in more comprehensively investigating rare, large CNV events (17). DISCUSSION We report evidence that rare, chr20p12.2 duplications increase risk for the major psychotic disorders, schizophrenia and bipolar disorder. Previous schizophrenia studies have identiﬁed at least eight rare CNVs of strong effect (genotypic relative risks of 4–20) (5). Most of these CNVs have been identiﬁed because they recur in the population, either as relatively large events im- plicating many genes and arising from nonallelic homologous recombination (NAHR), or through the accumulation of By haplotype analysis and sequencing, we established that the association is driven by a ≏149 kb duplication at the gene PAK7 with a likely common European ancestor. Inherited mutations have been described contributing to other neurological disorders, but their relative contribution to schizophrenia Human Molecular Genetics, 2014, Vol. 23, No. 12 3322 susceptibilityisunknown(21,22,36).Sucheventsarelikelytobe recent in origin and differentiated between populations making them difﬁcult to replicate across diverse collections using stand- ard gene association methods in large multicohort CNV studies (37–39). Although a single variant is unlikely to inﬂuence the overall population risk for a disorder, founder risk mutations can be identiﬁed through association analysis in speciﬁc popula- tions. By examining cases excluded, through relatedness QC, from our original Irish GWAS dataset, we identiﬁed an addition- al duplication carrier related at a distance of ﬁve meioses to one oftheindexcases.Withinthefamilyofthisadditionalcarrier,we found evidence of co-segregation of the duplication with psych- osis in two affected siblings. Identifying cryptic relationships between individuals in available GWAS datasets may be helpful in identifying extended pedigrees for analysis as a com- plementary approach to existing association methods. thephenotypicimplicationsofcarryingrareeventsinthegeneral population and this is one of future goals (e.g. The Generation Scotland Family Health Study, UK Biobank and ‘Growing Up in Ireland’ Study). Further clinical and genetic assessment of patient carriers and their families will also be important in quan- tifying the penetrance and range of phenotypic expression of the PAK7 duplication. p Schizophrenia is a neurodevelopmental disorder and adisease model involving aberrant synaptic network development has beenproposed(44).GroupIPAKsplayaroleinthedevelopment of these networks through a signaling pathway regulated by an established susceptibility gene (DISC1) (45). PAK7 is a brain- speciﬁc isoform, within the cell it is localized to ﬁlipodia, where it has been shown to promote the induction of neurite out- growth, ﬁlopodium formation and synaptic vesicle trafﬁcking (33). To clarify its mechanism of activation, we performed func- tional studies that conﬁrmed interaction between PAK7 and DISC1,suggestingthatthisgenemaybeundersimilarregulatory control to other PAK family members. DISCUSSION Furthermore, this inter- action occurs at the synapse and is particularly evidence in adult mouse brain suggesting a role in synaptic plasticity. The risk duplication identiﬁed in this study overlaps the ﬁrst two exons and enhancer and promoters regions in the known transcripts of PAK7. Because PAK7 is exclusively brain expressed, the impact of the duplication on gene expression and the genetic mechanism involved are yet to be determined. This is important as we identiﬁed other larger duplication events at the locus in two control samples, but do not know if these would have the same or different effects on gene function. From existing animal models of PAK7, we do know that knock- out mice are viable with no obvious developmental abnormal- ities but PAK6/PAK7 double knockout mice show behavioral and learning deﬁcits suggesting functional redundancy between these isoforms (40). Genomic studies in carriers of thefoundermutation willbeimportantasadditionalrisk variants or mutations (e.g. in DISC1 or PAK6) may be contributing to risk in this extended pedigree. In conclusion, we have identiﬁed a psychosis risk duplication at the gene PAK7, with evidence that it is inherited from a common ancestor. The gene functions in the development and maintenance of synaptic networks and may be under the regula- tory control of DISC1. Many of the cases segregating the DISC1 translocation in the original Scottish schizophrenia pedigree were affected with schizoaffective disorder or mood disorders as was the case in the largest PAK7 pedigree we investigated, and we also found evidence for association with bipolar disorder (46). This suggests a broader molecular risk mechanism for psychosis. A critical next step will be to understand how the du- plication impacts on gene expression and function so that it can be investigated in model systems. As PAK7 is exclusively brain-expressed, this will require further experimental work. The PAK7 risk mutation is rare, but based on data from the British Isles, we estimate the exposed attributable risk for car- riers is likely to be substantial (41). We attempted to date the origin of the duplication by examining haplotype sharing in this genomic region. After characterizing the haplotype patterns in the region, we were unable to make robust inference: further family data will be required to accurately phase haplotypes for this rare event. Study subjects Almost all of the risk CNVs identiﬁed to date in the schizo- phrenia literature also increase susceptibility to other develop- mental phenotypes including intellectual disability, autism and seizure disorder. Only four of our cases received a full cognitive assessment (including IQ measurement), and all were assessed as being within the normal range of cognitive function. One in- dividual had a history of co-morbid language delay and another had a history of seizure disorder. Although most of the cases had schizophrenia, seven had bipolar disorder or schizo- affective disorder. This is in keeping with evidence of shared genetic etiology between these disorders, although the evidence for CNV involvement in bipolar disorder has until now been more equivocal (42,43). Having identiﬁed carriers of the dupli- cation in control cohorts, we were interested in testing whether ‘control’ individuals had psychiatric or developmental pheno- types. In almost all studies (including the individual WTCCC1 studies), controls were not screened for mental disorder and little if any developmental information was available. Within theUKandIrelandhealthbiobank,dataarenowbecomingavail- able on a sufﬁciently large scale to allow us to directly examine The discovery sample included 1564 cases and 1748 controls from the Irish Schizophrenia Genomics Consortium/WTCCC2 GWAS study which has previously been described (47). Partici- pants, from the Republic of Ireland or Northern Ireland, were interviewed using a structured clinical interview and diagnosis of schizophrenia (n ¼ 1418) or a related disorder [schizoaffect- ive disorder (n ¼ 182); schizophreniform disorder (n ¼ 6)] was made by the consensus lifetime best estimate method using DSM-IV criteria. Control subjects were ascertained with written informed consent from the Irish GeneBank and repre- sented blood donors from the Irish Blood Transfusion Service. Cases and controls met the same ethnicity criteria (Irish origin with all four grandparents born in Ireland or the UK). To improve our estimate of the population frequency of what are rare events, we examined nominally signiﬁcant loci in an extended control sample (n ¼ 6944) including UK controls from WTCCC2 [2533 controls from the UK National Blood Service (NBS) and 2663 from the UK 1958 Birth Cohort (58BC)] (28). Human Molecular Genetics, 2014, Vol. 23, No. 12 3323 and Illumina 1.2M-Duo arrays as part of the wider WTCCC2 study. We called CNVs on the Illumina platform using Quan- tiSNP (51) and excluded those with log Bayes factor ,10, length ,100 kb or .10 Mb. Study subjects We excluded samples with more than 10 CNVs in total, with more than 10 Mb of total CNV length, or failed SNP quality control. This identiﬁed the same fourindividualsastheonlyPAK7duplicationcarriersacrossdif- ferent arrays and callingalgorithms. For theadditional European ancestry cohorts, CNV calls were made as described in the primary publications [see Supplementary Material, Table S1 and references (5,9,48,49)]. The CLOZUK calling method is described in Guha et al. (2013) (52). The ﬁrst phase of replication examined nonoverlapping sub- jects from the ISC dataset (8). To follow-up evidence of associ- ation in the British Isles, we performed further association analysis in bipolar disorder cases and controls from the UK/ Ireland. The WTCCC1 bipolar disorder sample included 1697 cases recruited throughout the UK. Most of the cases had a diag- nosis of bipolar I disorder (71%) or schizoaffective disorder (15%). Controls were nonpsychiatric disease case participants in the WTCCC1 study (n ¼ 10 259) (25). The UCL bipolar dis- order sample included 546 individuals (97% bipolar I disorder) and comparison subjects (n ¼ 510) with no personal or ﬁrst- degree history of any mental disorder and from a similar UK or Irish ancestry, based on the origin of all four grandparents (26). Finally, in an extended analysis, we included additional psychosis cases and controls from the UK. The additional psych- osiscaseswereofCaucasianoriginandcamefromtheCLOZUK sample (n ¼ 6223) (27). The CLOZUK sample consists of patients taking the antipsychotic medication Clozapine, a drug reserved for the treatment-resistant psychosis patients in the UK. The remaining control subjects were ascertained as described in previous WTCCC2 studies from the UK National Blood Service (NBS) (n ¼ 2533); the UK 1958 Birth Cohort (n ¼ 2663); the People of the British Isles (POBI) study and from(28,29).Wealsoinvestigated additionalEuropeanancestry cohorts for evidence of the PAK7 duplication and details on the samples tested, case and/or control numbers and genotyping platform are provided in Supplementary Material, Table S1 (5,9,48,49). CNV analysis Weconductedagene-basedanalysisusinggeneboundariesfrom UCSC Genome Browser refGene (hg18) and identiﬁed 2653 genes impacted (from the transcription start to end point) by a CNV (.100 kb but ,10 Mb) in at least one individual. Three hundred eighty-two genes had at least four overlapping CNVs (required to achieve P , 0.05) in the discovery data, represent- ing a smaller number of loci, as some CNVs overlap multiple genes. Fisher’s exact tests were used to calculate P-values in the discovery analysis and a ﬁxed-effects CMH test to assess the evidence across replication cohorts. Haplotype analysis We analyzed carriers of the ‘common’ PAK7 duplication at chr20:9,685,413–9,831,947, which is carried by 27 of 32 indivi- duals with duplications overlapping the PAK7 locus in the British Isles samples. Haplotype analysis used a core set of Affy- metrix SNPs that had been genotyped in23 ofthe samples and all genotype data were converted to the forward strand. The four samples omitted were from the CLOZUK sample genotyped on Illumina where no suitable proxy SNPs were available for analysis. From HapMap data, the start of the duplication sits in a haplotype block that extends from rs2423462 (9682770) to rs2423467 (9689876). The part of this block that is outside the region, termed Hap Block 1 is from rs2423462 (9682770) to rs742450 (9684493). The duplication ends in a region between haplotype blocks. Immediately, 3′ of the duplication is a haplo- type block that extends from rs6057009 (9840327) to rs6118819 (9867572) (Hap Block 2). We have focused on haplotypes outside the duplication region, to analyze diploid genotypes, and limited the analysis to regions of high LD where haplotypes could be phased. In the absence of family data, extended haplo- types outside these local blocks could not be accurately phased. Haplotype frequencies and phased haplotypes, with a probabil- ity .0.98, were estimated using the –hap and –hap-phase functions in Plink (see Supplementary Material, Tables S6 and S7). For either Hap Block, the chances that all 23 samples would carry at least one copy of a speciﬁc haplotype, i.e. be either heterozygous or homozygous for this haplotype q ¼ [q2 (probability of homozygous q) + 2∗q∗(1 2 q) (probability of homozygous q)]23 ¼ (2q 2 q2)23 where q is the haplotype fre- quency. This probability was calculated for both Hap Blocks and in the absence of LD between the blocks, these numbers were multiplied together to determine the combined probability Free ﬂoating immunohistochemistry Synaptoneurosome preparation was performed from full mouse brain at P8–10 as described elsewhere with modiﬁcations (53). Brieﬂy, brain tissue was glass/glass homogenized in ice-cold lysis buffer daily-fresh made until complete lysis. The protein extraction buffer contained 10 mM Tris Base, 0.1 M sodium chloride, 4 mM EDTA, 0.1% NP40 and was added with protease inhibitorcocktail(RocheDiagnostic).Thehomogenatewascen- trifuged at 1000g × 10 min at 48C inorder toseparate heavy cel- lular membranes. The resulting supernatant was passed through two 105 mm polypropylene mesh (Amazon, USA) and a 5 mm nitrocellulose ﬁlter (Millipore) and ﬁnally centrifuged at 1000 g × 15 min at 48C. The obtained synaptoneurosomal- depleted supernatant contains cytoplasmic proteins and was collected in fresh tubes; while the pellet, which is enriched in synaptoneurosomal proteins, was resuspended in protein extrac- tion buffer. Protein concentration was measured by spectropho- tometerquantiﬁcation(Nanodrop,ND8000)inbothcytoplasmic and synaptoneurosomal-enriched extracts for further analysis. Free ﬂoating immunohistochemistry Forhistologicalstaining,braintissuefrommiceatthreedifferent developmentalstageswasused:P7(postnatalday7)andP . 60. The brains, previously ﬁxed in 4%paraformaldehyde/PBS and kept in 20%sucrose/PBS at 48C, were sliced using a vibratome (Leica) to obtain 50 mm-thick coronal sections. After 5 min in- cubation with 0.1% hydrogen peroxide, sections were washed in PBS, and moved for 1 h into a PBS blocking buffer containing 10% goat serum and 3% Triton for tissue permeabilization. Sec- tions were then incubated for ≏36 h in blocking buffer added with the following primary antibodies: anti-PAK7 (polyclonal goat, Santa Cruz Biotechnology, sc-22155, 1:750), anti-DISC1 (polyclonal rabbit, Invitrogen, 40–6900, 1:500), anti-Synapsin 1/2 (polyclonal guinea pig, Synaptic Systems, 106 004, 1:1000). Primary antibody probing was followed by several PBS washes for a total time of 2 h. Sections were then moved to a secondary antibodies solution made of 0.05%Triton-X100 and added with the following ﬂuorofore-conjugated antibodies: DyLight 488 donkey anti-goat IgG (Jackson ImmunoResearch Labs, 705-485-003, 1:500), DyLight 649 goat anti-rabbit IgG (Jackson ImmunoResearch Labs, 111-495-003, 1:500) and Cy3 goat anti-guinea pig IgG (Jackson ImmunoResearch Labs, 106-165-003, 1:500). After 2 h, sections were washed for 15 min in PBS and mounted onto microscope slides using media containing DAPI (Vector H-1200). Co-immunostaining of PAK7 and DISC1 was conﬁrmed by confocal microscope imaging (Zeiss, ×63 objective) in hippocampus and brain cortex ﬁelds at each considered age. Western blot was carried out by standard procedures with the following speciﬁcations: protein samples were fractioned through 8 or 10% acrylamide gel and blotted onto PVDF mem- brane. CNV calling and validation We report a schizophrenia study investigating large (.100 kb), rare (,1% MAF in all samples) CNVs using data from the WTCCC2. All discovery samples were genotyped using the Affymetrix 6.0 platform either at the Affymetrix (Santa Clara, CA, USA) or at the Broad Institute (Cambridge, MA, USA) la- boratories.SampleswereprocessedusingtheWTCCC2pipeline and quality control details have been reported previously (27,47). CNV calls were created using Birdseye from Birdsuite (version 1.5.5) (50) for autosomes only, and we excluded calls where lengths were ,100 kb or .10 Mb, or LOD score ,10. We excluded CNVs with at least 50% overlap with a region copy-number variable in at least 1% of samples; individuals with .30 CNV calls or a total event length .10 Mb and calls for samples from plates containing fewer than 40 samples (Sup- plementaryMaterial,TableS2).Callswithacopy numberof0or 1wereconsideredtobedeletionsandcallswithacopynumberof 3 or 4 were considered duplications. For the replication analysis, the ISC data were called using the same calling protocol as in the discovery analysis. Details on genotyping platforms used for the other samples are provided in Supplementary Material, Table S1. For the identiﬁed PAK7 risk duplication, we performed standard qPCR validation of theCNVcalls inthediscoverysample (Supplementary Material, Table S4). Verifying the presence of CNVs by this method addresses sensitivity, but not the speciﬁcity of CNV calling. To test the speciﬁcity and sensitivity of calling across genotyp- ing platforms, we examined probe intensity data for 6542 control subjects who had been genotyped on Affymetrix 6.0 Human Molecular Genetics, 2014, Vol. 23, No. 12 3324 of all 23 carriers carry copies of the same haplotypes either side of the duplication. (Millipore) was applied and chemiluminescent signal was revealed by exposing photographic ﬁlm (Sigma, Z370398) to the membrane for ≏5 min. Co-immunoprecipitation Using an Agilent custom designed CGH microarray with high probe density in the PAK7 region, we conﬁrmed the duplication event in one individual and the breakpoints identiﬁed by CGH (chr20:9,684,902–9,833,151) map closely to those predicted by the SNP array analyses (chr20:9,685,413–9,831,947; Sup- plementary Material, Fig. S4). Based on these estimated break- points and assuming that this is a tandem duplication that is not inverted, we attempted to sequence from one copy of the du- plication into the second copy in order to identify the precise breakpoints. Using a forward primer positioned at the end of the duplication (TCTCTGTTGGATGGAGCTTCT) and a reverse primer positioned at the start of the duplication (CGATGTAAAAAGACACAAGAGAAA), we successfully PCR-ampliﬁed this unique region in a carrier sample. The PCR did not amplify in noncarrier samples. Using capillary se- quencing, we identiﬁed the event as being a tandem duplication of 148 951 bp at chr20:9,684,767–9,833,717(hg18) with a 15 bp sequence inserted between the ﬁrst and second copies of the repeated sequence (Fig. 2). p p Co-immunoprecipitationprocedurewas adaptedfromFellietal. (2005) (54) as described below. Synaptoneurosomal protein extracts were made from full brain tissue as described and PAK7 was immunoprecipitated. For both experiments, 500 ml-volume samples containing 1000 mg of synaptoneuroso- mal extracts were obtained by dilution in protein extraction buffer. Samples were immunoprecipitated with either 4 mg anti- body anti-PAK7 (polyclonal goat, S-16, Santa Cruz Biotechnol- ogy, sc-22155) or 4 mg antibody anti-DISC1 (polyclonal goat, N-16, Santa Cruz Biotechnology, sc-47990). Normal goat IgGs (Sigma, I5256) were used as a negative control. After 16 h, all samples were incubated with 25 ml of beads (protein A/G PLUS-Agarose, Santa Cruz Biotechnology, sc-2003) for 4 h and washed. Samples were then resolved by western blotting as described, and probed with antibody anti-DISC1 (rabbit poly- clonal antibody anti-DISC1 Mid, Invitrogen, 40–6900, 1:1500) or anti-PAK7 (polyclonal rabbit anti-PAK7, Sigma, SAB3500335, 1:1000). Primary antibody incubation was fol- lowed by incubation with biotinylated anti-rabbit IgG (Vector Labs, BA-1000, 1:3000) and ampliﬁcation with Vectastain ABC kit (Vector Labs, PK-6100). Free ﬂoating immunohistochemistry The membrane was probed overnight with antibody anti-MAP2 (mouse monoclonal, Millipore, MAB3418, 1:2500) and antibody anti-PSD95 (rabbit polyclonal, Cell Sig- naling Technology, 2507, 1:2500). Antibody anti-b-actin (rabbit polyclonal, Cell Signaling Technology, 4967, 1:5000) was used as a loading control. After 16 h, three washes of 5 min with 0.5% Tween/TBS were carried out followed by 1 h incubation with the following secondary antibodies: anti-mouse HRP-linked IgG (Cell Signaling Technology, 7076, 1:10 000) and anti-rabbit HRP-linked IgG (Cell Signaling Technology, 7074, 1:10 000). After washing as above, ECL substrate SUPPLEMENTARY MATERIAL Supplementary Material is available at HMG online. REFERENCES 19. Lichtenstein, P., Yip, B.H., Bjork, C., Pawitan, Y., Cannon, T.D., Sullivan, P.F. and Hultman, C.M. (2009) Common genetic determinants of schizophrenia and bipolar disorder in Swedish families: a population-based study. Lancet, 373, 234–239. 1. Xu,B.,Ionita-Laza,I.,Roos,J.L.,Boone,B.,Woodrick,S.,Sun,Y.,Levy,S., Gogos, J.A. and Karayiorgou, M. (2012) De novo gene mutations highlight patterns of genetic and neural complexity in schizophrenia. Nat. Genet., 44, 1365–1369. 20. Corvin, A. (2013) Schizophrenia at a genetics crossroads: where to now? Schizopr. Bull., 39, 490–495. 2. Girard, S.L., Gauthier, J., Noreau, A., Xiong, L., Zhou, S., Dionne-Laporte, A., Spiegelman, D., Henrion, E., Diallo, O., Thibodeau, P. et al. (2011) Increased exonic do novo mutation rate in individuals with schizophrenia. Nat. Genet., 43, 860–863. 21. Smith, B.N., Newhouse, S., Shatunov, A., Vance, C., Topp, S., Johnson, L., Miller, J., Lee, Y., Troakes, C., Scott, K.M. et al. (2013) The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder. Eur. J. Hum. Genet., 21, 102–108. 3. Lee, S.H., DeCandia, T.R., Ripke, S.,Yang, J., Schizophenia Psychiatric Genome-wide Association Study Consortium (PGC-SCZ), International Schizophrenia Consortium (ISC), Molecular Genetics of Schizophrenia Consortium(MGS),Sullivan,P.F.,Goddard,M.E.,Keller,M.C.etal.(2012) Estimatedproportionof variation insusceptibilityto schizophrenia captured by common SNPs. Nat. Genet., 44, 247–250. 22. Stoll, G., Pietilainen, O.P., Linder, B., Suvisarri, J., Brosi, C., Hennah, W., Leppa¨, V., Torniainen, M., Ripatti, S., Ala-Mello, S. et al. (2013) Deletions of TOP3b, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders. Nat. Neurosci., 16, 1228–1237. 23. Lupski, J.R., Belmont, J.W., Boerwinkle, E. and Gibbs, R.A. (2011) Clan genomics and the complex architecture or human disease. Cell, 147, 32–43. 4. Ripke, S., Sanders, A.R., Kendler, K.S., Levinson, D.F., Sklar, P., Holmans, P.A., Lin, D.Y., Duan, J., Ophoff, R.A., Andreassen, O.A. et al. (2011) Genome-wide association study identiﬁes ﬁve new schizophrenia loci. Nat. Genet., 43, 969–976. 24. Porteous, D.J., Millar, J.K., Brandon, N.J. and Sawa, A. (2011) DISC1 at 10: connecting psychiatric genetics and neuroscience. Trends Mol. Med., 17, 699–706. 5. Ripke, S., O’Dushlaine, C., Chambert, K., Moran, J.L., Kahler, A.K., Aakterin, S., Bergen, S.E., Collins, A.L., Crowley, J.J., Fromer, M. et al. (2012) Genome-wide association analysis identiﬁed 13 new risk loci for schizophrenia. Nat. Genet., 45, 1150–1159. 25. Wellcome Trust Case Control Consortium (2007) Genome-wide study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature, 447, 661–678. 26. McQuillin, A., Bass, N., Anjorin, A., Lawrence, J., Kandaswamy, R., Lawrence, J., Curtis, D., Sklar, P., Purcell, S.M. SUPPLEMENTARY MATERIAL Supplementary Material is available at HMG online. 3325 Human Molecular Genetics, 2014, Vol. 23, No. 12 332 FUNDING Funding for this study was provided by theWellcome Trust Case Control Consortium 2 project (085475/B/08/Z and 085475/Z/ 08/Z), the Wellcome Trust (072894/Z/03/Z, 090532/Z/09/Z and 075491/Z/04/B), NIMH grants (MH 41953 and MH083094) and Science Foundation Ireland (08/IN.1/B1916). We acknowledge use of the Trinity Biobank sample from the IrishBloodTransfusionService;theTrinity CentreforHighPer- formance Computing; British 1958 Birth Cohort DNA collec- tion funded by the Medical Research Council (G0000934) and the Wellcome Trust (068545/Z/02) and of the UK National Blood Service controls funded by the Wellcome Trust. Chris Spencer is supported by a Wellcome Trust Career Development Fellowship (097364/Z/11/Z). Funding to pay the Open Access publication charges for this article was provided by the Well- come Trust. 14. Levinson, D.F., Duan, J., Oh, S., Wang, K., Sanders, A.R., Shi, J., Zhang, N., Mowry, B.J., Olincy, A., Amin, F. et al. (2011) Copy number variants in schizophrenia: conﬁrmation of ﬁve previous ﬁndings and new evidence for 3q29 microdeletions and VIPR2 duplications. Am. J. Psychiatry, 16, 302–316. 15. Mefford, H.C., Sharp, A.J., Baker, C., Itsara, A., Jiang, Z., Buysse, K., Huang, S., Maloney, V.K., Crolla, J.A., Baralle, D., Collins, A. et al. (2008) Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes. N. Engl. J. Med., 359, 1685–1699. phenotypes. N. Engl. J. Med., 359, 1685–1699. 16. Sullivan,P.F.,Daly,M.J. andO’Donovan,M.(2012)Geneticarchitectureof psychiatric disorders: the emerging picture and its implications. Nat. Rev. Genet., 13, 537–551. 17. Cross Disorder Group of the Psychiatric Genomics ConsortiumSmoller, J.W., Craddock, N., Kendler, K.S., Lee, P.H., Neale, B.M., Nurnberger, J.I., Ripke, S., Santangelo, S. and Sullivan, P.F. (2013) Identiﬁcation of risk loci with shared effects on ﬁve major psychiatric disorders: a genome-wide analysis. Lancet, 381, 1379–1379. 18. Morgan, V.A., Croft, M.L., Valuri, G.M., Zubrick, S.R., Bower, C., McNeil, T.F. and Jablensky, A.V. (2012) Intellectual disability and other neuropsychiatric outcomes in high-risk children of mothers with schizophrenia, bipolar disorder and unipolar major depression. Br. J. Psychiatry, 200, 282–289. ACKNOWLEDGEMENTS Buizer-Voskamp, J.E. et al. (2008) Large recurrent microdeletions associated with schizophrenia. Nature, 455, 232–236. The authors sincerely thank all patients who contributed to this study and all staff who facilitated their involvement. We thank W. Bodmer and B. Winney for use of the People of the British Isles DNA collection, which was funded by the Wellcome Trust. We thank Akira Sawa and Koko Ishzuki for advice on the PAK7–DISC1 interaction experiment and Jan Korbel for discussions on mechanism of structural variation. 10. 10. Rujescu, D., Ingason, A., Cichon, S., Pietilainen, O.P., Barnes, M.R., Toulopoulo, T., Picchioni, M., Vassos, E., Ettinger, U., Braemon, E. et al. (2008) Disruption of the neurexin 1 gene is associated with schizophrenia Hum. Mol. Genet., 18, 988–996. 11. Kirov, G., Gumus, D., Chen, W., Norton, N., Georgieva, L., Sari, M., O’Donovan, M.C., Erdogan, F., Owen, M.J., Ropers, H.H. et al. (2008) Comparative genome hybridization suggests a role for NRXN1 and APBA2 in schizophrenia. Hum. Mol. Genet., 17, 458–465. 12. Kirov, G., Rujescu, D., Ingason, A., Collier, D.A., O’Donovan, M.C. and Owen, M.J. (2009) Neurexin 1 (NRXN1) Deletions in schizophrenia. Schizophr. Bull., 35, 851–854. Conﬂict of Interest statement. None declared. Conﬂict of Interest statement. None declared. 13. Vacic, V., McCarthy, S., Malhotra, D., Murray, F., Chou, H.H., Peoples, A., Makarov, V., Yoon, S., Bhandari, A., Corominas, R. et al. (2011) DuplicationsoftheneuropeptidereceptorgeneVIPR2confersigniﬁcantrisk for schizophrenia. Nature, 471, 499–503. 28. UK IBD Genetics Consortium, Barrett, J.C., Lee, J.C., Lees, C.W., Prescott, N.J., Anderson, C.A., Phillips, A., Wesley, E., Parnell, K., Zhang, H. et al. REFERENCES (2010) Disrupted-in–Schizophrenia-1 (DISC1) regulates spines of the glutamate synapse via Rac1. Nat. Neurosci., 13, 327–332. 31. Chen,S.Y.,Huang,P.H.andCheng,H.J.(2011)Disrupted-in-Schizophrenia 1-mediated axon guidance involves TRIO-RAC-PAK small GTPase pathway signaling. Proc. Natl Acad. Sci. USA, 108, 5861–5866. glutamate synapse via Rac1. Nat. Neurosci., 13, 327–332. 32. Peippo, M., Koivistro, A.M., Sarkamo, T., Sipponen, M., von Koskull, H., Ylisaaukko-oja, T., Rehnstrom, K., Froyen, G., Ignatuis, J. and Jarvela, I. (2007) PAK3 related mental disability: further characterization of the phenotype. Am. J. Med. Genet., 143A, 2406–2416. 46. St Clair, D., Blackwood, D., Muir, W., Carothers, A., Walker, M., Spowart, G., Godson, C. and Evans, H.J. (1990) Association within a family of a balanced translocation with major mental illness. Lancet, 336, 13–16. 47. Irish Schizophrenia Genomics Consortium and the Wellcome Trust Case Control Consortium 2 (2012) Genome-wide association study implicates HLA-C∗01:02 as a risk factor at the MHC locus in schizophrenia. Biol. Psychiatry, 72, 620–628. 33. Strochlic, T.I., Concilio, S., Viaud, J., Eberwine, R.A., Wong, L.E., Minden, A., Turk, B.E., Plomann, M. and Peterson, J.R. (2012) Identiﬁcation of neuronal substrates implicates Pak5 in synaptic vesicle trafﬁcking. Proc. Natl Acad. Sci. USA, 109, 4116–4121. 48. Cooper, G.M., Coe, B.P., Girirajan, S., Rosenfeld, J.A., Vu, T.H., Baker, C., Williams,C.,Stalker,H.,Hamid,R.,Hannig,V.etal..(2011)Acopynumber variation morbidity map of developmental delay. Nat. Genet., 43, 838–846. 34. Mills, R.E., Walter, K., Stewart, C., Handsaker, R.E., Chen, K., Alkan, C., Abyzov,A., Yoon, S.C., Ye, K., Cheetham, R.K. et al. (2011) Mappingcopy number variation by populations-scale genome sequencing. Nature, 470, 59–65. 49. The ARIC Investigators (1989) The atherosclerosis risk in communities (ARIC) study: design and objectives. Am. J. Epidemiol., 129, 687–702. 35. Hastings, P.J., Ira, G. and Lupski, L.R. (2009) A microhomology-mediated break-induced replication model for the origin of human copy number variation. PLOS Genet., 5, e1000327. 50. Korn, J.M., Kuruvilla, F.G., McCarroll, S.A., Wysoker, A., Nemesh, J., Cawley, S., Hubbell, E., Veitch, J., Collins, P.J., Darvishi, K. et al. (2008) Integrated genotype calling and association analysis of SNPs, common copy number polymorphisms and rare CNVs. Nat. Genet., 40, 1253–1260. 36. Magana, J.J., Gomez, R., Maldonado-Rodriguez, M., Velasquez-Perez, L., Tapia-Guerrero, Y.S., Cortes, H., Levya-Garcia, N., Hernandez-Hernandez, O. and Cisneros, B. (2013) Origin of the Spinocerebellar ataxia type 7 mutation in Mexican population. Cerebellum, 12, 902–905. 51. Collela,S.,Yau,C.,Taylor,J.M.,Mirza,G.,Butler,H.,Clouston,P.,Bassett, A.S., Seller, A., Holmes, C.C. and Ragoussis, J. (2007) QuantiSNP: an objective Bayes hidden Markov model to detect and accurately map copy numbervariation using SNP genotyping data. REFERENCES and Gurling, H.M. (2011) Analysis of genetic deletions and duplication in the University College London bipolar disorder case control sample. Eur. J. Hum. Genet., 19, 588–592. 6. Shi,Y.,Li,Z.,Xu,Q.,Wang,T.,Li,T.,Shen,J.,Zhang,F.,Chen,J.,Zhou,G., Ji, W. et al. (2011) Common variants on 8p12 and 1q24.2 confer risk of schizophrenia. Nat. Genet., 43, 1224–1227. 7. Karyiorgou, M., Flint, J., Gogos, J.A. and Malenka, R.C. and Genetic and Neural Complexity in Psychiatry 2011 Working Group (2012) The best of times, the worst of times for psychiatric disease. Nat. Neurosci., 15, 811–812. 27. Hamshere, M.L., Walters, J.T., Smith, R., Richards, A.L., Green, E., Grozeva, D., Jones, I., Forty, L., Gordon-Smith, K., Riley, B. et al. (2013) Genome-wide signiﬁcant associations in schizophrenia to ITIH3/4, CACNA1C and SDCCAG8, and extensive replication of associations reported by the Schizophrenia PGC. Mol. Psychiatry, 18, 708–712. 8. International Schizophrenia Consortium (2008) Rare chromosomal deletions and duplications increase risk of schizophrenia. Nature, 455, 237–241. 28. UK IBD Genetics Consortium, Barrett, J.C., Lee, J.C., Lees, C.W., Prescott, N.J., Anderson, C.A., Phillips, A., Wesley, E., Parnell, K., Zhang, H. et al. 9. Stefansson, H., Rujescu, D., Cichon, S., Pietila¨inen, O.P., Ingason, A., Steinberg, S., Fossdal, R., Sigurdsson, E., Sigmundsson, T., 3326 Human Molecular Genetics, 2014, Vol. 23, No. 12 (2009) Genome-wide association study of ulcerative colitis identiﬁes three new susceptibility loci, including the HNF4A region. Nat. Genet., 41, 1330–1334. number variants: a point of rarity in genetic risk for bipolar disorder and schizophrenia. Arch. Gen. Psychiatry, 67, 318–372. number variants: a point of rarity in genetic risk for bipolar disorder and schizophrenia. Arch. Gen. Psychiatry, 67, 318–372. 43. Malhotra, D., McCarthy, S., Michaelson, J.J., Vacic, V., Burdick, K.E., Yoon, S., Cichon, S., Corvin, A., Gary, S., Gershon, E.S. et al. (2011) Hig frequencies ofde novoCNVsinbipolardisorderandschizophrenia.Neuron 72, 951–963. 29. Winney, B., Boumertit, A., Day, T., Davison, D., Echeta, C., Evseeva, I., Hutnik, K., Leslie, S., Nicodemus, K., Royrvik, E.C. et al. (2012) People of the British Isles: preliminary analysis of genotypes and surnames in a UK-control population. Eur. J. Hum. Genet., 20, 203–210. UK-control population. Eur. J. Hum. Genet., 20, 203–210. 44. Meyer-Lindenberg, A. (2010) From maps to mechanisms through neuroimaging of schizophrenia. Nature, 468, 194–202. 30. Arias-Romero, L.E. and Chernoff, J. (2008) A tale of two Paks. Biol. Cell., 100, 97–108. 45. Hayashi-Takagi, A., Takaki, M., Graziance, N., Seshadri, S., Murdoch, H., Dunlop, A.J., Makino, Y., Seshadri, A.J., Ishizuka, K., Srivastava, D.P. et al. REFERENCES NucleicAcids Res., 35, 2013– 2025. 37. 1000 Genomes Project ConsortiumAbecasis, G.R., Auton, A., Brooks, L.D., DePristo, M.A., Durbin, R.M., Handsaker, R.E., Kang, H.M., Marth, G.T. andMcVean,G.A.(2012)Anintegratedmapofgeneticvariationfrom1,092 human genomes. Nature, 491, 56–65. 52. Guha, S., Rees, E., Darvasi, A., Ivanov, D., Ikeda, M., Bergen, S.E., Magnusson, P.K., Cormican, P., Morris, D., Gill, M. et al. (2013) Implications of a rare deletion at distal 16p11.2 in schizophrenia. JAMA Psychiatry, 70, 252–260. 38. Gravel, S., Henn, B.M., Gutenkunst, R.N., Indap, A.R., Marth, G.T., Clark, A.G., Yu, F. and Gibbs, R.A. and 1000 Genomes Project, Bustamante, C.D. (2011) Demographic history and rare allele sharing among human populations. Proc. Natl Acad. Sci. USA, 108, 11983–11988. populations. Proc. Natl Acad. Sci. USA, 108, 11983–11988. 53. Hollingsworth, E.B., McNeal, E.T., Burton, J.L., Williams, R.J., Daly, J.W. and Creveling, C.R. (1985) Biochemical characterization of a ﬁltered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3′:5′-monophosphate-generating systems, receptors, and enzymes. J. Neurosci., 5, 2240–2253. 39. Mathieson, I. and McVean, G. (2012) Differential confounding of rare and commonvariantsinspatiallystructuredpopulations.Nat.Genet.,44,243–246. 40. Nekrasova, T., Jobes, M.L.,Ting, J.H., Wagner,G.C.and Minden,A. (2008) Targeted disruption of the Pak5 and Pak6 genes in mice leads to deﬁcits in learning and locomotion. Dev. Biol., 322, 95–108. 54. Felli, M.P., Vacca, A., Calce, A., Bellavia, D., Campese, A.F., Grillo, R., Giovine, M., Checquolo, S., Talora, C., Palermo, R. et al. (2005) PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia. Oncogene, 24, 992–1000. 41. Benichou, J. (2007) Biostatistics and epidemiology: measuring the risk attributableto an environmental or genetic factor. C. R. Biol., 330, 281–298. 42. Grozeva, D., Kirov, G., Ivanov, D., Jones, I.R., Jones, L., Green, E.K., St Clair, D.M., Young, A.H., Ferrier, N., Farmer, A.E. et al. (2010) Rare copy
https://openalex.org/W4388041248	https://www.mdpi.com/2413-8851/7/4/115/pdf?version=1698736512	English	null	Developing a Qualitative Urban Green Spaces Index Applied to a Mediterranean City	Urban science	2,023	cc-by	10,981	Article Developing a Qualitative Urban Green Spaces Index Applied to a Mediterranean City ,2,3,* , Faiza Khebour Allouche 1,2, Aude Nuscia Taîbi 3 and Safa Bel Fekih Boussema 1,2 Rania Ajmi 1,2,3,* , Faiza Khebour Allouche 1,2, Aude Nuscia Taîbi 3 and Safa Bel Fekih B 1 Department of Horticulture Sciences and Landscape, High Institute of Agronomic Science of Chott Mariem (ISA CM) Sousse University, 4042 Chott Mariem, Tunisia 2 Lr GREEN TEAM (LR17AGR01), National Agronomic Institute of Tunis, Carthage University, 1082 Carthage, Tunisia 3 Espace SOciété (ESO), UMR 6590, 5bis BD, Lavoisier, Angers University, 49100 Angers, France * Correspondence: ajmi.rania.ep.ismail@gmail.com; Tel.: +216-58-840-467 ( ) y, , 2 Lr GREEN TEAM (LR17AGR01), National Agronomic Institute of Tunis, Carthage University, 1082 Carthage, Tunisia 3 Espace SOciété (ESO) UMR 6590 5bis BD Lavoisier Angers University 49100 Angers France ( ) y 2 Lr GREEN TEAM (LR17AGR01), National Agronomic Institute of Tunis, Carthage University, 1082 Carthage, Tunisia g 3 Espace SOciété (ESO), UMR 6590, 5bis BD, Lavoisier, Angers University, 49100 Angers, France * Correspondence: ajmi.rania.ep.ismail@gmail.com; Tel.: +216-58-840-467 g 3 Espace SOciété (ESO), UMR 6590, 5bis BD, Lavoisier, Angers University, 49100 Angers, France * Correspondence: ajmi.rania.ep.ismail@gmail.com; Tel.: +216-58-840-467 3 Espace SOciété (ESO), UMR 6590, 5bis BD, Lavoisier, Angers University, 49100 Angers, France * Correspondence: ajmi.rania.ep.ismail@gmail.com; Tel.: +216-58-840-467 Espace SOciété (ESO), UMR 6590, 5bis BD, Lavoisier, Angers University, 49100 Angers, Fran * Correspondence: ajmi.rania.ep.ismail@gmail.com; Tel.: +216-58-840-467 Abstract: As a primary goal, urban green spaces (UGSs) have been linked to several aspects of inhabitants’ wellbeing. Quality could be a way to intervene in the UGS–human health interaction. For that purpose, we developed an urban green space quality index (UGS QIndex) applied to a Mediterranean region, Sousse City. This index was based on a set of criteria, indicators, and elements chosen after bibliographical research related to UGS quality assessment tools and their contribution to the Sustainable Development Goals. Then, we evaluated the quality of the Sousse Ramparts Gardens using the UGS QIndex. In fact, this index includes 41 elements grouped into 23 indicators covering seven thematic criteria: environmental regulating capacity, functional amenities, aesthetic amenities, landscape features, integration in its surroundings, development policy objectives, and space issues. According to the UGS QIndex, Bab El Gharbi garden exceeds Bab El Finga garden in terms of its scenery, aesthetics, and functionality. This index could be used by city planners to improve their UGS’s capacity to satisfy the inhabitants’ requirements. Keywords: urban green spaces; quality; index; wellbeing; Mediterranean Citation: Ajmi, R.; Allouche, F.K.; Taîbi, A.N.; Boussema, S.B.F. Developing a Qualitative Urban Green Spaces Index Applied to a Mediterranean City. Urban Sci. 2023, 7, 115. https://doi.org/10.3390/ urbansci7040115 Article Developing a Qualitative Urban Green Spaces Index Applied to a Mediterranean City Otherwise, it needs to be enhanced and tailored to various types of UGSs and then applied to other Mediterranean cities, as well as cities suffering from UGS degradation. 1. Introduction In the ﬁeld of urban planning, the relevance of urban green spaces (UGSs) has been widely discussed [1–3]. Aside from its aesthetic impact, urban green spaces offer recre- ational activities, as well as a variety of environmental and health beneﬁts [4–6]. UGSs are known for all the green paved, open, and burial places. It is also related to sports ﬁelds, private gardens, formal and informal green forests, road verges, derelict land, and horticulture within a city [7,8]. They provide a natural meeting point for the residents and facilitate social interaction, as well as community integration. Moreover, they promote physical activities, as well as mental and psychological relaxation, provide oxygen for breathing, and purify air pollutants [9]. Previous research has demonstrated that growing urbanization threatens both mental health and biodiversity [10,11]. Academic Editors: Luis Hernández-Callejo and Biao Zeng Received: 3 August 2023 Revised: 25 October 2023 Accepted: 26 October 2023 Published: 31 October 2023 According to Van Den Berg et al. [25] and Sugiyama et al. [26], this tendency conﬂicts with common values of restorative environments, as people prefer relatively natural environments, which they feel offer them psychological restoration and suitable places for physical activity. Urban densiﬁcation processes and emphasis on the compact city as a model for future cities have created needs within planning to address issues related to the recognition and prioritization of urban green space qualities in the urban fabric [27]. Additionally, parks and gardens have been proven to help people cope with stress by promoting social support. Overall, neighborhood greenery has a direct stress-relieving beneﬁt, but this can be offset via its detrimental inﬂuence on social support [28]. According to Littke et al. [29], park areas have a greater beneﬁcial inﬂuence on health and wellbeing than the total amount of neighborhood greenery if their effect sizes are likened. Similarly, Fan et al. [28] recommended that policymakers should focus on developing organized green spaces, with public leisure and sociability possibilities instead of merely protecting green areas in the community. In this way, the greening process for health beneﬁts will be more intentional, concentrating on various components of UGSs rather than just providing additional green areas of different aspects of UGSs, and not simply focusing on providing more green spaces [30]. Many recent studies have aimed to identify the aspects of the landscape and environ- ment of UGSs that interact to enable the relationship with health and wellbeing [8,31–33]. For example, Lee et al. [34], in their study, assumed that walking pathways, shade, water features, irrigated lawns, birds, illumination, athletic facilities, playgrounds, the nature of neighboring roadways, and the availability of water were all used to characterize quality. Their study also found that various user groups have varied needs for public open spaces; for example, some people may ﬁnd water features relaxing and appealing, while parents of small children may consider them to be a safety threat. In general, park visitors appreciate a mix of biotic, abiotic, and man-made park infrastructure components, as well as attributes. For that reason, Voigt et al. [17] suggested that these three elements of structural variety have an impact on how people utilize and appreciate urban parks. This is why there is a broader link between the streetscape’s aesthetics or beauty and particular sorts of activities. On the one hand, many researchers, such as Macintyre and Ellaway [12], have studied the link between neighborhood features and individual wellbeing. Traditionally, sociologi- cal and psychological elements, such as social cohesiveness, social capital, and feeling of the community, have been the focus of their studies. On the other hand, physical neighborhood conditions are increasingly recognized as both sources of stress and resources that might help inhabitants manage them [13]. Subsequently, according to Van Den Berg [14], re- searchers and politicians have increasingly focused their attention on UGSs as a potentially signiﬁcant physical neighborhood resource. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/urbansci Urban Sci. 2023, 7, 115. https://doi.org/10.3390/urbansci7040115 Urban Sci. 2023, 7, 115 2 of 17 UGSs, as public spaces, are important contributions to the resiliency of urban sys- tems as they have a variety of health, physical, and social beneﬁts. They also have ecosystem services, as well as sustainability that help to offset the negative effects of urbanization [11,15,16]. People perceive the feelings and emotions produced in parks and gardens as extremely essential contributions to their wellbeing, according to the ﬁndings. Direct advantages include their restoration of psychophysical balance, relaxation, an escape from the daily routine, and a promotion of spiritual connection with nature. All of these emotional and psychological advantages create a signiﬁcant difference in people’s lives [17]. Due to changes in global urbanization, the United Nations (UN) has included sustain- able urban development in its recent Sustainable Development Goals (SDGs) agenda [18]. This recent modiﬁcation addressed the planning and sustainable management of cities that foster inclusive social and economic growth and face climate change [19]. This detailed the creation of multi-modal transportation networks, green spaces, green designs, and sustain- able approaches to the utilization of the environment’s resources (land, water, etc.) [20]. Thus, UGSs can have an immense impact on the Sustainable Development Goals (SDGs) [21]. Well-managed UGSs can both directly and indirectly help achieve 15 of the 17 SDGs [22]. In consequence, sustainable management, along with the use of UGSs, can be a pragmatic and cost-effective way to achieve the SDGs’ beneﬁts [23]. Meanwhile, the quality of green spaces is decreasing, and nature areas are shifting far away from city centers, increasingly alienating inhabitants from nature [24]. 2. Materials and Methods 2.1. Study Area The study area, the municipality of Sousse, which covers an area of 2669 km2, is situ- ated in the eastern coastal zones at 35◦49’32” N and 10◦38’28” E of Tunisia. It includes ﬁve boroughs: Sousse Medina, Sousse Khzema, Sousse Sidi Abd Elhamid, Sousse Jawhara, and Sousse Riadh (Figure 1). Sousse is one of the colonial North African cities currently under pressure from both within (densiﬁcation, destruction and rebuilding, gentriﬁcation, pau- perization, etc.) and from the areas outside them, where fast urbanization has profoundly changed the environment. The ever-receding or vanishing urban vegetation in many parts of these cities is the consequence of the new connections that the public and decisionmakers retain with this “heritage”. Although extensively recognized in the North, where they have been incorporated into public policy, the issues of urban vegetation, particularly in terms of context, quality of life, and ecological services have only recently started to gain attention in cities in southern nations, such as French colonial countries. However, in these cities, which are typically hot and dirty, with a sizeable working-class population, and beset via mismanaged urban expansion, the advantages of plants, sometimes known as ecosystem services, may be more substantial. Although they are uniquely adapted to their social, climatic, environmental, and urban surroundings and problems, the functions (or ecosystem services) of vegetation in the city are still underappreciated in these cities today [43]. y The municipality of Sousse beneﬁts from a both stable and mild climate, offering its inhabitants a context that is particularly conducive to living in public spaces. It belongs to the lower semi-arid bioclimatic stage. This implies a low level of precipitation, varying between 200 mm/year and 300 mm/year. In addition, evaporation often exceeds precip- itation, which favors short, bushy vegetation strewn with grasses or shrubs [44]. As for temperature, August is the warmest month, with an average of 26 ◦C, while January is the coldest month, with an average of 11.5 ◦C. According to Bousemma et al. [45], this city is well known for its diverse cultures and beautiful sceneries, which have boosted tourism. The occupation of the soil is dominated by olive trees, particularly in suburban areas on the outskirts of the city, which are notable for their economic prosperity and scenic landscapes. Attractiveness was linked to people’s walking behavior [35], exercise [36], and leisure [37]. The aspects of quality, absence of annoyance, and decent pathways of a neighborhood’s green space have also been linked to increased walking time [38]. Therefore, Chen et al. [39] and Knobel et al. [24] considered that each quality aspect is related to a particular element of the quality tool, Urban Sci. 2023, 7, 115 3 of 17 3 of 17 such as the availability of amenities and services, accessibility, safety, or biodiversity. However, recent studies have tended, while developing the UGS assessment tool, to focus on several dimensions (e.g., accessibility, facilities, amenities, aesthetics, and incivilities), while others are neglected (e.g., surroundings, activities, policies, and vegetal biodiversity) [40]. As an example, the Public Open Space Tool (POST) [41] only considers green spaces’ accessibility, aesthetics, and amenities related to physical activity and recreation. The Natural Environment Scoring Tool (NEST) [42] and urban green space quality assessment tool (RECITAL) [33] include many quality dimensions (surroundings, biodiversity, facilities, amenities, aesthetics, incivilities, safety, etc.) but neglect the policies dimension. Thus, there is a need to conduct a study to develop a comprehensive UGS evaluation multi-dimensional tool that assesses all dimensions equally, including the “Development policy objectives” dimension, and to simultaneously understand the relationship between the SDGs and UGS and its amenities. The aims of this paper were: (1) to develop and implement an urban green space quality index (UGS QIndex) related to the SDGs, and (2) to apply the tool developed for the city of Sousse (Tunisia) in order to evaluate the quality of the Sousse Ramparts gardens. 2. Materials and Methods 2.1. Study Area Indeed, the municipality, which serves as the governorate’s capital, has been a recognized “city garden” since December 2009, and, as a result, has several green spaces and green-lined thoroughfares, the distribution of which may be studied. 4 of 17 Urban Sci. 2023, 7, 115 Figure 1. Localization of the study area. Figure 1. Localization of the study area. Figure 1. Localization of the study area. Figure 1. Localization of the study area. 2.2. Methodology As the ﬁrst step, we identiﬁed the Sustainable Development Goals related to urban public green spaces and then classiﬁed them according to seven thematic criteria. Then, the UGS quality index was developed, as well as the scoring tool. Following this, the UGS QIndex was used to assess the Rampart Gardens in Sousse. Finally, the study areas were visited to assess the aesthetic quality and the other quality tool item, which is a trustwor- thy manner. In addition to that, we used Google Earth Pro to measure items that should be conducted via remote technique assessments to have an eﬃcient result (Figure 2). The 1895 French occupation of Sousse resulted in the embellishment of the new European city, with spacious avenues, squares, and tree-lined pathways. Consequently, under the French protectorate, efﬁcient landscape designs enabled the establishment of functional and aesthetic green facilities. Since the 1990s, there has been increasing awareness of the signiﬁcant contribution that green spaces can make to enhancing living circumstances in the municipality of Sousse. It thus rebuilt the green areas along the Ramparts, Sahloul area, and Erriadh area after the revolution. The lack of monitoring and evaluation are among the biggest challenges in the management of green spaces in Sousse, despite the initiatives that the municipality has taken to preserve its green spaces, resulting in the emergence of the importance of developing a tool to facilitate the UGSs’ assessment in terms of quantity and quality. 2.2. Methodology As the ﬁrst step, we identiﬁed the Sustainable Development Goals related to urban public green spaces and then classiﬁed them according to seven thematic criteria. Then, the UGS quality index was developed, as well as the scoring tool. Following this, the UGS QIndex was used to assess the Rampart Gardens in Sousse. Finally, the study areas were visited to assess the aesthetic quality and the other quality tool item, which is a trustworthy manner. In addition to that, we used Google Earth Pro to measure items that should be conducted via remote technique assessments to have an efﬁcient result (Figure 2). VIEW 5 of 18 Figure 2. A conceptual framework for research methodology. Figure 2. A conceptual framework for research methodology. Figure 2. A conceptual framework for research methodology. Figure 2. A conceptual framework for research methodology. Urban Sci. 2023, 7, 115 5 of 17 5 of 17 2.2.1. Tool Development Three existing quality assessment tools were selected and used as our primary ref- erences for developing the UGS QIndex: Public Open Space Tool (POST) [41], Natural Environment Scoring Tool (NEST) [42], and urban green space quality assessment tool (RECITAL) [33]. Furthermore, as green spaces exhibit a signiﬁcant impact on the social, en- vironmental, and economic dimensions of sustainable development, which form the basis of the SDGs [18], we took into consideration four goals: SDG 11—sustainable cities and communities, SDG 13—climate action, SDG 15—life on land, and SDG 3—good health and wellbeing [46,47]. These green areas serve as recreational areas regulating the bioclimate, which is its main local role [48]. Based on existing reviews of UGS quality assessment tools by Lee et al. [34] and their contribution to the SDGs, a set of scientiﬁcally validated thematic criteria and indicators were selected, with a total of 41 items divided into 23 indicators organized into 7 quality criteria: “Environmental regulating capacity”, “Functional amenities”, “Aesthetic ameni- ties”, “Landscape features”, “Space issues” [49], and “Integration in its surroundings” [50]. We added the “Development policy objectives” criteria, which are important, but there is no tool for assessing it (Table 1). Table 1. Description of the thematic criteria. Sustainable Development Goal Thematic Criteria Description SDG 13 SDG 15 SDG 11 Environmental regulating capacity Based on temperature, energy, etc., it depends on the surface area and the physiological state of the vegetation, as well as the plant life composition. SDG 3 SDG 11 Functional amenities Corresponds to the different functional facilities in the UGSs (urban furniture, entrances, playgrounds for children, etc.) SDG 3 SDG 11 Aesthetic amenities Depends on the beautiﬁcation and the requaliﬁcation functions. SDG 3 SDG 13 SDG 11 Landscape features Based on the history, economy, and biodiversity of the UGSs. SDG 3 SDG 11 Integration in its surroundings Based on the spatial and social integration of the space in its environment (demands of people, the framework of the space, etc.). SDG 3 SDG 11 Development policy objectives Corresponds to the planning process about the Sustainable Development Goals and the participatory approach of the inhabitants. SDG 3 SDG 11 Space issues Based on the disadvantages of the space, like the existence of quality-of-life offenses, unpleasant smells, dogs, mosquitoes, noise nuisance, etc. For example, goal 11 of the SDGs refers to urban development, and target 11.7 of goal 11 especially aims to develop urban green spaces. 2.2.2. Tool Application We used the UGS QIndex to characterize the quality of the Rampart Gardens, located in the middle east of Sousse in Sousse Medina (Figure 3). The Rampart Gardens, namely Bab El Jabli garden, Bab El Finga garden, and Bab El Gharbi garden, cover an area of 8230 m2, 3950 m2, and 6824 m2, respectively. These French-style gardens, used for commercial activities before the colonization, were maintained and improved in the 2000s and then redeveloped in 2010. Figure 3. Localization maps of Bab El Gharbi garden, Bab El Finga garden, and Bab El Jabli garden. Figure 3. Localization maps of Bab El Gharbi garden, Bab El Finga garden, and Bab El Jabli garden. A straightforward additive method was applied to determine a coefﬁcient for all the characteristics and items measured. We used the developed index at three different levels: global scores, thematic scores, and scores via indicators. Google Earth Pro software 7.3.6 was used to assess two items, namely tree cover and vegetation cover rate. For the other items, ﬁeld visits, interviews with public decision makers, and surveys among the inhabitants were carried out, and the archives were consulted. 2.2.1. Tool Development This explains why goal 11 is connected to all the UGS Quality Index’s criteria. By giving residents access to areas where they can practice physical activities, target 11.7 will help in achieving SDG 3 [51]. Moreover, all urban green spaces, regardless of kind, help mitigate the effects of climate change. Liu and Shen [52] provided evidence that urban green spaces alter the microclimate and air pollutants that directly cause climate change related to SDG 13 and the “Environmental regulating capacity” criteria. Additionally, urban green spaces offer adequate habitats for species, which helps conserve the local ones [53,54]. This contribution of urban green spaces is related to SDG target 15.5: take immediate and meaningful action to reduce the degradation of habitat and biodiversity loss. The “Functional amenities” and “Aesthetic amenities” criteria enhance residents’ physical and mental wellbeing, as well as improve Table 1. Description of the thematic criteria. For example, goal 11 of the SDGs refers to urban development, and target 11.7 of goal 11 especially aims to develop urban green spaces. This explains why goal 11 is connected to all the UGS Quality Index’s criteria. By giving residents access to areas where they can practice physical activities, target 11.7 will help in achieving SDG 3 [51]. Moreover, all urban green spaces, regardless of kind, help mitigate the effects of climate change. Liu and Shen [52] provided evidence that urban green spaces alter the microclimate and air pollutants that directly cause climate change related to SDG 13 and the “Environmental regulating capacity” criteria. Additionally, urban green spaces offer adequate habitats for species, which helps conserve the local ones [53,54]. This contribution of urban green spaces is related to SDG target 15.5: take immediate and meaningful action to reduce the degradation of habitat and biodiversity loss. The “Functional amenities” and “Aesthetic amenities” criteria enhance residents’ physical and mental wellbeing, as well as improve Urban Sci. 2023, 7, 115 6 of 17 6 of 17 the quality of life in their neighborhoods. Thus, the contribution of green spaces to reducing mortality and the maternal mortality rate [55] is linked with SDG 3 (Table 1). the quality of life in their neighborhoods. Thus, the contribution of green spaces to reducing mortality and the maternal mortality rate [55] is linked with SDG 3 (Table 1). 3.1. UGS QIndex Development and Scoring The UGS QIndex includes seven criteria, namely environmental regulating capacity, functional amenities, aesthetic qualities, landscape features, integration in its surroundings, development policy objectives, and space issues. For the environmental regulating capacity criterion, it is based on temperature, energy, etc. This depends on the surface area and the physiological state of the vegetation, as well as the plant life composition. The functional amenities criterion corresponds to the different functional facilities in the urban green spaces, such as urban furniture, entrances, parking, etc. The integration in its surroundings criterion is based on the spatial and social integration of the space in its environment, including the demands of locals, as well as the framework of the space, etc. The space issues criterion bears on the disadvantages of the space, such as the existence of quality-of- life offenses, unpleasant smells, dogs, mosquitoes, noise pollution, etc. 1 According to the green spaces’ development goal, these criteria might be weighted differently: one for a desired criterion, two for a required criterion, and three for an obligatory criterion for quality development. The weighting of the thematic criteria is only useful for public policymakers when there is a speciﬁc development objective to Urban Sci. 2023, 7, 115 7 of 17 7 of 17 be achieved. Each criterion may contain a range from one to ﬁve indicators. In all, the evaluation tool includes 23 indicators divided into 41 items. Table 1 explains how each criterion was divided into the indicators and items. It also shows the type of scoring for each one. For example, the functional amenities criterion contains ﬁve indicators, such as urban furniture development, path layout, entrance layout, security, and multifunctionality. The urban furniture development indicator contains two items, like benches, seats, kiosks, and water protection with their related facilities, which are weighted based on quantity and quality. The path layout indicator includes three items, namely the presence of user tracks, path diversity, and path compliance with width standards. p y p p Each item has its source of information. For example, to ﬁnd out whether the green space meets the needs of the population or not, surveys must be carried out among the inhabitants. However, in order to know if the space has experienced a striking historical event or not, we must refer to historical documents. Other sources of information exist, such as interviews with decision makers and ﬁeld visits. 3.1. UGS QIndex Development and Scoring For example, benches, seats, kiosks, visibility from ground level, children’s play facilities, colors of ﬂowering shrubs, trees, plant beds, public art, and cleanliness, among other items, are evaluated during the ﬁeld visit. Regarding the embellishment of a neighborhood or city criteria, the information provided regarding the means of access, schedules, activities, differentiated management, etc., is evaluated based on interviews with decision makers (Table 2). Table 2. Description of the UGS QIndex’s items, scoring, and source of information. Thematic Criterion Indicators Items Type of Scoring Source of Information Environmental regulating capacity Surface area and physiological condition of plant life. Surface area and physiological condition of the planted surface Quantity and quality Remote sensing and/or ﬁeld visit Surface area and physiological condition of tree cover Surface area and physiological condition of tree cover Quantity and quality Remote sensing and/or ﬁeld visit Plant life composition Number of species Quantity Field visit Functional amenities Urban furniture development Benches, seats, and kiosks Quantity and quality Field visit Water protection and related facilities Quantity and quality Field visit Path layout Presence of users’ tracks Reverse quantity Field visit Path diversity Quantity and quality Field visit Paths comply with width standards Standard Field visit Entrance layout Entrance diversity Quantity and quality Field visit Entrances comply with width standards Standard Field visit Security Security guard Yes/no Field visit Space enclosure Quantity and quality Field visit Surveillance cameras Quantity and quality Field visit The lighting of the space Quantity and quality Field visit Multifunctionality Children’s play facilities Quantity and quality Field visit Sports facilities Quantity and quality Field visit An open area for activities (sports, socio-cultural, etc.) Quantity and quality Field visit Biodiversity conservation facilities (botanical gardens, wild gardens, urban forests, etc.) Quantity Field visit Table 2. Description of the UGS QIndex’s items, scoring, and source of information. 8 of 17 Urban Sci. 2023, 7, 115 Table 2. Cont. 3.1. UGS QIndex Development and Scoring Thematic Criterion Indicators Items Type of Scoring Source of Information Aesthetic qualities Embellishment function Diversity of vegetation (type, colors, textures, etc.) Quantity and quality Field visit Field visit Waterpoint Quantity and quality Field visit Public art Quantity and quality Field visit Cleanliness Quantity Field visit Shade Quantity Field visit Requaliﬁcation function Valuation of world heritage or historical element Yes/no Field visit Landscape features History and heritage Striking historic event/heritage and/or historical elements Yes/no Historical documents Past agricultural biodiversity Vestiges of past agriculture Quantity and quality Field visit Historical documents Natural biodiversity Species of pollinators Quantity and quality Field visit Socio-economic interest Private services with an economic interest Quantity and quality Field visit Atmospheres of the space Special atmosphere or several types of atmospheres Quantity Field visit Topography (hill/gutter/slope lowering or raising an artiﬁcial surface) Yes/no Field visit Integration in its surroundings Connection of the space with its surroundings Linear connections with the outside Quantity and quality Field visit Visibility of the space Surrounded by primary or secondary pathways Quantity Field visit Guidance signs Quantity and quality Field visit The functionality of the space Meeting the needs of the population Quantity Surveys among the inhabitants People feel at home in the space Yes/no Surveys among the inhabitants Development policy objectives Planning and sustainable development Development objectives included in the Sustainable Development Goals Yes/no Interviews with public decision makers Participatory approach in urban design Surveys and comments/ meetings with residents/ ﬁeld observations Yes/o Interviews with public decision makers Provision of information related to the project carried out Information provided about means of access, schedules, and activities Yes/no Interviews with public decision makers Space issues Quality-of-life offenses Vandalism Reverse quantity Interviews with public decision makers/ﬁeld visit Drug use/alcohol use/sexual activity Reverse quantity Interviews with public decision makers Other drawbacks Unpleasant odors/noise pollution/visual impact/harmful plants (toxic, allergenic, etc.) Reverse quantity Field visit Table 2. Cont. The tool weighting was based on a percentage scale (0%, 25%, 50%, 75%, and 100%). With the various scoring systems, we established a guideline that included all the item types (Table 3). Quantity scoring evaluates things whose signiﬁcance is contingent on their existence, such as the number of species. In this situation, reversed quantity evaluates things whose importance is determined by their existence. However, in this case, less presence was preferable (e.g., unpleasant odors or noise). 3.1. UGS QIndex Development and Scoring Table 4 shows that the Bab El Gharbi garden has a higher environmental regulation capacity than the other two gardens, with a score of about 78%. It also has more aesthetic appeal (76%) and few disadvantages (83.75%). Bab El Finga garden is a space with a strong landscape character (34.37%) compared to Bab El Gharbi garden (26.87%) and Bab El Jabli garden (30.62%). Regarding the other criteria, the Rampart Gardens had the same score regarding the integration in its surroundings (62%) and development policies objectives (60%) items. Table 4. Rampart Gardens’ thematic evaluation in Sousse, 2021. Thematic Criteria Bab El Jabli Garden Bab El Finga Garden Bab El Gharbi Garden Environmental regulating capacity 58.75 73.75 78.12 Functional amenities 51.62 65 65 Aesthetic qualities 61.62 70.62 75.62 Landscape features 30.62 34.37 26.87 Integration in its surroundings 62 62 62 Development policy objectives 60 60 60 Space issues 79.37 83.75 83.75 Table 3. Guidelines for scoring the UGS QIndex items. 0% 25% 50% 75% 100% Quantity No presence Almost no presence Somewhat present Mostly present Sufﬁciently present Reverse quantity Very present Mostly present Somewhat present Almost no presence No presence at all Quality Bad Poorly maintained and aesthetically unpleasant Poorly maintained or aesthetically unpleasant Well-maintained and aesthetically pleasing Exceptionally well maintained and aesthetically pleasing Quantity and quality Not present Not ﬁt for purpose Suitable but in need of repair or insufﬁcient quantity Suitable and sufﬁcient Suitable, sufﬁcient, and aesthetically pleasing Standard Not met Almost not met Somewhat met Mostly met Met Yes/no No ----------------- --------------- ----------------- Yes Coverage rate 5% or less From 5% to 25% From 25% to 50% From 50% to 75% 75% and above 3 2 Rampart Gardens Assessment with the UGS QIndex Table 3. Guidelines for scoring the UGS QIndex items. Table 3. Guidelines for scoring the UGS QIndex items. 3.2. Rampart Gardens Assessment with the UGS QIndex The results show that the three gardens, which share the same history as the ﬁrst green spaces during the colonial period, are of “good quality”, as the total score was above 50%. Bab El Gharbi gardens reported the best score for several aesthetic and functional reasons. According to ﬁeld observations, it is the most frequented space of the three gardens. However, more precisely, they have a different score for each criterion. 3.1. UGS QIndex Development and Scoring Quality type, on the other hand, refers to objects whose use is determined via their level of upkeep and visual appeal, such Urban Sci. 2023, 7, 115 9 of 17 as the physiological condition or visibility of planted areas. The yes/no question scoring type is for questions that must be answered with yes or no, such as the presence of historical elements and differentiated management. As for the standard scoring type, this evaluates aspects where the standards have not been met, almost not met, somewhat met, mostly met, or met. For example, the entrances comply with width standards, while the paths comply with width standards items. Regarding coverage scoring type, the evaluated items were quantitative, such as planted surface area and tree cover surface area. UGS QIndex data may be used at four different levels: single-item scores, scores by indicators, thematic scores, and global scores. data may be used at four different levels: single item scores, scores by indicators, thematic scores, and global scores. Table 3. Guidelines for scoring the UGS QIndex items. 0% 25% 50% 75% 100% Quantity No presence Almost no presence Somewhat present Mostly present Sufﬁciently present Reverse quantity Very present Mostly present Somewhat present Almost no presence No presence at all Quality Bad Poorly maintained and aesthetically unpleasant Poorly maintained or aesthetically unpleasant Well-maintained and aesthetically pleasing Exceptionally well maintained and aesthetically pleasing Quantity and quality Not present Not ﬁt for purpose Suitable but in need of repair or insufﬁcient quantity Suitable and sufﬁcient Suitable, sufﬁcient, and aesthetically pleasing Standard Not met Almost not met Somewhat met Mostly met Met Yes/no No ----------------- --------------- ----------------- Yes Coverage rate 5% or less From 5% to 25% From 25% to 50% From 50% to 75% 75% and above 3.2. Rampart Gardens Assessment with the UGS QIndex The results show that the three gardens, which share the same history as the ﬁrst green spaces during the colonial period, are of “good quality”, as the total score was above 50%. Bab El Gharbi gardens reported the best score for several aesthetic and functional reasons. According to ﬁeld observations, it is the most frequented space of the three gardens. However, more precisely, they have a different score for each criterion. It shows the weaknesses of each area according to the criteria, which led to the development of guidelines to improve the area as a whole and particularly its weaknesses. 3.1. UGS QIndex Development and Scoring It shows the weaknesses of each area according to the criteria, which led to the development of guidelines to improve the area as a whole and particularly its weaknesses. Table 4 shows that the Bab El Gharbi garden has a higher environmental regulation capacity than the other two gardens, with a score of about 78%. It also has more aesthetic appeal (76%) and few disadvantages (83.75%). Bab El Finga garden is a space with a strong landscape character (34.37%) compared to Bab El Gharbi garden (26.87%) and Bab El Jabli garden (30.62%). Regarding the other criteria, the Rampart Gardens had the same score regarding the integration in its surroundings (62%) and development policies objectives (60%) items. Table 4. Rampart Gardens’ thematic evaluation in Sousse, 2021. Table 4. Rampart Gardens’ thematic evaluation in Sousse, 2021. Thematic Criteria Bab El Jabli Garden Bab El Finga Garden Bab El Gharbi Garden Environmental regulating capacity 58.75 73.75 78.12 Functional amenities 51.62 65 65 Aesthetic qualities 61.62 70.62 75.62 Landscape features 30.62 34.37 26.87 Integration in its surroundings 62 62 62 Development policy objectives 60 60 60 Space issues 79.37 83.75 83.75 Urban Sci. 2023, 7, 115 10 of 17 To uncover more details, an evaluation of the three gardens, based on the indicators, was carried out. Figure 4 shows that the Bab El Jabli and Bab El Gharbi gardens have a higher plant life surface area and better physiological condition (75%) than that of Bab El Finga (62.5%). However, the tree cover physiological conditions of the Bab El Finga and Beb El Gharbi gardens (62.5%) were much better than Bab El Jabli’s (50%). The Bab El Finga garden’s ﬂoristic composition was more heterogeneous (100%) than that of the Bab El Gharbi garden (70%). Regarding the Bab El Jabli garden, its ﬂoristic composition is somewhat homogeneous (50%). As a result, the sanitary condition of plant life and tree cover should be improved. As illustrated in Figure 5, the urban furniture development score of Bab El Gharbi garden (60%) is much better than the other gardens. The Bab El Gharbi garden has much better-quality street furniture. The three gardens’ paths and entrance layout are in good condition, sufﬁcient, and varied. The Rampart Gardens are not very safe and protected (40%). They are not protected by a security guard, nor equipped with cameras. 3.1. UGS QIndex Development and Scoring The three spaces are not multifunctional, with a score of 30% for the Bab El Finga and Bab El Jabli gardens and 37.5% for the Bab El Gharbi garden. Indeed, these gardens lack facilities for sports and children’s play. Bab El Jabli garden’s function of aesthetics was not fulﬁlled (22.5%), since the vegetation was not varied in terms of color and texture, nor was there any shade along the pathway. However, the other two gardens responded moderately to the function of embellishment (Figure 6). VIEW 12 of 18 life oﬀenses reverse score equal to 93.75%. On the other hand, we noticed the presence of some unpleasant smells, noise, and visual nuisances (70%). In the future, it is advised that some actions must be made, including improving the quality and quantity of the street furniture, adding other amenities (such as sports ﬁelds and playgrounds) to create multi- functional areas, and increasing the area’s security. The UGS QIndex is extremely useful, since it allows users to extract data at four diﬀerent levels: a top-level overview, a second- level look at the thematic criteria, a third-level look at the indicators for each criterion, and ﬁnally a fourth-level look at the indicator elements. However, this tool must be applied to other UGSs of diﬀerent sizes and functions to measure the reliability and eﬀectiveness of the tool. VIEW 12 of 18 life oﬀenses reverse score equal to 93.75%. On the other hand, we noticed the presence of some unpleasant smells, noise, and visual nuisances (70%). In the future, it is advised that some actions must be made, including improving the quality and quantity of the street furniture, adding other amenities (such as sports ﬁelds and playgrounds) to create multi- functional areas, and increasing the area’s security. The UGS QIndex is extremely useful, since it allows users to extract data at four diﬀerent levels: a top-level overview, a second- level look at the thematic criteria, a third-level look at the indicators for each criterion, and ﬁnally a fourth-level look at the indicator elements. However, this tool must be applied to other UGSs of diﬀerent sizes and functions to measure the reliability and eﬀectiveness of the tool Figure 4. Environmental regulating capacity’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 4. Environmental regulating capacity’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 4. 3.1. UGS QIndex Development and Scoring Environmental regulating capacity’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 4. Environmental regulating capacity’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 4. Environmental regulating capacity’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 4. Environmental regulating capacity’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 5. Functional amenities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 5. Functional amenities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 5. Functional amenities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 5. Functional amenities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 5. Functional amenities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 5. Functional amenities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Urban Sci. 2023, 7, 115 Urban Sci. 2023, 7, x FO 11 of 17 13 of 18 Figure 6. Aesthetic qualities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 6. Aesthetic qualities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. VIEW 13 Figure 6. Aesthetic qualities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 6. Aesthetic qualities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. The score of the history and heritage indicator of the three gardens was 100%. The Rampart Gardens have a signiﬁcant history since the pre-colonial period, being, in the past, a space where the cattle market is held every Sunday. In addition, these gardens aim to enhance a world heritage site, which is the ramparts of the medina (Figure 7). On the other hand, there are no vestiges of past agriculture (e.g., olive trees and eucalyptus) (0%), nor species of melliferous interest (0%), that do not offer private services of any economic interest (0%). Bab El Finga has the best atmosphere among the three gardens, with a score of 62.5%. In contrast, Bab El Gharbi has the worst atmosphere among the three gardens, with a score of 12.5%. Bab El Finga garden has a unique ambiance due to its topography, unlike Bab El Gharbi, which lacks any atmosphere. Figure 6. Aesthetic qualities’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 7. Landscape features’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 7. Landscape features’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 7. 3.1. UGS QIndex Development and Scoring Landscape features’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 7. Landscape features’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 7. Landscape features’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 8. Integration in its surroundings’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 8 shows that the three gardens have linear connections with the outside envi- ronment (100%). Being located on main roads, their visibility is good (90%). However, it seems that the inhabitants’ requirements are not fulﬁlled in these areas (12.5%). According to Figure 9, the Rampart Gardens’ development goals do not align with the Sustainable Development Goals (0%). After meeting with representatives from the municipality of Sousse, we found that observations and interviews with residents had been conducted before the development of these areas (100%). Sources of information on the three spaces are available to city inhabitants (85%), as their development comprises the objectives to attract tourists and residents to visit the Medina. As can be noticed in Figure 10, the gardens are safe, where the practice of drugs or sex, for example, is absent, with a quality-of-life offenses reverse score equal to 93.75%. On the other hand, we noticed the presence of some Urban Sci. 2023, 7, 115 12 of 17 unpleasant smells, noise, and visual nuisances (70%). In the future, it is advised that some actions must be made, including improving the quality and quantity of the street furniture, adding other amenities (such as sports ﬁelds and playgrounds) to create multifunctional areas, and increasing the area’s security. The UGS QIndex is extremely useful, since it allows users to extract data at four different levels: a top-level overview, a second-level look at the thematic criteria, a third-level look at the indicators for each criterion, and ﬁnally a fourth-level look at the indicator elements. However, this tool must be applied to other UGSs of different sizes and functions to measure the reliability and effectiveness of the tool. Figure 7. Landscape features’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 8. Integration in its surroundings’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 8. Integration in its surroundings’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. EVIEW 14 of 18 VIEW 14 of 18 Figure 8. Integration in its surroundings’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 8. 4. Discussion The UGS QIndex was introduced and developed as a striking multidimensional instrument for measuring various quality aspects of UGSs that are important for the urban residents’ wellbeing. In terms of the number of aspects and items assessed, the UGS QIndex’s components are more detailed in this study compared to others mentioned in different studies [17,33,45]. It includes 23 indicators divided into 41 items. The single- item ratings of the tool could give information that can be used to directly target the characteristics of UGSs interacting with human health. It was used to assess the Rampart Gardens of Sousse, whose surface area does not exceed 9000 m2. The ﬁndings show that Bab El Jabli garden meets its environmental regulation, aesthetic qualities, and development policy objectives. It integrates into the surroundings, but with far less aesthetic and functional design criteria. When comparing the Bab El Finga and Bab El Gharbi gardens, the latter exceeds the other in terms of scenery, aesthetics, and functionality. Based on these results, we recommend that the municipality’s green space department focus on the problem of these spaces to ensure better-quality UGSs in Sousse City. The purpose is to evaluate the quality of the city’s green spaces, identify their weaknesses to improve them, and monitor their long-term condition. Over time, the number of tools available has increased. Several large studies needed a multidimensional evaluation instrument for urban green areas; hence, they created their own, suited to their needs and resources, for example, RECITAL, POST, EARPS, and BRAT- DO [33,56–58]. However, future works should focus on the reliability of the tool and its items. According to Knobel et al. [33], the assessment of quality items is very sensitive and exposed to subjectivity, for example, aesthetic aspects, as well as quality-of-life offenses. The existing quality-of-life offense items serve as a proxy for speciﬁc undesirable behaviors (e.g., vandalism, drug use, or sex work). While it is difﬁcult to be uniformly objective, these acts and the aesthetic items could be more objectively measured and help to mitigate their impact. The use of clear and concise deﬁnitions is required for the tool’s validity, comparability, and replicability, as it creates a shared foundation from which to operate [40]. Thus, we have deﬁned the UGS QIndex, which was created for assessing urban parks and gardens. 3.1. UGS QIndex Development and Scoring Integration in its surroundings’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 9. Development policy objectives’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 9. Development policy objectives’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 9. Development policy objectives’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 9. Development policy objectives’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 9. Development policy objectives’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 9. Development policy objectives indicators evaluation of the Rampart Gardens in Sousse, 2021. Figure 10. Space issues’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. 4 Di i Figure 10. Space issues’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. 4 Discussion Figure 10. Space issues’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 10. Space issues’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 10. Space issues’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Figure 10. Space issues’ indicators’ evaluation of the Rampart Gardens in Sousse, 2021. Urban Sci. 2023, 7, 115 13 of 17 5. Conclusions The UGS QIndex is a multidimensional instrument assessing several quality features of UGSs that are signiﬁcant to urban residents’ health. It has forty-one elements grouped into seven thematic criteria. In today’s increasingly diverse population, UGSs must cater to a wide range of needs and demands. Therefore, it must be planned accordingly. Thus, this tool’s aims include assessing the quality of the city green areas, identifying their shortcom- ings so they may be improved, and keeping track of their long-term maintenance. As the ﬁndings suggest, several features of green spaces are contributing to the use of these spaces, such as biotic and abiotic features, as well relaxation facilities, security, and/tranquility. Our study has focused on three elements which are different from the recent studies: (i) we have reﬁned and detailed deﬁnitions, such as the deﬁnition of urban green spaces, adapted to Maghreb countries. It is necessary for the comparability and replicability of the tool; (ii) by adding the policy dimension, we increased the comprehensiveness of the tool. All the tools that have been constructed have focused on the dimension, description, item, and type of scoring. We have added important information, which is the “source of information” of each item, (iii) and we have considered the SDGs when developing our tool so that urban planners and policymakers can better meet the needs of the citizens and achieve the SDGs, contributing to a sustainable city. Having such detailed information about the UGSs’ features and characteristics via many different quality dimensions will enable us to better understand the importance of the quality aspect. It can be used by urban planners and policymakers to enhance their UGS’s ability to better meet their inhabitants’ needs and according to the SDGs. This tool can also be used to study other aspects of the UGSs, such as urban vegetation management or spatial disparity in the management of green areas. The UGS QIndex was created to ﬁt the features of the Tunisian and Maghreb cities, but its items and scoring may be tweaked to ﬁt different environments and the SDGs. After applying the tool for the different types of UGSs, researchers will be able to estimate the value of each item and tailor them to various goals. We acknowledge a few limitations, which were mostly for practical reasons. First, only one type of green space was evaluated. 4. Discussion However, it must be adjusted to be applied to other types of green spaces such as public squares and green spaces around buildings. p q g p g The UGS QIndex combines structural measures (functional and aesthetical amenities), biodiversity measures (tree cover, planted area, and number of species), and social measures (history, geography, and policies), which allows for a richer characterization of quality. Indeed, the survey conducted with the residents in parallel to assess their perceptions and the aspects of the UGSs that are essential to them adds value to our research. It has been used to improve the measurement of some items, such as the physiological condition of plant life, space multifunctionality, meeting the needs of the population, etc. The development of a green space quality index is important for policymakers to monitor the condition and evolution of UGSs. The high-quality data provided via the UGS QIndex can help public health researchers better understand the link between urban green spaces and human wellbeing through the index quality and its link with the SDGs. This would allow for urban planners and policymakers to tweak the UGSs in their cities to meet their SDGs and the needs of the target population. However, the use of a parallel evaluation approach, such as in situ repeated observation assessments through ﬁeldworker training, is necessary for further studies on the tool’s reliability [59]. The lower size used for the tool’s application was chosen for practical reasons not related to the tool’s capabilities. The tool can be used with larger and smaller sizes. However, some items should be adjusted to be applied to different types of UGSs such as squares. For example, assessing biodiversity-related items, such as “Surface area and physiological condition of tree cover”, can be complicated for non-expert ﬁeldworkers. We suggest conducting a workshop to train the technicians on how to utilize this tool. The tool was designed to be applied in other Mediterranean countries, particularly the Maghreb countries, but its items and scores may be adjusted Urban Sci. 2023, 7, 115 14 of 17 to ﬁt diverse contexts and the SDGs. Using this tool with its detailed information, urban planners, and policymakers might be able to support and encourage the management of UGSs towards achieving Agenda 2020. 4. Discussion The ﬁndings of this study will help promote the conservation and development of UGSs in rapidly developing Northeast Africa, as well as the management and uses of UGSs. References Macintyre, S.; Ellaway, A. Ecological approaches: The rediscovery of the role of the physical and soci Epidemiology; Berkman, L., Kawachi, I., Eds.; Oxford University Press: Oxford, UK, 2000; ISBN 9780195 12. Macintyre, S.; Ellaway, A. Ecological approaches: The rediscovery of the role of the physical and social environment. In Social Epidemiology; Berkman, L., Kawachi, I., Eds.; Oxford University Press: Oxford, UK, 2000; ISBN 9780195083316. 13. Diez-Roux, A.V. Bringing context back into epidemiology: Variables and fallacies in multilevel analysis. Am. J. Public Health 1998, 13. Diez-Roux, A.V. Bringing context back into epidemiology: Variables and fallacies in multilevel analysis. 88, 216–222. [CrossRef] 14. Van den Berg, A.E.; Maas, J.; Verheij, R.A.; Groenewegen, P.P. Green space as a buffer between stressful life events and health. Soc. Sci. Med. 2010, 70, 1203–1210. [CrossRef] [PubMed] 15. Jennings, V.; Larson, L.; Yun, J. Advancing sustainability through urban green space: Cultural ecosystem services, equity, and social determinants of health. Int. J. Environ. Res. Public Health 2016, 13, 196. [CrossRef] [PubMed] 16. Lee, S.A. Coronavirus Anxiety Scale: A brief mental health screener for COVID-19 related anxiety. Death Stud. 2020, 44, 393–401. [CrossRef] [PubMed] 17. Voigt, A.; Kabisch, N.; Wurster, D.; Haase, D.; Breuste, J. Structural Diversity: A Multi-dimensional Approach to Assess Recreational Services in Urban Parks. AMBIO 2014, 43, 480–491. [CrossRef] 18. United Nations. Transforming Our World: The 2030 Agenda for Sustainable Development; United Nations: N 18. United Nations. Transforming Our World: The 2030 Agenda for Sustainable Development; United Nations: New York, NY, USA, 2015. 19. Teklemariam, N. Sustainable Development Goals and Equity in Urban Planning: A Comparative Analysis of Chicago, São Paulo, and Delhi. Sustainability 2022, 14, 13227. [CrossRef] y 20. Grêt-Regamey, A.; Celio, E.; Klein, T.M.; Hayek, U.W. Understanding ecosystem services trade-offs with interactive procedural modeling for sustainable urban planning. Landsc. Urban Plan. 2013, 109, 107–116. [CrossRef] 21. Hyder, M.; Haque, T. Understanding the linkages and importance of urban greenspaces for achieving sustainable development goals 2030. J. Sustain. Dev. 2022, 15, 144. [CrossRef] ender, N. The beneﬁts of trees for livable and sustainable communities. Plants People Planet 2019, 1, 323–335 22. Turner-Skoff, J.; Cavender, N. The beneﬁts of trees for livable and sustainable communities. Plants Peop [CrossRef] 23. Ghani, F.; Tan, D. The role of green urban spaces in enhancing population health and achieving the sustainable development goals Malays. J. Med. Health Sci. 2020, 16, 380–383. 24. 5. Conclusions Thus, further applications of this tool on other UGS types must be conducted. Second, the technicians, with the help of experts, must be trained before using the tool, especially when assessing biodiversity-related items. This tool ﬁt the Maghreb cities. Therefore, we suggest applying this tool on other cities (e.g., Alger, Morrocco) and consider other UGS types. Author Contributions: R.A.: Conceptualization, Methodology, Data curation, Formal analysis, Writing—original draft. S.B.F.B.: Formal analysis review & editing. A.N.T.: Conceptualization, Methodology, Writing—review & editing, Supervision. Writing—review & editing. F.K.A.: Conceptu- alization, Methodology, Writing—review & editing, Supervision. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: This work was supported by members of the project “Partenariat Hubert Curien Maghreb” 20MAG38. Conﬂicts of Interest: The authors declare no conﬂict of interest. 15 of 17 15 of 17 Urban Sci. 2023, 7, 115 References 1. Maurani, T.; Amit-Cohen, I. Open Space Planning Models: A Review of Approaches and Methods. Landsc. Urban Plan. 2007, 81, 1–13. [CrossRef] 2. Springgate, L. Deﬁning Parks and Park Systems—From Recreation to Re-Creation: New Directions in Parks and Open Space System Planning; American Planning Association: Chicago, IL, USA, 2008. 2. Springgate, L. Deﬁning Parks and Park Systems—From Recreation to Re-Creation: New Directions in Par Planning; American Planning Association: Chicago, IL, USA, 2008. 3. Pakfegrat, A.; Taghvaei, M.; Zarrabi, A. A comprehensive approach in green space site planning: An ap multi-criteria decision support system. Urban Res. Pract. 2020, 13, 45–76. [CrossRef] 4. Marilyn, M. Decision Making in Allocating Metropolitan Open Space: State of the Art. Trans. Kans. Acad. Sci. 1975, 78, 149–153. [CrossRef] 5. Dahmann, N.; Wolch, J.; Joassart-Marcelli, P.; Reynolds, K.; Jerrett, M. 2010. The Active City? Disparities in provision of urban public recreation resources. Health Place 2010, 16, 431–445. [CrossRef] 6. Wolch, J.R.; Byrne, J.; Newell, J.P. Urban green space, public health, and environmental justice: The Challenge of Making Cities ‘just green enough’. Landsc. Urban Plan. 2014, 125, 234–244. [CrossRef] 7. Wang, Y.; Akbari, H. The effects of Street Tree Planting on Urban Heat Island Mitigation in Montreal. Sustain. Cities Soc. 2016, 27, 122–128. [CrossRef] M.M.; Shaﬁe, A. Assessing the role of urban green spaces for human well-being: A systematic review 4405–4423. [CrossRef] 8. Jabbar, M.; Yussof, M.M.; Shaﬁe, A. Assessing the role of urban green spaces for human well- GeoJournal 2022, 97, 4405–4423. [CrossRef] 9. Dadvand, P.; Nieuwenhuijsen, M.J.; Esnaola, M.; Forns, J.; Basagaña, X.; Alvarez-Pedrerol, M.; Rivas, I.; López-Vicente, M.; De Castro Pascual, M.; Su, J.; et al. Green spaces and cognitive development in primary schoolchildren. Proc. Natl. Acad. Sci. USA 2015, 112, 7937–7942. [CrossRef] 10. Gascon, M.; Mas, M.; Martínez, D.; Dadvand, P.; Forns, J.; Plasència, A.; Nieuwenhuijsen, M. Mental health beneﬁts of long-term exposure to residential green and blue spaces: A systematic review. Int. J. Environ. Res. Public Health 2015, 12, 4354–4379. [CrossRef] [ ] 11. Callaghan, A.; McCombe, G.; Harrold, A.; McMeel, C.; Moore-Cherry, N.; Cullen, W. The impact of green spaces on mental health in urban settings: A scoping review. J. Ment. Health 2020, 30, 179–193. [CrossRef] 12. Macintyre, S.; Ellaway, A. Ecological approaches: The rediscovery of the role of the physical and social environment. In Social Epidemiology; Berkman, L., Kawachi, I., Eds.; Oxford University Press: Oxford, UK, 2000; ISBN 9780195083316. 12. References Perceived environmental aesthetics and convenience and company are associated with walking for exercise among Australian adults. Prev. Med. 2001, 33, 434–440. [CrossRef] g g 37. Owen, A.L.; Twist, C.; Ford, P.R. Small-sided games: The physiological and technical effect of altering pitch size and player numbers. OR Insight 2004, 7, 50–53. 38. Sugiyama, T.; Thompson, C.W. Outdoor Environments, Activity and the Well-Being of Older People: C mental Support. Environ. Plan. A Econ. Space 2007, 39, 1943–1960. [CrossRef] pp p 39. Chen, S.; Sleipness, O.; Xu, Y.; Park, K.; Christensen, K. A systematic review of alternative protocols for evaluating non-spatial dimensions of urban parks. Urban For. Urban Green. 2020, 53, 126–718. [CrossRef] p , , [ ] 40. Knobel, P.; Dadvand, P.; Maneja-Zaragoza, R. A systematic review of multidimensional quality assessment tools for urban green spaces. Health Place 2019, 59, 102–198. [CrossRef] 41. Taïbi, A.N.; El Hannani, M.; Boujrouf, S.; Allouche Khebour, F.; Bensaid, S.; Slimani, H.; Bara, H.; Ajmi, R.; Salhi, S. The place of colonial vegetation in Malagasy and Maghrebian African cities, between abandonment and mimetic reproduction. In Proceedings of the 4th Biennal Conference African Studies Association of Africa (ASAA), Cape Town, South Africa, 11–16 April 2022. Available online: https://hal.science/hal-03704911v1 (accessed on 5 May 2022). p y 42. Allouche Khebour, F.; Ajmi, R.; Bel Fekih Boussema, S.; Serban, G. Ecosystem behaviour face to climatic ch actions. Case study: A north-eastern urban wetland, Tunisia. Forum Geogr. 2023, 21, 182–185. [CrossRe p y Allouche Khebour, F.; Ajmi, R.; Bel Fekih Boussema, S.; Serban, G. Ecosystem behaviour face to climatic chang ti C t d A th t b tl d T i i F G 2023 21 182 185 [C R f] 42. Allouche Khebour, F.; Ajmi, R.; Bel Fekih Boussema, S.; Serban, G. Ecosystem behaviour face to climatic changes and anthropogenic actions. Case study: A north-eastern urban wetland, Tunisia. Forum Geogr. 2023, 21, 182–185. [CrossRef] 43. Bousemma Bel Fekih, S.; Khebour Allouche, F.; Saidane, I.; Mechmech, Y.; Bettaieb, T. State of the art of greenway concept application in Tunisian green policy: A case study of an urban landscape in Sousse city. Int. J. Environ. Geoinformat. 2018, 5, 36–50. [CrossRef] 44. Giles-Corti, B.; Broomhall, M.H.; Knuiman, M.; Collins, C.; Douglas, K.; Ng, K.; Lange, A.; Donovan, R.J. Increasing walking: How important is distance to, attractiveness, and size of public open space? Am. J. Prev. Med. 2005, 28, 169–176. [CrossRef] 45. References Knobel, P.; Maneja, R.; Bartoll, X.; Alonso, L.; Bauwelinck, M.; Valentin, A.; Zijlema, W.; Borrell, C.; Nieuwenhuijsen, M.; Davdand, P. Quality of urban green spaces inﬂuences residents’ use of these spaces, physical activity, and overweight/obesity. Environ. Pollut. 2021, 271, 116–393. [CrossRef] 25. Van Den Berg, E.A.; Harting, T.; Staats, H. Preference for Nature in Urbanized Societies: Stress, Restoration, and the Pursuit of Sustainability. J. Soc. Issues 2007, 63, 79–96. [CrossRef] 26. Sugiyama, T.; Leslie, E.; Giles-Corti, B.; Owen, N. Associations of neighbourhood greenness with physical and mental health: Do walking, social coherence and local social interaction explain the relationships? J. Epidemiol. Community Health 2008, 62, e9. [CrossRef] [ ] 27. Swanwick, C.; Dunnett, N.; Woolley, H. Nature, Role and Value of Green Space in Towns and Cities: An Overview. Built Environ. 2003, 29, 94–106. [CrossRef] Urban Sci. 2023, 7, 115 16 of 17 28. Fan, Y.; Das, K.V.; Chen, Q. Neighborhood green, social support, physical activity, and stress: Assessing the cumulative impact. Health Place 2011, 17, 1202–1211. [CrossRef] [ ] 29. Littke, H.; Locke, R.; Haas, T. Taking the High Line: Elevated parks, transforming neighbourhoods, and the ever-changing relationship between the urban and nature. J. Urban Int. Res. Placemaking Urban Sustain. 2015, 9, 353–371. [CrossRef] p g 30. Zhang, L.; Tan, P.Y.; Richards, D. Relative importance of quantitative and qualitative aspects of urban green spaces in promoting health. Landsc. Urban Plan. 2021, 213, 104–131. [CrossRef] 31. Littke, H. Planning the Green Walkable City: Conceptualizing Values and Conﬂicts for Urban Green Space Strategies in Stockholm. Sustainability 2015, 7, 11306–11320. [CrossRef] y 32. Rojas-Rueda, D.; Nieuwenhuijsen, M.J.; Gascon, M.; Perez-Leon, D.; Mudu, P. Green Spaces and Mortal and Meta-Analysis of Cohort Studies. Lancet Planet Health 2019, 3, 469–477. [CrossRef] 33. Knobel, P.; Dadvand, P.; Alonso, L.; Costa, L.; Espanol, M.; Maneja, R. Development of the urban green tool (RECITAL). Urban For. Urban Green. 2021, 57, 126895. [CrossRef] 34. Lee, A.C.K.; Jordan, H.C.; Horsley, J. Value of urban green spaces in promoting healthy living and wellbeing: Prospects for planning. Risk Manag. Healthc. Policy 2015, 8, 131–137. [CrossRef] p g g y [ ] 35. Pikora, T.; Giles-Corti, B.; Bull, F.; Jamrozik, K.; Donovan, R. Developing a framework for assessment of the environmental determinants of walking and cycling. Soc. Sci. Med. 2003, 56, 1693–1793. [CrossRef] 36. Ball, K.; Bauman, A.; Leslie, E.; Owen, N. References Gidlow, C.; Van Kempen, E.; Smith, G.; Triguero-Mas, M.; Kruize, H.; Grazuleviciene, R.; J Ellis, N.; Hurst, G.; Masteron, D.J.; Cirach, M.; et al. Development of the natural environment scoring tool (NEST). Urban For. Urban Green. 2018, 29, 322–333. [CrossRef] [ ] 46. Lorenzo-Sáez, E.; Lerma-Arce, V.; Coll-Aliaga, E.; Oliver-Villanueva, V. Contribution of green urban areas to the achievement of SDGs. Case study in Valencia (Spain). Ecol. Indic. 2021, 131, 108246. [CrossRef] 47. Csigene Nagypal, N. Valuation challenges of urban green infrastructure. Ecocycles 2022, 8, 1–7. [CrossR g gyp g g y 48. Wey, Y.E.; Sarma, V.; Lechner, A.M.; Nath, T.K. Malaysians’ perception on the contribution of urban green spaces to the UN sustainable development goals. Urban For. Urban Green. 2022, 78, 127792. [CrossRef] 49. Miralles-Guasch, C.; Dopico, J.; Delclos-Alio, X.; Knobel, P.; Marquet, O.; Maneja- Zaragoza, R.; Schipperijn, J.; Vich, G. Natural landscape, infrastructure, and health: The physical activity implications of urban green space composition among the elderly. Int. J. Environ. Res. Public Health 2019, 16, 3986. [CrossRef] 50. Senetra, A.; Krzywnicka, I.; Mielke, M. An analysis of the spatial distribution, inﬂuence and quality of urban green space- a case study of the Polish city of Tczew. Bulletin of Geography. Socio-Econ. Ser. 2018, 42, 129–149. [CrossRef] netra, A.; Krzywnicka, I.; Mielke, M. An analysis of the spatial distribution, inﬂuence and quality of urban g udy of the Polish city of Tczew. Bulletin of Geography. Socio-Econ. Ser. 2018, 42, 129–149. [CrossRef] 51. Goal 11—Cities Will Play an Important Role in Achieving the SDGs. Available online: https://www.un.org/en/chronicle/ article/goal-11-cities-will-play-important-role-achieving-sdgs (accessed on 1 April 2023). 51. Goal 11—Cities Will Play an Important Role in Achieving the SDGs. Available online: http article/goal-11-cities-will-play-important-role-achieving-sdgs (accessed on 1 April 2023). 52 Li H L Sh YS Th i f h i ll i d i li A 52. Liu, H.L.; Shen, Y.S. The impact of green space changes on air pollution and microclimates: A case study of the taipei metropolitan area. Sustainability 2014, 6, 8827–8855. [CrossRef] 53. Aronson, M.F.J.; La Sorte, F.A.; Nilon, C.H.; Katti, M.; Goddard, M.A.; Lepczyk, C.A.; Warren, P.S.; Williams, N.S.G.; Cilliers, S.; Clarkson, B.; et al. A global analysis of the impacts of urbanization on bird and plant diversity reveals key anthropogenic drivers. Proc. R. Soc. B Biol. Sci. 2014, 281, 20133330. [CrossRef] 54. Shukuroglou, P.; McCarthy, M.A. Modelling the occurrence of rainbow lorikeets (Trichoglossus haematodus) in Melbourne. Austral. Ecol. 2006, 31, 240–253. 55. Hu, Z.; Liebens, J.; Ranga, K.R. Linking stroke mortality with air pollution, income, and greenness in northwest Florida: An ecological geographical study. Int. J. Health Geogr. 2008, 7, 1–22. [CrossRef] g g g p y g 56. Bird, M.; Datta, G.; Van Hulst, A.; Kestens, Y.; Barnett, T. A reliability assessment of a direct-observation park evaluation tool: The Parks, activity, and recreation among kids (PARK) tool. BMC Public Health 2015, 15, 906. [CrossRef] [PubMed] y g 57. Bedimo-Rung, A.L.; Gustat, J.; Bradley, J.T.; Rice, J.; Thomson, J. Development of a Direct Observatio Environmental Characteristics of Parks for Physical Activity. J. Phys. Act. Health 2006, 3, 176–189. [Cro References [CrossRef] Urban Sci. 2023, 7, 115 17 of 17 55. Hu, Z.; Liebens, J.; Ranga, K.R. Linking stroke mortality with air pollution, income, and greenness in northwest Florida: An ecological geographical study. Int. J. Health Geogr. 2008, 7, 1–22. [CrossRef] 55. Hu, Z.; Liebens, J.; Ranga, K.R. Linking stroke mortality with air pollution, income, and greenness in northwest Florida: An ecological geographical study. Int. J. Health Geogr. 2008, 7, 1–22. [CrossRef] g g g p y J g 56. Bird, M.; Datta, G.; Van Hulst, A.; Kestens, Y.; Barnett, T. A reliability assessment of a direct-observation park evaluation tool: The Parks, activity, and recreation among kids (PARK) tool. BMC Public Health 2015, 15, 906. [CrossRef] [PubMed] y g 57. Bedimo-Rung, A.L.; Gustat, J.; Bradley, J.T.; Rice, J.; Thomson, J. Development of a Direct Observation Instrument to Measure Environmental Characteristics of Parks for Physical Activity. J. Phys. Act. Health 2006, 3, 176–189. [CrossRef] y y y 58. Saelens, B.E.; Frank, L.D.; Auffrey, C.; Whitaker, R.C.; Burdette, H.L.; Colabianchi, N. Measuring Physical Environments of Parks and Playgrounds: EAPRS Instrument Development and Inter-Rater Reliability. J. Phys. Act. Health 2006, 3, 190–207. [CrossRef] [PubMed] [ ] 59. Kaczynski, A.T.; Robertson-Wilson, J.; Decloe, M. Interaction of perceived neighborhood walkability and self-efﬁcacy on physical activity. J. Phys. Act. Health 2012, 9, 208–217. [CrossRef] [PubMed] [ ] 59. Kaczynski, A.T.; Robertson-Wilson, J.; Decloe, M. Interaction of perceived neighborhood walkability and self-efﬁcacy on physical activity. J. Phys. Act. Health 2012, 9, 208–217. [CrossRef] [PubMed] [ ] 59. Kaczynski, A.T.; Robertson-Wilson, J.; Decloe, M. Interaction of perceived neighborhood walkability and self-efﬁcacy on physical activity. J. Phys. Act. Health 2012, 9, 208–217. [CrossRef] [PubMed] 59. Kaczynski, A.T.; Robertson-Wilson, J.; Decloe, M. Interaction of perceived neighborhood walkability and self-efﬁcacy on physical activity. J. Phys. Act. Health 2012, 9, 208–217. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W4389919270	https://ensaiospioneiros.usf.edu.br/ensaios/article/download/313/200	Portuguese	null	BIOSSOLUBILIZAÇÃO DE ROCHAS E RESÍDUOS DE ROCHAS COMO FONTES ALTERNATIVAS DE FERTILIZANTES PARA A AGRICULTURA	Revista Ensaios Pioneiros	2,023	cc-by	5,810	BIOSSOLUBILIZAÇÃO DE ROCHAS E RESÍDUOS DE ROCHAS COMO FONTES ALTERNATIVAS DE FERTILIZANTES PARA A AGRICULTURA NASCIMENTO, Mariana Ruiz Frazão1,4; ROCHA, Daniele Leonel2,4; VIDEIRA, Sandy Sampaio3; DE SOUZA, Ivana Miguel4; CUNHA, Cláudia Duarte4 1Pesquisadora PCI - CNPq - Centro de Tecnologia Mineral 2Pesquisadora - FINEP - Centro de Tecnologia Mineral 3Pesquisadora - FAPERJ - Centro de Tecnologia Mineral 4 Graduanda – BIC - CNPq - Centro de Tecnologia Mineral 4Pesquisadora do Centro de Tecnologia Mineral msantiago@cetem.gov.br NASCIMENTO, Mariana Ruiz Frazão1,4; ROCHA, Daniele Leonel2,4; VIDEIRA, Sandy Sampaio3; DE SOUZA, Ivana Miguel4; CUNHA, Cláudia Duarte4 1Pesquisadora PCI - CNPq - Centro de Tecnologia Mineral 2Pesquisadora - FINEP - Centro de Tecnologia Mineral 3Pesquisadora - FAPERJ - Centro de Tecnologia Mineral 4 Graduanda – BIC - CNPq - Centro de Tecnologia Mineral 4Pesquisadora do Centro de Tecnologia Mineral msantiago@cetem.gov.br RESUMO. O potássio (K) é um nutriente essencial para todos os organismos vivos, sendo um macronutriente fundamental para o desenvolvimento das plantas. As rochas que possuem elevados teores de potássio podem ser aproveitadas como fontes alternativas de fertilizantes para aplicação direta no solo, com o objetivo de atender a alta demanda agrícola brasileira, reduzindo assim, a dependência da importação de fertilizantes. Além disso, é sabido que muitos microrganismos presentes no solo têm a capacidade de solubilizar o potássio contido nos minerais. Nesse contexto, o presente estudo buscou avaliar o potencial de bactérias isoladas de um solo tropical, na solubilização do potássio presente em rochas e resíduos de rocha. Foram isoladas 72 estirpes bacterianas, das quais 5 apresentaram potencial para biossolubilização de potássio, a partir do teste de halo. Os ensaios de biossolubilização de potássio in vitro utilizando as estirpes selecionadas foram realizados em meio Aleksandrov, com a adição do pó de rocha como única fonte de potássio no meio. Os resultados mostraram que todas as estirpes bacterianas selecionadas foram capazes de solubilizar o potássio presente nos pós de rocha. Dentre as estirpes testadas, a IA13 se destacou por ter apresentado o maior percentual de extração de potássio em relação ao controle abiótico (259,82% para a Amostra 8, 300% para a Amazonita e 216,13% para o Kamafugito). Dessa forma, as estirpes bacterianas avaliadas neste estudo demonstraram potencial para serem testadas em processos biohidrometalúrgicos. Esses resultados são considerados promissores, frente aos desafios e a complexidade da remoção de potássio por rota biológica. INTRODUÇÃO O potássio (K) é um dos 16 elementos essenciais para o crescimento e desenvolvimento de animais, seres humanos e plantas. No processo de nutrição vegetal é um macronutriente importante para o desenvolvimento, pois desempenha papéis significativos na ativação de vários processos metabólicos, incluindo fotossíntese, síntese de proteínas e enzimas, bem como na resistência a doenças, pragas, entre outros (MEENA et al., 2016; SATTAR et al., 2018; NAIN et al., 2023). É considerado para as plantas o terceiro nutriente mais importante depois do nitrogênio (N) e fósforo (P) (SOUMARE; SARR; DIÉDHIOU, 2023). Em geral, os solos contêm quantidades substanciais de potássio, porém, apenas de 1% a 2% do potássio total presente no solo encontra-se prontamente disponível (SOUMARE; SARR; DIÉDHIOU, 2023; NAIN et al., 2023). Como a maior parte do potássio contido nos minerais presentes no solo é insolúvel, este nutriente se encontra indisponível para a planta. A liberação ocorre naturalmente por meio da decomposição desses minerais, sendo um processo que demanda um tempo considerável para atender às necessidades agrícolas. Consequentemente, a baixa solubilidade limita a capacidade de fornecimento direto de nutrientes às plantas, tornando a aplicação de fertilizantes no solo uma prática necessária (ALVES et al., 2010; SCHUELER et al., 2021). O Brasil é responsável por cerca de 6% do consumo global de fertilizantes, sendo o segundo maior consumidor do mundo. O crescimento da demanda por fertilizantes no Brasil tem superado a média de crescimento mundial, resultando em um aumento significativo das importações. A situação se torna ainda mais preocupante quando analisamos fertilizante potássico, pois, de acordo com os dados do Ministério de Agricultura e Pecuária (MAPA) do ano de 2020, o país importou cerca de 94% do potássio utilizado na agricultura nacional. Portanto, o Brasil como grande consumidor de fertilizantes, precisa direcionar suas pesquisas para a exploração de fontes alternativas de potássio, com o objetivo de reduzir a dependência das importações de fertilizantes químicos, visando atender à crescente demanda agrícola (BRASIL, 2023). Nesse contexto, o desenvolvimento de novos processos que visem obter nutrientes, especificamente potássio a partir de rochas existentes no território nacional seria benéfico tanto para o setor agrícola quanto para o setor mineral (SWOBODA; DORING; HAMER, 2022). Palavras-chave: Biossolubilização; pó de rocha; potássio; microrganismos. ABSTRACT. Potassium (K) is an essential nutrient for all living organisms and serves as a fundamental macronutrient for plant development. Rocks abundant in potassium can serve as alternative sources for direct soil application as fertilizers, aiming to reduce Brazil's agricultural demand, and reduce reliance on imported fertilizers. Additionally, it is well established that numerous soil microorganisms have the capacity to solubilize potassium found in minerals. In this context, this study sought to evaluate the potential of bacteria isolated from a tropical soil, in solubilizing potassium present in rocks and waste rock. A total of 72 bacterial strains were isolated, of which 5 showed potential for potassium biosolubilization, as determined through the halo test conducted on plates. In vitro potassium biosolubilization assays employing the selected strains were conducted in Aleksandrov’s medium, with rock powder as the sole source of potassium in the culture medium. The results demonstrated that all tested bacterial strains were capable of solubilizing the potassium present in the rock powder. Among the strains tested, IA13 exhibited the highest percentage of potassium extraction in comparison to the abiotic control (259.82% increase for Sample 8, 300% for Amazonite and 216.13% for Kamafugito). Consequently, the bacterial strains tested in this study exhibit potential for further application in biohydrometallurgical processes. These findings hold promise, considering the challenges and complexity associated with biological potassium extraction. Keywords: Biosolubilization; rock powder; potassium; microorganisms. configurar uma alternativa capaz de auxiliar na obtenção de fertilizantes a serem utilizados na agricultura (EL-SHABRAWY et al., 2019) configurar uma alternativa capaz de auxiliar na obtenção de fertilizantes a serem utilizados na agricultura (EL-SHABRAWY et al., 2019) No grupo dos microrganismos solubilizadores de potássio podem ser encontrados tanto bactérias, dos gêneros Pseudomonas, Burkholderia, Acidothiobacillus, Bacillus, Paenibacillus, Klebsiella, Enterobacter, Pantoea, Microbacterium, Agrobacterium, Rhizobium quanto fungos dos gêneros Aspergillus e Penicillium, já descritos na literatura (MEENA et al., 2015, PADHAN et al., 2019, BASAK et al., 2020). Os microrganismos envolvidos na biossolubilização operam através de diversos mecanismos para disponibilizar o potássio, entre eles a liberação de ácidos orgânicos, ácidos inorgânicos, produção de exopolissacarídeos (EPS) e formação de biofilme (PADHAN et al., 2019; NAIN et al., 2023). O presente estudo teve como objetivo avaliar a eficácia de diferentes estirpes bacterianas isoladas de solo agrícola na solubilização in vitro de potássio presente em diferentes rochas e resíduos de rochas brasileiras, e identificar as condições de processo mais adequadas para promover uma maior liberação desse elemento específico. Rochas e resíduos de rochas potássicas utilizadas nos ensaios As amostras de rochas e resíduos minerais utilizados no experimento foram: Amazonita, que é uma variedade do feldspato microclínio, proveniente de Potiraguá, BA; Kamafugito, uma rocha de origem vulcânica, máfica e ultramáfica, e a Amostra 8 (pó de brita), um resíduo obtido de uma mineradora localizada na região Centro-Oeste do Brasil. As amostras continham 5.28%, 3% e 1.18% de teor de K2O em suas composições, respectivamente. INTRODUÇÃO No entanto, devido a dificuldade de liberação desse elemento a partir das rochas, faz-se necessário a escolha de uma rota adequada, face às diferentes estruturas mineralógicas existentes, para estabelecer um processo eficiente de disponibilização dos nutrientes nelas encontrados (BAGHEL et al., 2020). ( ) Assim, o desenvolvimento de novas tecnologias para a solubilização de potássio a partir de agrominerais utilizando rotas biológicas surge como uma alternativa interessante na busca por soluções para o suprimento de fertilizantes no mercado nacional (ALI et al., 2019). Além do aproveitamento direto das rochas, como fontes de nutrientes para as plantas, o desenvolvimento de um processo biohidrometalúrgico para a solubilização de potássio pode 38 38 Isolamento e seleção de estirpes bacterianas da rizosfera de sorgo forrageiro para ensaios de solubilização de potássio Inicialmente foi realizado um ensaio agronômico, conduzido em casa-de-vegetação, utilizando vasos contendo 8 kg de terra proveniente do campo experimental da Embrapa Agrobiologia, que apresentava baixa fertilidade natural, principalmente baixos teores de fósforo e potássio (obtida no âmbito do Projeto Universal do CNPq intitulado “Respostas do microbioma de solo agrícola à aplicação de remineralizadores como fontes alternativas de nutrientes para as plantas”). Neste experimento foram realizados 4 tratamentos em quadruplicata nas seguintes condições: (i) não fertilizado (controle), (ii) fertilizado com resíduo mineral (Amostra 8 - Pó de brita), (iii) fertilizado com resíduos da moagem de Amazonita e (iv) fertilizado com Kamafugito, pelo período de aproximadamente 145 dias que incluiu a incorporação dos compostos com a terra por 15 dias, plantio de crotalária (Crotalaria juncea L.) e corte da parte aérea destas plantas para deposição das mesmas sob a terra por mais 45 dias, incorporação e manejo por mais 15 dias e por fim o plantio das sementes de sorgo forrageiro com a colheita final em 70 dias após o plantio. A partir deste ensaio, foi realizado o isolamento de estirpes bacterianas utilizando amostras de solo rizosférico obtidas da última etapa do processo (ao final dos 145 dias). Amostras de 5 g de solo proveniente dos diferentes tratamentos foram adicionadas em frascos erlenmeyer de 250 mL contendo 100 mL de solução salina. Os frascos foram agitados à 150 rpm e 30 oC por 2 horas. A partir destas soluções, foram realizadas diluições seriadas até 10-6 e 0.1 ml da cada diluição foi adicionado em placas de Petri contendo meio TSA (Tryptic Soy Agar) utilizando a técnica Spread Plate. As placas foram incubadas em estufa à 30 ± 1 °C por 24/48 horas e as colônias foram selecionadas de acordo com suas características morfológicas. 39 39 As estirpes bacterianas selecionadas foram testadas quanto a sua pureza após esgotamento e repicadas para tubo de ensaio contendo o mesmo meio, sendo armazenadas a 4 °C para uso posterior. Para detecção do potencial de solubilização de potássio pelas estirpes isoladas foi realizado o teste do halo (RAJAWAT et al., 2016) em placas de Petri contendo meio Aleksandrov modificado (composição em g/L: 5.0 glicose; 0.5 MgSO4⋅7H2O; 0.005 FeCl3; 0.1 CaCO3; 2.0 CaH4PO4), contendo 15 g/L de Agar nutriente e 2.0 g/L de cada pó de rocha como fonte de potássio (HU et al., 2006). Isolamento e seleção de estirpes bacterianas da rizosfera de sorgo forrageiro para ensaios de solubilização de potássio O corante azul de bromotimol (concentração de 0.25%) foi adicionado ao meio antes da esterilização. Foi adicionada em cada placa uma microgota da suspensão bacteriana preparada a partir de um pré-inóculo de 24 horas das estirpes selecionadas. As placas foram incubadas em estufa a 30 ± 1 °C por 2–3 dias. A detecção do potencial de solubilização do potássio pelas estirpes isoladas foi baseada na mudança da coloração do meio, de azul esverdeado para amarelo. Ensaios de biossolubilização de potássio As estirpes isoladas que apresentaram resposta no teste do halo foram testadas em ensaios de biossolubilização, em meio Aleksandrov (HU et al., 2006). Além das estirpes isoladas foi testada também uma estirpe do gênero Paenibacillus proveniente do banco de coleção de culturas do Laboratório de Biotecnologia (Labiotec) do CETEM, conhecidamente solubilizadora de potássio. O pH do meio de cultura foi ajustado para 7 antes da esterilização. Os pós de rochas testados (1.0 % p/v) foram esterilizados separadamente antes da adição ao meio de cultura, como única fonte de potássio. Os experimentos foram conduzidos em frascos erlenmeyer de 250 mL contendo 100 mL de meio de cultura. O inóculo foi padronizado em 10% (v/v) para cada estirpe após a realização das suas curvas de crescimento. Os frascos foram agitados por 10 dias a 30 °C e 150 rpm. Ensaios controle foram realizados contendo apenas o meio de cultura e o pó de rocha e todas as condições (bióticas e abióticas) foram feitas em triplicata. Ao final do experimento, as amostras foram centrifugadas a aproximadamente 4500 rpm à 4 °C por 20 minutos e os sobrenadantes foram filtrados utilizando membranas de 0.22 μm. A concentração de potássio em solução (mg.L-1) foi determinada por espectrometria de absorção atômica (LIU et al., 2006). Isolamento e seleção de bactérias com potencial de biossolubilização de potássio Foram isoladas 72 estirpes bacterianas a partir dos 4 tratamentos estudados, sendo 16 do tratamento sem adição de fertilizante, controle (i), 14 do tratamento com fertilização utilizando resíduo mineral – Pó de brita (Amostra 8) (ii), 17 do tratamento com fertilização utilizando resíduos da moagem de Amazonita (iii) e 25 do tratamento com fertilização utilizando pó de rocha Kamafugito (iv). Foi realizado o teste do halo para triagem rápida de microrganismos com capacidade de solubilização de potássio, com base na melhor visualização da formação dos halos ao redor da colônia. A utilização do corante azul de bromotimol no meio Aleksandrov (PARMAR; SINDHU, 2018; RAJI; THANGAVELU, 2021) permite visualizar a mudança de coloração do meio, de verde azulado para amarelo, que é um indicativo da produção de ácidos orgânicos pelos microrganismos, como mecanismo para solubilizar principalmente P e K contidos nas rochas e resíduos de rochas (ARIA et al., 2010; BASHIR et al., 2018). 40 Das 72 estirpes avaliadas, 5 apresentaram potencial para solubilização de potássio. Vale ressaltar que após o crescimento das estirpes isoladas da amostra proveniente do plantio sem adição de pó de rocha (tratamento i), nenhuma apresentou mudança na coloração do meio. Em contrapartida, 2 estirpes bacterianas isoladas de solo contendo a rocha Amazonita (tratamento iii) e 3 estirpes bacterianas isoladas de solo contendo a rocha Kamafugito (tratamento iv) apresentaram mudança de coloração. Apenas para a condição em que o solo foi fertilizado com resíduo de mineração (Amostra 8 – tratamento ii), cujo teor de K2O era muito baixo (1,8%), não houve estirpe isolada que tivesse ocasionado mudança de coloração no meio, assim como foi observado no tratamento sem fertilização. A Tabela 1 mostra os resultados referentes ao tamanho do halo formado pelos 5 microrganismos que apresentaram potencial para biossolubilização de K e a estirpe Paenibacillus polymyxa, utilizando os 3 pós de rocha em estudo. Os microrganismos que apresentaram os maiores tamanhos de halos foram os isolados IA4 e IA13, e a estirpe Paenibacillus polymyxa, para todos os pós de rocha testados. Tabela 1 – Tamanhos dos halos formados pelas estirpes bacterianas selecionadas. Isolamento e seleção de bactérias com potencial de biossolubilização de potássio Pó de rocha Estirpes bacterianas Halo Amostra 8 IA4 ++ IA13 ++ IK7 + IK10 + IK16 + Paenibacillus + Amazonita IA4 ++ IA13 + IK7 + IK10 + IK16 + Paenibacillus + Kamafugito IA4 ++ IA13 ++ IK7 + IK10 + IK16 + Paenibacillus ++ +, halos < 0,1 cm ao redor da colônia ++, halos > 0,1 cm ao redor da colônia Fonte: Próprio autor Na Figura 1 é possível visualizar a imagem de uma placa de Petri com o meio de cultivo contendo o indicador azul de bromotimol. É possível notar a mudança na coloração após o período de incubação de 48h. O uso do corante azul de bromotimol demonstrou ser eficaz na identificação preliminar de microrganismos capazes de solubilizar potássio, o que também foi corroborado por diversos estudos anteriores (PARMAR; SINDHU, 2013; RAJAWAT et al., 2016; AZIZAH et al., 2020; BOUBEKRI et al., 2021; ONYEWENJO et al., 2021). Tabela 1 – Tamanhos dos halos formados pelas estirpes bacterianas selecionadas. Pó de rocha Estirpes bacterianas Halo Amostra 8 IA4 ++ IA13 ++ IK7 + IK10 + IK16 + Paenibacillus + Amazonita IA4 ++ IA13 + IK7 + IK10 + IK16 + Paenibacillus + Kamafugito IA4 ++ IA13 ++ IK7 + IK10 + IK16 + Paenibacillus ++ +, halos < 0,1 cm ao redor da colônia ++, halos > 0,1 cm ao redor da colônia Fonte: Próprio autor Tabela 1 – Tamanhos dos halos formados pelas estirpes bacterianas selecionadas. Na Figura 1 é possível visualizar a imagem de uma placa de Petri com o meio de cultivo contendo o indicador azul de bromotimol. É possível notar a mudança na coloração após o período de incubação de 48h. O uso do corante azul de bromotimol demonstrou ser eficaz na identificação preliminar de microrganismos capazes de solubilizar potássio, o que também foi corroborado por diversos estudos anteriores (PARMAR; SINDHU, 2013; RAJAWAT et al., 2016; AZIZAH et al., 2020; BOUBEKRI et al., 2021; ONYEWENJO et al., 2021). 41 Figura 1 – Mudança na coloração do meio após o período de incubação da estirpe IA13 por 48h. Figura 1 – Mudança na coloração do meio após o período de incubação da estirpe IA13 por 48h. Figura 1 – Mudança na coloração do meio após o período de incubação da estirpe IA13 por 48h. Isolamento e seleção de bactérias com potencial de biossolubilização de potássio Figura 1 – Mudança na coloração do meio após o período de incubação da estirpe IA13 por 48h Fonte: Próprio autor Fonte: Próprio autor Esses resultados mostram que as 5 estirpes isoladas apresentam potencial para uma possível utilização como inoculantes em solos agrícolas, assim como em ensaios biohidrometalúrgicos para obtenção de potássio em solução, que corresponde a uma alternativa tecnológica sustentável para a produção de fertilizantes potássicos. A partir dos resultados obtidos, foram realizados ensaios de biossolubilização para quantificar o potássio extraído dos pós de rochas. Ensaios prospectivos de biossolubilização Os resultados da solubilização de potássio pelas estirpes bacterianas estão apresentados na Tabela 2. É possível observar que todos os microrganismos testados foram capazes de remover potássio das três rochas utilizadas nos ensaios. As concentrações de K obtidas pelos microrganismos variaram de 200 a 500 mg.kg-1. Os maiores valores de solubilização foram obtidos pela bactéria isolada da rocha Amozonita (IA13) nos três pós de rochas testados, alcançando valores de solubilização de 500 mg.kg-1 para a Amazonita, 490 mg.kg-1 para o Kamafugito e 340 mg.kg-1 para a Amostra 8, após 10 dias de ensaio utilizando meio Aleksandrov. Em geral, os mecanismos de biossolubilização de potássio presente em alguns minerais podem envolver a capacidade de alguns microrganismos em produzir ácidos orgânicos, sideróforos, polissacarídeos capsulares e ligantes orgânicos. A diminuição do pH promove a liberação de prótons que facilita o processo de solubilização (ZARJANI et al., 2013; PARMAR; SINDHU, 2013; GROUDEV, 1987; SATTAR et al., 2018). Os polissacarídeos podem adsorver fortemente os ácidos orgânicos e aderir à superfície do mineral, resultando em uma área de alta concentração de ácidos (LIU et al., 2012). No presente estudo, em todos os ensaios houve diminuição do valor pH e, consequentemente aumento das concentrações de potássio, sugerindo produção de ácidos orgânicos pelas estirpes testadas. Os resultados obtidos foram satisfatórios quando comparados aos resultados recentes obtidos na literatura (AZIZAH et al., 2020; SUN et al., 2020; BOUBEKRI et al., 2021; YONGMEI; PANPAN 2021; CHINACHANTA; SHUTSRIRUNG, 2021). Tabela 2 – Concentração e percentual de solubilização de potássio pelas estirpes isoladas e pela estirpe de Paenibacillus polymyxa após 10 dias de incubação em meio Aleksandrov. Tabela 2 – Concentração e percentual de solubilização de potássio pelas estirpes isoladas e pela estirpe de Paenibacillus polymyxa após 10 dias de incubação em meio Aleksandrov. la 2 – Concentração e percentual de solubilização de potássio pelas estirpes isoladas e pela estirpe d bacillus polymyxa após 10 dias de incubação em meio Aleksandrov. Ensaios prospectivos de biossolubilização p y y p ç Pó de rocha Estirpes bacterianas K (mg.kg-1) Extração (%) pH Amostra 8 Controle 94,5 ± 07,77 0,96 ± 0,00 7 IA4 270,0 ± 14,14 2,76 ± 0,14 6 IA13 340,0 ± 00,00 3,47 ± 0,00 6 IK7 245,0 ± 21,21 2,50 ± 0,08 6 IK10 205,0 ± 21,21 2,09 ± 0,21 6 IK16 280,0 ± 70,71 2,86 ± 0,72 6 Paenibacillus polymyxa 210,0 ± 14,14 2,14 ± 0,02 5 Amazonita Controle 125,0 ± 07,07 0,26 ± 0,01 7 IA4 395,0 ± 07,07 0,82 ± 0,01 6 IA13 500,0 ± 14,14 1,04 ± 0,03 6 IK7 295,0 ± 21,21 0,61 ± 0,04 6 IK10 310,0 ± 14,14 0,64 ± 0,03 6 IK16 250,0 ± 42,42 0,52 ± 0,09 6 Paenibacillus polymyxa 200,0 ± 28,28 0,41 ± 0,06 5 Kamafugito Controle 155,0 ± 07,07 0,62 ± 0,03 7 IA4 330,0 ± 00,00 1,33 ± 0,00 6 IA13 490,0 ± 42,42 1,97 ± 0,17 6 IK7 270,0 ± 00,00 1,08 ± 0,00 6 IK10 275,0 ± 07,07 1,10 ± 0,03 6 IK16 285,0 ± 35,35 1,14 ± 0,14 6 Paenibacillus polymyxa 260,0 ± 28,28 1,04 ± 0,11 5 Isolado Amazonita 4: IA4; Isolado Amazonita 13: IA13; Isolado kamafugito 7: IK7; Isolado kamafugito 10: IK10; Isolado kamafugito 16: IK16. Fonte: Próprio autor Isolado Amazonita 4: IA4; Isolado Amazonita 13: IA13; Isolado kamafugito 7: IK7; Isolado kamafugito 10: IK10; Isolado kamafugito 16: IK16. Fonte: Próprio autor Dentre os valores de concentração de potássio obtidos nos ensaios de biossolubilização com o isolado IA13, o maior valor (3,47% p/p) foi alcançado na presença da Amostra 8. Em um estudo realizado por Matias et al. (2019) foi avaliado o desempenho do isolado bacteriano Acidithiobacillus thiooxidans FG-01 na biossolubilização de potássio, utilizando 20 g/L de rocha verdete, por 49 dias, em meio de cultura 9 K (SILVERMAN; LUNDGREN, 1959). Nesse estudo foram obtidos 6,6% de potássio a partir da rocha verdete. Embora o resultado tenha sido superior aos obtidos pelas estirpes bacterianas deste estudo, é importante notar que o tempo de ensaio foi cinco vezes superior, com o dobro da densidade de polpa (pó de rocha no meio de cultura), o que aumenta significativamente os custos do processo. ) q g p Além disso, em um estudo realizado por Schueler et al. (2021) foram avaliadas as estirpes bacterianas Burkholderia sp., Bacillus sp., C. Glathei, P. Ensaios prospectivos de biossolubilização caribensis isoladamente além de um consórcio contendo todas, nos ensaios de biossolubilização utilizando meio Aleksandrov, e a rocha verdete, por 15 dias. Os resultados do percentual de extração de potássio foram de 0,68%, 0,61%, 0,48%, 0,50% e 0,58%, respectivamente. Estes resultados mostram que, apesar do tempo de processo ter sido maior, os valores percentuais de extração de potássio foram inferiores aos obtidos no presente estudo. Vale ressaltar, que apesar de o maior valor de concentração de potássio no meio (500 mg.kg-1) ter sido obtido para o isolado IA13 na presença da rocha Amazonita, o percentual de 43 43 extração foi o menor (1,04%) em função do maior teor de K2O presente nesta amostra, quando comparado a Amostra 8. Da mesma forma, observou-se que com o uso da Amazonita, os menores percentuais de extração de potássio foram obtidos para todos os microrganismos testados. Isto pode ter ocorrido devido às diferentes composições mineralógicas das amostras testadas. Em geral, os feldspatos possuem uma estrutura de rede tridimensional de Si e Al, coordenada tetraedricamente com oxigênio, que precisa ser destruída para a liberação do potássio. Dessa forma, é sugerido que feldspatos necessitam de escalas de tempo mais longas para a biossolubilização de potássio. Tais informações demonstram que ensaios abordando as mesmas condições físico-químicas e biológicas, podem apresentar diferentes resultados de solubilização, devido à diferença de estrutura dos minerais. extração foi o menor (1,04%) em função do maior teor de K2O presente nesta amostra, quando comparado a Amostra 8. Da mesma forma, observou-se que com o uso da Amazonita, os menores percentuais de extração de potássio foram obtidos para todos os microrganismos testados. Isto pode ter ocorrido devido às diferentes composições mineralógicas das amostras testadas. Em geral, os feldspatos possuem uma estrutura de rede tridimensional de Si e Al, coordenada tetraedricamente com oxigênio, que precisa ser destruída para a liberação do potássio. Dessa forma, é sugerido que feldspatos necessitam de escalas de tempo mais longas para a biossolubilização de potássio. Tais informações demonstram que ensaios abordando as mesmas condições físico-químicas e biológicas, podem apresentar diferentes resultados de solubilização, devido à diferença de estrutura dos minerais. Na Figura 2, é apresentado o gráfico contendo a concentração de potássio no meio para todas as condições testadas em relação ao ensaio controle abiótico. CONCLUSÃO A partir das 72 estirpes bacterianas isoladas de solo rizosférico, 5 foram capazes de extrair o potássio das rochas em 10 dias de processo, utilizando meio Aleksandrov, e diferentes pós de rocha como fonte única de potássio. De acordo com os resultados obtidos, os ensaios de biossolubilização de potássio foram considerados positivos quando comparados ao ensaio controle, em relação a disponibilização de potássio. Todas as estirpes bacterianas apresentaram potencial nos ensaios in vitro, ressaltando que os melhores resultados encontrados ocorreram na presença da estirpe isolada IA13, onde foram alcançados 500 mg.kg-1 de potássio em solução para a amostra de Amazonita, 490mg.kg-1 para a amostras de Kamafugito e 340mg.kg-1 para a Amostra 8. A partir destes resultados, novos ensaios complementares deverão ser realizados com o intuito de otimizar o processo para obtenção de maiores valores de remoção. Ensaios prospectivos de biossolubilização O maior percentual de extração de potássio em relação ao controle (aumento de 300%) foi obtido com a utilização da rocha Amazonita na presença do microrganismo IA13. Figura 2 – Extração de K(%) em meio de cultura contendo o pó de rocha e os microrganismos em relação ao % de extração do controle abiótico (somente o pó de rocha). Figura 2 – Extração de K(%) em meio de cultura contendo o pó de rocha e os microrganismos em relação ao % de extração do controle abiótico (somente o pó de rocha). Fonte: Próprio autor Fonte: Próprio autor Fonte: Próprio autor Ainda de acordo com o gráfico é possível observar que todos os ensaios conduzidos na presença dos diferentes microrganismos, apresentaram percentuais de extração de potássio superiores a 50% quando comparados ao ensaio controle. O menor aumento percentual na extração de potássio em relação ao controle (aumento de 60%) foi obtido com o microrganismo Paenibacillus polymyxa e a rocha Amazonita. Em comparação com os demais microrganismos, os menores valores percentuais de extração de potássio em relação ao controle foram obtidos quando a rocha Kamafugito foi utilizada. Em um estudo conduzido por Sarikhani et al. (2018), o potencial de biossolubilização de potássio por um isolado bacteriano do gênero Pseudomonas foi avaliado em meio de cultura Aleksandrov, utilizando os minerais moscovita e biotita. Nesse 44 estudo foram observados aumentos na extração de potássio de 127% e 188% em relação ao controle, respectivamente. Neste contexto, o presente trabalho demonstrou a potencialidade de diferentes microrganismos isolados de um solo suplementado com pó de rocha, na biossolubilização de potássio in vitro. Foi observado que os valores de remoção de potássio obtidos estão de acordo com os encontrados na literatura. Com isso, tais resultados impulsionam a adoção de novas estratégias e rotas de biossolubilização para obter maiores valores de remoção de potássio a partir das rochas. AGRADECIMENTOS Os autores agradecem ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), a Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) e a Financiadora de Estudos e Projetos (FINEP) pelo apoio financeiro, ao Centro de Tecnologia Mineral (CETEM) pela infraestrutura e aos pesquisadores Marisa Bezerra de Mello Monte e Luiz Carlos Bertolino pelo fornecimento das amostras utilizadas no estudo. (Iris kashmiriana) rhizosphere. The Pharma Innovation Journal. v. 7, n. 4, p. 747-750, 2018. BAGHEL, V.; THAKUR, J. K.; YADAV, S. S.; MANNA, M. C.; MANDAL, A.; SHIRALE, A. O.; SHARMA, P.; SINHA, N. K.; MOHANTY, M.; SINGH, A. B.; PATRA, A. K. Phosphorus and potassium solubilization from rock minerals by endophytic Burkholderia sp. strain FDN2-1 in soil and shift in diversity of bacterial endophytes of corn root tissue with crop growth stage. Geomicrobiology Journal.v.37, n.6, p. 1-13, 2020. BAGHEL, V.; THAKUR, J. K.; YADAV, S. S.; MANNA, M. C.; MANDAL, A.; SHIRALE, A. O.; SHARMA, P.; SINHA, N. K.; MOHANTY, M.; SINGH, A. B.; PATRA, A. K. Phosphorus and potassium solubilization from rock minerals by endophytic Burkholderia sp. strain FDN2-1 in soil and shift in diversity of bacterial endophytes of corn root tissue with crop growth stage. Geomicrobiology Journal.v.37, n.6, p. 1-13, 2020. BASAK, B. B.; MAITY, A.; RAY, P.; BISWAS, D. R.; ROY, S. Potassium supply in agriculture through biological potassium fertilizer: A promising and sustainable option for developing countries. Archives of Agronomy and Soil Science. v. 68, p. 101-114, 2020. BOUBEKRI, K.; SOUMARE, A.; MARDAD, I.; LYAMLOULI, K.; HAFIDI, M.; OUHDOUCH, Y.; KOUISNI, L. The screening of potassium- and phosphate- solubilizing actinobacteria and the assessment of their ability to promote wheat growth parameters. Microrganisms. v. 9, n. 470, p. 1-16, 2021 RASIL. Ministério da Indústria Comércio Exterior e Serviços. Governo Federal. Comex Stat. Disponível em: http://comexstat.mdic.gov.br/pt/comex-vis. Acesso em: mai. 2023. CHINACHANTA, K.; SHUTSRIRUNG, A. Screening for P- and K- solubilizing and siderophore producing capacity of rhizobacteria from Khao Dawk Mali 105 Aromatic Rice. IOP Conf. Series: Earth Environmental Science. p. 1-13, 2021. EL-SHABRAWY, R.M.I., ALY., M. E.; EL-HOSEINY, O.H.M.; ISMAIL, S. A. Improving of feldspar efficiency as potassium fertilization for potato (Salenum Tuberosumi) by using silicate dissolving bacteria and compost. Annals of Agricultural Sciences, v. 57, n. 1, p. 185- 196, 2019. GROUDEV, S. N. Use of heterotrophic microorganisms in mineral biotechnology. Acta Biotechnology. v.17, p. 299-306, 1987. LIU, D.; LIAN, B.; DONG, H. Isolation of Paenibacillus sp. and assessment of its potential for enhancing mineral weathering. Geomicrobiology Journal. v. 29, p. 413-421, 2012. MANNING, D. A. C., BAPTISTA, J., SANCHEZ LIMON, M., & BRANDT, K. Testing the ability of plants to access potassium from framework silicate minerals. Science of The Total Environment, v. 574, p. 476–481, 2017. MATIAS, P. C.; MATTIELLO, E. M.; SANTOS, W. O.; JADEL, J. L.; ALVAREZ, V. H. Solubilization of a K-silicate rock by Acidithiobacillus thiooxidans. REFERÊNCIAS ALI, A. M.; AWAD, M.; HEGAB, S. A.; ABD EL-GAWAD, A. Promoting effect of potassium solubilizing bacteria (Bacillus cereus) on nutrients availability and yield of potato. Archives of Agricultural Sciences Journal. v. 2, n. 2, p. 43-54, 2019. ALVES, L.; OLIVEIRA, V. L.; SILVA FILHO, G. N. Utilization of rocks and ectomycorrhizal fungi to promote growth of eucalypt. Brazilian Journal of Microbiology. v. 41, p. 676-684, 2010. ALVES, L.; OLIVEIRA, V. L.; SILVA FILHO, G. N. Utilization of rocks and ectomycorrhizal fungi to promote growth of eucalypt. Brazilian Journal of Microbiology. v. 41, p. 676-684, 2010. ARIA, M.M.; LAKZIAN, A.; HAGHNIA, G.H.; BERENJI, A.R.; BESHARATI, H.; FOTOVAT, A. Effect of Thiobacillus, sulfur, and vermicompost on the water-soluble phosphorus of hard rock phosphate. Bioresource Technology. v. 101, p. 551-554, 2010. AZIZAH, H.; RAHAJENG, S. M.; JATMIKO, Y. D. Isolation and screening of phosphate and potassium solubilizing endophytic bacteria in Maize (Zea mays L.). The Journal of Experimental Life Science. v. 10, n. 3, p. 165- 170, 2020. BASHIR, Z.; ZARGAR, M. Y.; BABA, Z. A.; MOHIDDIN, F. A.; PEER, F. A.; MIR, S. A.; NAJAR, G. R. Solubilization of potassium containing mineral by bacteria from Kashmir iris 45 (Iris kashmiriana) rhizosphere. The Pharma Innovation Journal. v. 7, n. 4, p. 747-750, 2018. (Iris kashmiriana) rhizosphere. The Pharma Innovation Journal. v. 7, n. 4, p. 747-750, 2018. Minerals Engineering. v. 132, p. 69-75, 2019. MEENA, V. S.; MAURYA, B. R.; VERMAC, J. P.; AEROND, A.; KUMARA, A.; KIM, K.; BAJPAIF, V. K. Potassium solubilizing rhizobacteria (KSR): isolation, identification, and K- release dynamics from waste mica. Ecological Engineering. v. 81, p. 340-347, 2015. MEENA V. S., BAHADUR I., MAURYA B. R., KUMAR A., MEENA R. K., MEENA S. K., VERMA J. P. Potassium-solubilizing microorganism in evergreen agriculture: An overview. In: Meena V, Maurya B, Verma, J, Meena R (eds). Potassium solubilizing microorganisms for sustainable agriculture. p. 1-20, 2016. 46 NAIN, A.; CHAUDHARY, K.; SHARMA, C.; SHEORAN, H. Potassium solubilization in soils. Ecofarming. v. 3, n. 1, p. 58-63, 2023. NATH, D.; MAURYA, B. R.; KHAN, S. In-vitro solubilization of waste minerals and morphological characteristics of potassium and phosphorus solubilizing bacteria. Journal of Pharmacognosy and Phytochemistry. v. 7, n. 2, p. 1626-1632, 2018. PARMAR, P.; SINDHU, S. S. Potassium solubilization by rhizosphere bacteria: influence of nutritional and environmental conditions. Journal of Microbiology Research. v. 3, n. 1, p. 25-31, 2013. PARMAR, P.; SINDHU, S. S. The novel and efficient method for isolating potassium solubilizing bacteria from rhizosphere soil. Geomicrobiology Journal. v.36, n.2, p. 1-7, 2018. PADHAN, D.; SEN, A.; KUNDU, R.; YADAV, V. K. Potassium solubilisation in soils: mechanisms, effect on plant growth and future prospects. Current Research in soil fertility. AkiNik Publications. Chapter 3. p. 37-59, 2019. RAJAWAT, M. V. S.; SINGH, S.; TYAGI, S. P.; SAXENA, A. K. A modified plate assay for rapid screening of potassium-solubilizing bacteria. Pedosphere. v. 26, n. 5, p. 768–773, 2016. RAJI, M..; THANGAVELU, M. Isolation and screening of potassium solubilizing bacteria from saxicolous habitat and their impact on tomato growth in different soil types. Archives of Microbiology. p. 1-15, 2021. SATTAR, A.; NAVEED, M.; ALI, M.; ZAHIR Z. A.; NADEEM, S. M.; YASEEN, M.; MEENA, V. S.; FAROOQ, M.; SINGH, R.; RAHMANF, M.; MEENA, H. N. Perspectives of potassium solubilizing microbes in sustainable food production system: A review. Applied Soil Ecology. p. 1-14, 2018. SARIKHANI, M. R.; OUSTAN, S.; EBRAHIMI, M.; ALIASGHARZAD, N. Isolation and identification of potassium-releasing bacteria in soil and assessment of their ability to release potassium for plants. European Journal of soil Science. p. 1-9, 2018. SCHUELER, T. A.; DOURADO, M. L.; VIDEIRA, S. S.; CUNHA, C. D.; RIZZO, A. C. L. Biosolubilization of verdete: An alternative potassium source for agriculture fertilizer. Biocatalysis and Agricultural Biotechnology. v. (Iris kashmiriana) rhizosphere. The Pharma Innovation Journal. v. 7, n. 4, p. 747-750, 2018. 34, p.1-6, 2021. SILVERMAN, M.P.; LUNDGREN, D.G. Studies on the chemoautotrophic iron bacterium Ferrobacillus ferrooxidans. I. An improved medium and a harvesting procedure for securing high cell yields. Journal of Bacteriology. v. 77, p. 642–647, 1959. SOUMARE, A.; SARR, D.; DIÉDHIOU, A. G. Potassium sources, microorganisms and plant nutrition: Challenges and future research directions. Pedosphere. v. 33, n. 1, p. 105-115, 2023. SUN, F.; OU, Q.; WANG, N.; GUO, Z. X.; OU, Y.; Li, N.; PENG, C. Isolation and identification of potassium-solubilizing bacteria from Mikania micrantha rhizospheric soil and their effect on M. micrantha plants. Global Ecology and Conservation. v. 23, p. 1-9, 2020. SUN, F.; OU, Q.; WANG, N.; GUO, Z. X.; OU, Y.; Li, N.; PENG, C. Isolation and identification of potassium-solubilizing bacteria from Mikania micrantha rhizospheric soil and their effect on M. micrantha plants. Global Ecology and Conservation. v. 23, p. 1-9, 2020. ZARJANI, J. K.; ALIASGHARZAD, N.; OUSTAN, S.; EMADI, M.; AHMADI, A. Isolation and characterization of potassium solubilizing bacteria in some Iranian soils. Archives of Agronomy and Soil Science. v. 59, n.12, p. 1713-1723, 2013. ZARJANI, J. K.; ALIASGHARZAD, N.; OUSTAN, S.; EMADI, M.; AHMADI, A. Isolation and characterization of potassium solubilizing bacteria in some Iranian soils. Archives of Agronomy and Soil Science. v. 59, n.12, p. 1713-1723, 2013. ZARJANI, J. K.; ALIASGHARZAD, N.; OUSTAN, S.; EMADI, M.; AHMADI, A. Isolation and characterization of potassium solubilizing bacteria in some Iranian soils. Archives of Agronomy and Soil Science. v. 59, n.12, p. 1713-1723, 2013. Publicado em 18/12/2023.

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.